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(Journal of Leukocyte Biology. 2001;70:551-558.)
© 2001 by Society for Leukocyte Biology

Crotalus durissus terrificus snake venom regulates macrophage metabolism and function

S. C. Sampaio*, M. C. C. Sousa-e-Silva*, P. Borelli2, R. Curi3 and Y. Cury*

* Laboratory of Pathophysiology, Butantan Institute;
{dagger} Laboratory of Haematology, Department of Clinical Analyses, Faculty of Pharmaceutics Science; and
{ddagger} Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Brazil

Correspondence: Prof. Y. Cury, Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900, São Paulo, Brazil. E-mail: yarac{at}attglobal.net


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we examined the effect of Crotalus durissus terrificus venom on rat macrophage metabolism and function. Two hours after subcutaneous injection of the venom, peritoneal resident (unstimulated), elicited (thioglycollate-stimulated), and activated Mycobacterium bovis strain bacille Calmette Guérin (BCG) macrophages were collected, and their functional and metabolic parameters were analyzed. The venom inhibited spreading and phagocytosis of macrophages. On the other hand, this treatment increased H2O2 and NO production, candidacidal activity, and the activities of key enzymes of glycolysis and glutaminolysis. We also investigated whether the venom could affect macrophage activation by thioglycollate or BCG. The administration of venom 2 h before injection of thioglycollate and BCG or 2 or 3 days after injection of the thioglycollate or BCG, respectively, did not modify the previous observations. These findings suggest that crotalic venom leads the macrophage to an activated state, with high production of oxygen- and nitrogen-reactive species. This cell activation state does not include inflammatory properties of spreading and phagocytosis.

Key Words: glycolysis • glutaminolysis Krebs cycle • phagocytosis • nitric oxide • oxidative metabolism • candidacidal activity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The venom of most viperid snakes is highly phlogistic in humans [1 , 2 ]. However, there is an exception, the venom of the South American rattlesnake Crotalus durissus terrificus. This venom does not induce an inflammatory response at the site of the bite. Studies in animals have shown that this venom inhibits the immune and inflammatory responses and causes antinociception [3 4 5 6 ]. The venom or crotoxin, the main neurotoxic component of this venom composed of a phospholipase A2 and a polypeptide named crotapotin [7 ], has an inhibitory effect on the humoral immune response by interfering with the synthesis of immunoglobulin G (IgG) antibodies [3 ]. An anti-inflammatory activity is described for crotapotin, based on the observation of an inhibitory activity of this polypeptide on the oedematogenic response induced by carrageenin [4 ]. In spite of the intriguing observations that crotalic venom inhibits inflammatory response, there is only one study concerning the effect of this venom on macrophage function. Sousa-e-Silva et al. [5 ] reported that C. durissus terrificus venom (Cdt), either in vivo or in vitro, inhibits spreading and the phagocytic capacity of mouse peritoneal macrophages.

Macrophages are terminally differentiated end cells [8 ] characterized as motile, highly secretory, and phagocytic cells [9 , 10 ]. These cells play a central role for the inflammatory and immunological responses. They are found in several organs and tissues such as the serous cavities. In the tissues or organs, these cells remain quiescent as resident cells with low functional activities [11 ]. Nevertheless, macrophages become elicited or activated by inflammatory or immunologic stimuli [for review, see ref. 11 ]. In this case, newly recruited macrophages with markedly different secretory and endocytic properties can accumulate in large numbers at specific sites [12 , 13 ]. Experimentally, elicited macrophages can be obtained by injection of sterile thioglycollate [14 ]. These cells are high in secretory activity and phagocytic capacity but present low microbicidal or tumoricidal activity [15 , 16 ]. Activated macrophages express low secretory activity but present high capacity in secreting reactive-oxygen or -nitrogen species and important microbicidal and tumoricidal activity. Activated cells can be experimentally obtained by the injection of Mycobacterium bovis strain BCG [17 18 19 20 ]. Besides these important functional actions, macrophages present marked metabolic activity with high capacity for utilization of glucose and glutamine [21 , 22 ]. These metabolites provide ATP and are precursors for DNA, RNA, protein, and lipid synthesis important for the secretory activity of these cells and for the synthesis of new cell membrane after phagocytic activity [23 ]. In fact, glucose and glutamine metabolism is markedly stimulated both in elicited and activated macrophages as compared with resident cells [24 ].

The information above led us to carry out a systematic study on the effect of Cdt venom on macrophage metabolism and function. The aim was to answer the following question: does the crotalic venom inhibit all functional activities and the metabolism of macrophage or lead to a particular activation stage of these cells that does not exhibit inflammatory properties? To investigate the effects of the venom on glucose and glutamine metabolism, key enzyme activities of several metabolic pathways were measured in resident (unstimulated), elicited (thioglycollate-stimulated), and activated bacille Calmette Guérin (BCG)-stimulated macrophages. The maximum activities of hexokinase, a quantitative index of maximum glycolytic flux [22 , 25 ]; glucose-6-phosphate dehydrogenase (G6PDH), a quantitative index of maximum flux through the pentose-phosphate pathway; and phosphate-dependent glutaminase, a quantitative index of maximum flux through glutaminolysis [22 ], were determined. Citrate synthase activity was also determined, although it provides only a qualitative index of the flux through the Krebs cycle [26 ]. The oxidation of [U-14C]glucose and [U-14C]glutamine was also measured as an additional approach to study macrophage metabolism. The following aspects of macrophage function were also analyzed: spreading, phagocytic activity of opsonized zymosan or Candida albicans, candidacidal activity, and production of hydrogen peroxide and nitric oxide. The treatment with the venom was performed following three different protocols: (1) the venom was administered 2 h before collecting resident, elicited, or activated macrophages; (2) the venom was given 2 or 4 days after thioglycollate or BCG injections, respectively, and (3) the administration of the venom was performed 2 h before thioglycollate or BCG injections. These three protocols allowed us to compare the effect of the venom with those well-known for thioglycollate and BCG and to examine whether the venom could interfere with the response to these stimuli.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male Wistar rats weighing 170–190 g were used throughout this study. These animals were obtained from the Central Animals’ House of the Butantan Institute, São Paulo, Brazil.

Venom
Lyophilized venom of C. durissus terrificus was obtained from the Laboratory of Herpetoloy, Butantan Institute, São Paulo, Brazil, and stored at -20°C. The venom (30 µg/rat) was dissolved in sterile physiological saline [0.85% (w/v) NaCl solution] at the moment of use and administered by the subcutaneous (s.c.) route. The venom was given 2 h before collecting the peritoneal cells, 2 or 4 days after thioglycollate or BCG injections, respectively, or 2 h before thioglycollate or BCG injections. The doses of venom used did not cause clinical signs of crotalic envenomation, such as neurotoxic facies, external and internal ophthalmoplegia, and respiratory paralysis [1 ].

Peritoneal cell preparation
Animals were anaesthetized with ether and sacrificed by exsanguination by sectioning the cervical vessels. The peritoneal cavity was washed with 10 mL of cold phosphate-buffered saline (PBS), pH 7.4. After a gentle massage of the abdominal wall, the peritoneal fluid containing resident macrophages was collected. Elicited macrophages were obtained by peritoneal lavage 4 days after injection of 3 mL of thiogycollate broth (4%) in the peritoneal cavity. Activated cells were obtained by the lavage of the cavity 7 days after intraperitoneal administration of 40 mg of heating inactivated ONCO-BCG (bacille Calmette Guérin, supplied by the Butantan Institute). Cell viability was assessed by the Trypan blue exclusion test (>95%). Total peritoneal cells were determined in a Neubauer’s chamber, and the differential counts were performed in smears stained with modification of Wright’s and May Giemsa stains [27 ]. For all measurements, samples of individual animals were used. The assays were always performed in duplicates.

Spreading of peritoneal macrophages
The spreading capacity of macrophages was estimated according to the method described by Rabinovitch and DeStefano [28 ]. Briefly, 200 µL of cell suspension in PBS (106 of total cells) were placed onto glass cover slips and left to adhere for 15 min at room temperature. The cover slips were washed with PBS and incubated in 1640 RPMI medium at 37°C for 2 h. Cells were fixed in a 2.5% glutaraldehyde solution, and the index of spread cells was determined by examination under phase-contrast microscopy. The spreading activity index was defined as the ratio between spread cells and 100 cells counted.

Preparation of zymosan (Saccharomyces cerevisiae)
The zymosan particles were resuspended in PBS containing Ca2+ and Mg2+ ions providing a concentration of particles of 11.4 mg/2 mL. For opsonization, 2 mL of zymosan particles (11.4 mg/2 mL in PBS) were mixed with 2 mL of normal rat serum and incubated for 30 min at 37°C [29 ]. The opsonized zymosan particles were then washed, resuspended in PBS at a concentration of 11.4 mg/2 mL.

Phagocytic activity of peritoneal macrophages
Macrophages (106 cells/mL) were incubated with 1 mL of PBS containing 2% bovine serum albumin, glucose (5 mM), glutamine (2 mM), and opsonized zymosan for 40 min at 37°C, in an atmosphere containing 5% CO2. The percentage of phagocytosis was determined by counting (in a Neubauer’s chamber) the percentage of cells that had phagocytosed more than three particles of zymosan.

Phagocytosis of C. albicans and candidacidal activity
C. albicans (ATCC Y-537) were cultured in 20% Sabouraud’s dextrose broth (Microbiology and Mycology Laboratories, Department of Clinical Analyses, Faculty of Pharmaceutics Science, University of São Paulo) at 30°C for 4–5 days. The fungi were centrifuged, washed twice with Dulbecco PBS, suspended to a concentration of 2–4 x 107/mL. Viability was determined by exclusion of methylene blue 0.05% (>98%), and the number of Candida bacteria was determined in a Neubauer’s chamber. Phagocytosis and candidacidal activity was studied by the method described by Sasada and Johnston [30 ], modified by Corazzini [31 ]. Macrophages (2 x 106 cells/mL) were incubated in sterile plastic tubes (to avoid cell adherence) at 37°C with opsonized C. albicans (2 x 107 cells/mL), in the presence of Dulbecco’s PBS, pH 7.4, 20% fetal calf serum, and 1640 RPMI medium. After 30, 60, 90, 120, or 180 min of incubation, an aliquot of 100 µL of this suspension were adhered to cover slip glass by cytocentrifugation. After centrifugation, cover slips were stained with Wright’s and May-Giemsa stains. The percentage of phagocytosis was determined by counting the percentage of macrophages that had phagocytosed more than three particles of C. albicans. For each rat, two cover slips were prepared and 100 cells per cover slip were counted. For candidacidal activity determination, cell viability of phagocytosed particles was assessed by the dye exclusion (Rosenfeld) test. Different scores were given to the number of macrophages that had killed no Candida cells (x0); 1 or 2 cells (x1); 3 or 4 cells (x2); or >4 cells (x3). The index of candidacidal activity was calculated by the sum of the scores obtained per rat.

Hydrogen peroxide production
The production of H2O2 was measured as described by Pick and Mizel [32 ], modified by Russo et al. [33 ]. The peritoneal cells were adjusted to 4 x 106 cells/mL, centrifuged for 10 min, and resuspended in 1 mL of phenol red solution (PRS), containing 140 mM NaCl, 10 mM potassium-phosphate buffer, pH 7.0, 5 mM dextrose, 0.28 mM phenol red, and 8.5 U/mL of horseradish peroxidase for H2O2 detection. The final volume of 7.4 mL was obtained with Hank’s solution. One hundred microliters of the cell suspension were plated onto each well of 96-well flat-bottomed tissue culture plates (Corning, NY) and incubated in a humidified atmosphere at 37°C for 1 h. Vertical row no. 1 was left without cells and filled with 100 µL per well of PRS. The second and third vertical rows were used for the establishment of H2O2 standard curves. These wells were covered with 100 µL of PRS, added to 10 µL of H2O2 solution, resulting in a final concentration of H2O2 ranging from 5 to 40 µM. The subsequent rows contained wells covered with 100 µL of PRS in the absence (basal H2O2 production) or the presence of phorbol myristate acetate (20 ng). After 60 min of incubation at 37°C, the reaction was stopped by the addition of 10 µL 1 N NaOH solution. Hydrogen peroxide-dependent phenol red oxidation was measured spectrophotometrically at 620 nm, in a Titertek Multiscan apparatus. The concentration of H2O2 was calculated from absorbance measurements, as described by Pick and Mizel [32 ], and expressed as nanomoles of H2O2 per milligram of cell protein.

Nitric oxide production
For determination of nitric oxide, the production of nitrite was measured in the supernatants of cultured macrophages based on the method described by Ding et al. [34 ]. Macrophages (106 cells/mL) obtained as described above were maintained in RPMI 1640 culture medium at 37°C and an environment of 5% CO2 for 48 h. At the end of the culture period, 50 or 100 µL of the supernatant were removed and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylene diamine dihydrochloride, 2.5% H3PO4) at room temperature for 10 min. The absorbance at 550 nm was determined in a Titertek Multiscan apparatus. Nitrite was determined by using sodium nitrite as a standard. Cell-free medium alone contained 0.2–0.3 nmol of NO2-/well; this value was determined in each experiment and subtracted from the value obtained with cells.

Enzyme assays
Macrophages (107 cells) were resuspended in 300 µL of the extraction medium of each enzyme and sonicated for disruption of cellular membrane. The cells were then centrifuged at 1,000 rpm, and the supernatant was used for enzyme assays.

Enzyme activities were determined as previously described [35-38]. The extraction medium for hexokinase (E.C. 2.7.1.1) contained 25 mM Tris-HCl, 1 mM EDTA, and 30 mM ß-mercaptoethanol at pH 7.4 and that for glutaminase (EC 3.5.1.2) contained 150 mM potassium phosphate, 1 mM EDTA, and 50 mM Tris-HCl at pH 8.6. The extraction medium for citrate synthase (E.C. 4.1.3.7) and G6PDH (E.C.1.1.1.49) contained 50 mM Tris-HCl and 1 mM EDTA; the final pH values were 7.4 for citrate synthase and 8.0 for G6PDH. For all enzyme assays, 0.05% (v/v) Triton X-100 was added to the assay system to complete the extraction of the enzymes. For the assay of MgCl2 hexokinase, the following medium was used: 75 mM Tris-HCl, 7.5 mM MgCl2, 0.8 mM EDTA, 1.5 mM KCl, 4.0 mM ß-mercaptoethanol, 0.4 mM creatine phosphate, 1.8 U of creatine kinase, 1.4 U of G6PDH, 0.4 mM NADP, pH 7.5. The assay of citrate synthase consisted of 100 mM Tris-HCl, 0.2 mM 5,5'-dithiobis-2-nitrobenzoic acid, 15 mM acetyl-coenzyme A, and 0.5 mM oxaloacetate, pH 8.1. The assay medium for G6PDH consisted of 86 mM Tris-HCl, 6.9 mM MgCl2, 0.4 mM NADP, 1.2 mM glucose-6-phosphate, 1.2 U of 6-phosphogluconate dehydrogenase, pH 7.6. The assay medium for glutaminase consisted of 50 mM phosphate buffer (equimolar mixture of KH2PO4 and K2HPO4), 0.2 mM EDTA, 50 mM Tris-HCl, and 20 mM glutamine, pH 8.6. The final volume of the assay mixture was 1.0 mL. Citrate synthase was assayed by following the rate of change in absorbance at 412 nm, and the remainder enzymes were assayed by monitoring the rate of change in absorbance at 340 nm. All spectrophotometric measurements were performed using a Gilford (Response) recording spectrophotometer at 25°C, except for glutaminase, which was determined at 37°C. Preliminary experiments established that extraction and assay procedures were such as to produce maximum activities [39 ] for all enzymes studied.

Metabolite measurements
Macrophages were incubated (0.5 x 107 cells/flask) at 37°C for 1 h, in PBS, pH 7.4, with 1.5% defatted bovine serum albumin in the presence of [U-14C]glucose (5.6 mM/0.2 µCi/mL) or [U-14C]glutamine (2 mM/0.2 µCi/mL). After incubation, the cells were disrupted with 0.4 mL of 25% trichloroacetic acid. The 14CO2 produced from [U-14C]glucose and [U-14C]glutamine was collected as previously described [40 ], and the radioactivity was counted in a Beckman-LS 5000 TD liquid scintillator (Beckman Instruments, Fullerton, CA).

Expression of results
The enzyme activities are expressed as nanomoles of substrate utilized/minute per 107 cells. The rates of consumption and production of metabolites and of decarboxylation are presented as nanomoles per hour per 107 cells.

Statistical analysis
Results are presented as the means ± SE. Statistical evaluation of the results was carried out by analysis of variance and sequential differences among means according to Bonferroni contrast analysis at P < 0.05 [41 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of venom administration 2 h before macrophage collection
Peritoneal mononuclear cell number
The s.c. injection of the venom 2 h before macrophage collection did not alter the number of mononuclear cells present in the peritoneal cavity of untreated rats (resident cells) or of rats pretreated with thioglycollate or BCG (Fig. 1A ).



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  		Figure 1. Effect of Cdt venom on the number of peritoneal mononuclear cells (A), and spreading activity (B) and phagocytic capacity (C) of peritoneal macrophages. The cell counts were determined in the peritoneal fluid obtained from untreated rats (resident) or rats injected 4 or 7 days before the experiments with thioglycollate (elicited) or BCG (activated), respectively. The venom (30 µg/rat) or saline (control group) was s.c. injected 2 h before peritoneal cells were collected. Particles of zymosan opsonized with serum of untreated rats were used for phagocytosis studies. The results are expressed as means ± SE for six animals per group. *P < 0.05, significantly different from control group.

 
Spreading and phagocytosis index
Cdt venom induced a significant inhibition of spreading activity of peritoneal resident (25%), elicited (18%), or activated (33%) macrophages (Fig. 1B) . As shown in Fig. 1C , inhibitory effect was also observed on the phagocytic activity of these cells (34% for resident, 20% for elicited, and 13% for activated cells).

Hydrogen peroxide and nitrite production
The crotalic venom caused an increase in the stimulated [by phorbol myristate acetate (PMA)] production of H2O2 of peritoneal resident (88%), elicited (50%), or activated (13%) macrophages and the induction of NO2- of peritoneal resident (252%), elicited (155%), or activated (37%) macrophages (Table 1 ).


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  		Table 1. Effect of Crotalus durissus terrificus Venom on Hydrogen Peroxide and Nitrite Production by Peritoneal Macrophages

 
Candidacidal activity
As presented in Table 2 , the crotalic venom increased the killing of opsonized C. albicans by resident (after 30–120 min of incubation), elicited (after 90–180 min of incubation), and activated (after 30–90 min of incubation) macrophages, as compared with control animals treated with saline. In this experiment, the index of phagocytosis was also determined. The results showed that the Candida phagocytosis by macrophages was also inhibited by the venom.


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  		Table 2. Effect of Crotalus durissus terrificus Venom on Candidacidal and Phagocytic Activities of Peritoneal Macrophage

 
Metabolism of glucose and glutamine
The crotalic venom increased the maximal activity of hexokinase, G6PDH, citrate synthase, and phosphate-dependent glutaminase of resident (68%, 57%, 66%, and 63%, respectively), elicited (58%, 25%, 20%, and 15%, respectively), and activated (40%, 68%, 38%, and 40%, respectively) macrophages (Table 3 ).


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  		Table 3. Effect of the Crotalus durissus terrificus Venom on Maximal Activities of Hexokinase (HK), Citrate Synthase (CS), Glucose-6-Phosphate Dehydrogenase (G6PDH), and Phosphate-Dependent Glutaminase (GLUNASE) of Peritoneal Macrophages

 
The production of 14CO2 from [U-14CO2]glucose and [U-14CO2]glutamine was also raised by Cdt venom in resident (117% and 58%, respectively), elicited (45% and 19%, respectively) and activated (34% and 45%, respectively) macrophages (Table 4 ).


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  		Table 4. Effect of Crotalus durissus terrificus Venom on 14CO2 Production from [U-14C]Glucose and [U-14C]Glutamine by 1-h-incubated peritoneal macrophages

 
Effect of the venom administered before and during macrophage activation by either thioglycollate or BCG
The administration of the venom before and during macrophage activation, by either thioglycollate or BCG, caused effects as those found when it was given just 2 h before collecting the cells. The venom reduced spreading activity and phagocytosis capacity and raised hydrogen peroxide production and the enzyme activities (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Data presented herein showed that Cdt snake venom inhibits spreading and phagocytic activities of rat peritoneal macrophages. On the other hand, this venom stimulated hydrogen peroxide and nitric oxide production, candidacidal activity, and the metabolism of glucose and glutamine. These effects were not modified by the macrophage activation stage, because they are observed in resident, elicited, and activated cells.

The inhibitory effects of the venom are not a consequence of alterations in cell viability because (1) >95% of the cells are viable and (2) variations in the number of cells present in the peritoneal cavity of the animals treated with the venom could not be detected.

The inhibitory effect of crotalic venom on the spreading activity of macrophages may contribute to the altered phagocytic activity of these cells. It is well established that spreading is an important mechanism for the interaction between the phagocyte and the ingestible particle [42 ]. Both processes use the actin cytoskeleton to achieve a controlled advance of pseudopodia across a surface [for review, see ref. 43 ]. Therefore, substances that inhibit macrophage [44 45 46 ] spreading interfere in the same way with the phagocytic process [28 , 42 , 45 , 47 ].

Similar results were obtained by the pioneer work of Sousa-e-Silva et al. [5 ]. These authors found lower spreading and phagocytosis capacities of macrophage obtained from mice injected, either intraperitoneally or s.c., with Cdt venom. The same was observed when the venom was added into the medium of 1-h-incubated macrophages, that is 50% inhibition in the presence of 0.25 µg/mL. Our results and those obtained by Sousa-e-Silva et al. [5 ] indicate that the effect of crotalic venom on the phagocytic process is not related to the type of membrane receptor because the inhibitory effect was observed for both C3b (opsonized zymosan and C. albicans)- and Fc (opsonized sheep erythrocytes)-mediated phagocytosis. Nevertheless, an effect of the venom on cell membrane receptors can not be ruled out.

The process of phagocytosis stimulates the respiratory burst of the phagocytic cells [48 ] in phagosomes via NADPH oxidase [for review, see ref. 49 ]. Despite the inhibition of spreading and phagocytosis observed in our studies, the venom stimulated the production of hydrogen peroxide. This effect was observed only in the presence of PMA. This tumor-promoting phorbol diester stimulates the oxidative burst, bypassing the need for receptor-ligand coupling, stimulating a Ca2+-dependent protein kinase C [50 , 51 ]. Therefore, the mechanism of the venom action remains unknown, but it might potentiate the PMA effect.

The venom also increased the production of nitric oxide. This result indicates an action of this venom on the iNOS, because this enzyme is the main source for the production of NO in macrophages [34 , 52 , 53 ]. Recent data have shown that NO is an important effector of the actions of the crotalic venom because its peripheral analgesic action is mediated by stimulation of the NO-cyclic-GMP pathway [54 ].

The stimulation of H2O2 and NO production by the venom could explain the enhanced microbicidal (candidacidal) activity of the macrophages. Toxic products derived from the oxidative-burst and reactive-nitrogen species mediate the microbicidal and tumoricidal activity of macrophages [55 56 57 ]. Also, these reactive species play an important role in the killing of C. albicans and C. parapsilosis by macrophages [55 56 57 58 ].

The stimulatory effect of the venom on production of H2O2 and NO was observed in the three cell types, but it was clearly more pronounced in resident cells. Resident macrophages are activated on appropriate inflammatory or immunological stimuli, with acquisition of different capacities and functions. This process distinguishes different stages and characterizes the macrophages as elicited or activated cells [8 , 9 , 12 , 13 ]. Our results suggest that crotalic venom, at least for the capacities of H2O2 and NO production and microbicidal function, acts as an inductor of macrophage activation. Under 2 h of venom stimulation, the resident macrophage, a quiescent cell, acquires specific metabolic and functional properties. It is important that, different from thioglycollate or BCG, which must be injected 4 or 7 days before cell harvest, respectively, for full expression of their capacity of activating macrophage, the stimulatory effect of the venom was detected just 2 h after its administration. The activation state of the macrophage induced by the venom presented particular characteristics of stimulated respiratory burst but suppressed spreading and phagocytic activities.

The present results of a dual effect of Cdt venom on macrophage function are in agreement with data showing that substances able to inhibit the phagocytic capacity of macrophages, like carrageenin, simultaneously activate the production of cytokines and NO and the ability of these cells to kill yeast cells. This candidacidal activity has been correlated with the increased production of NO [59 ; for review, see ref. 60 ].

Macrophages utilize glucose and glutamine at high rates [10 , 61 ]. It has been demonstrated that this metabolism is related to the specific function of these cells in the inflammatory and immunological responses [24 ]. Glucose and glutamine metabolism provides ATP and precursors for biosynthetic pathways [23 , 62 ]. Macrophages and their metabolism are highly influenced by the activation stage of these cells [24 ]. Cdt venom stimulates the maximal activities of hexokinase, G6PDH, phosphate-dependent glutaminase, and citrate synthase. The increased activity of these enzymes explains the augmented consumption of glucose and glutamine, as indicated by the increased oxidation of [U-14C]glucose and [U-14C]glutamine.

The increased G6PDH activity suggests an augmented flux of substrates through the pentose-phosphate pathway that might enhance the production of NADPH, which is the substrate for NADPH oxidase. In macrophages, as in other leukocytes, production of hydrogen peroxide involves a membrane-associated NADPH oxidase that removes electrons from NADPH to reduce O2 into O2-, which is rapidly dismuted to H2O2 [63 ].Therefore, a high production of NADPH may comprise the mechanism for the increased production of H2O2 induced by the venom. Correlation between increased G6PDH activity and H2O2 production has been previously reported in macrophages activated by BCG [24 ].

High flux of substrates through the pentose-phosphate pathway and the production of NADPH might also contribute to the increased production of NO. Various cytosolic electron donors such as NADPH, flavin adenine dinucleotide, flavin mononucleotide, and glutathione [for review, see ref. 53 ] are required for full iNOS activity [64 , 65 ]. On the other hand, macrophage metabolism can be stimulated by the products of iNOS [64 ]. The activity of iNOS has been correlated with increased consumption of glucose, higher glycolysis and hexose-monophosphate shunt activities, activation of G6PDH, and decreased flux of glucose through the tricarboxylic acid cycle [64 , 65 ]. Therefore, the increased production of NO induced by crotalic venom may contribute to the stimulation of glucose consumption. However, a direct stimulatory effect of the venom on key enzymes of glucose and glutamine metabolism can not be ruled out.

The inhibitory effect of Cdt venom on the spreading and phagocytic capacity of macrophage and the stimulatory action on the production of H2O2 and NO and activity of key enzymes of glycolysis and glutaminolysis are also observed when the venom is administered before or during macrophage activation by either thioglycollate or BCG. Cell mobilization to the peritoneal cavity in response to injection of thioglycollate or BCG was not modified by the venom. These data are not in agreement with those obtained by Sousa-e-Silva et al. [5 ], who observed that injection of crotalic venom in mice inhibits leukocyte migration induced by intraperitoneal injection of thioglycollate. Also, Sousa-e-Silva et al. [5 ] showed in in vitro assays that the inhibitory effect of the venom on macrophage spreading and phagocytosis was not detected when the cells were already elicited by thioglycollate. Differences in the dose of the venom and the animal used may contribute to explain these distinct results.

The inhibitory and stimulatory actions of crotalic venom on macrophage metabolism and function are long lasting effects. Similar changes were observed even after 7 days of venom administration. This conclusion was obtained from experiments in which the venom was administered 2 h prior to the injection of thioglycollate or BCG. In this case, the cells were harvested 4 or 7 days after the venom injection. These data are in agreement with those obtained by Sousa -e-Silva et al. [5 ], who observed that the inhibitory effect of the venom on spreading and phagocytosis persists for 16 days after venom administration. The low turnover of resident peritoneal macrophages [66 ] may partially explain the long lasting effect of the venom on these cells. The total number of blood monocytes was not changed by venom administration (data not shown). This fact however does not exclude the possibility that circulating monocytes are already modified even before migrating to the peritoneal cavity. In addition to a prolonged action of the venom on macrophage metabolism and function, recent study has shown that the analgesic effect of this venom is also detected even 5 days after oral administration of a unique dose [54 ].

In conclusion, the data herein presented show that Cdt venom induces a dual effect on macrophage function. The stimulatory effect of the venom on H2O2 and NO production, candidacidal activity, and glucose and glutamine metabolism and the inhibition of spreading and phagocytosis suggest that crotalic venom induces a unique activation stage of the macrophage. The significance and the molecular mechanisms for paradoxical responses of the macrophages caused by venom as well as characterization of the substances present in the venom responsible for the observed effects are under investigation. Further studies might also be carried out to correlate this unique activation state of the macrophage induced by the venom with the outcome of the snakebite.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Pronex and Fundação Butantan. The authors are grateful to Stella Regina Zamunér for help with the nitric oxide production assay.

Received July 20, 2000; revised May 14, 2001; accepted May 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Rosenfeld, G. (1971) Symptomatology, pathology and treatment of snake bites in South America Bëcherl, W. Buckley, E. eds. Venomous Animals and Their Venoms 2nd ed. New York NY.
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