Induction of Mast-Cell Accumulation by Promutoxin, an Arg-49 Phospholipase A2

Local inflammation is a prominent characteristic of snakebite wound, and snake-venom phospholipase A2s (PLA2s) are some of the main component that contribute to accumulation of inflammatory cells. However, the action of an R49 PLA2s, promutoxin from Protobothrops mucrosquamatus venom, on mast-cell accumulation has not been previously examined. Using a mouse peritoneal model, we found that promutoxin can induce approximately-6-fold increase in mast-cell accumulation, and the response lasts at least for 16 h. The promutoxin-induced mast cell accumulation was inhibited by cyproheptadine, terfenadine, and Ginkgolide B, indicating that histamine and platelet-activating factor (PAF) is likely to contribute to the mast-cells accumulation. Preinjection of antibodies against adhesion molecules ICAM-1, CD18, CD11a, and L-selectin showed that ICAM-1, and CD18, CD11a are key adhesion molecules of promutoxin-induced mast-cell accumulation. In conclusion, promutoxin can induce accumulation of mast cells, which may contribute to snake-venom wound.

Mast cells are primarily located in mucosal and perivascular areas of various tissues, which play an important role in body-defense processes. Mast cells can be activated by snake-venom and release carboxypeptidase A and possibly other proteases, which can degrade venom components [21,22]. Several snake-venom PLA 2 s were reported to be able to activate the rat mast cells and to induce microvascular leakage and in�ammatory-cell accumulation at the sites of in�ammation [15][16][17][18][19][20]. Our previous studies showed that TM-N49, an N49 PLA 2 puri�ed from Protobothrops mucrosquamatus venom, induces skin edema and mast-cell activation and accumulation [23], and promutoxin, an R49 PLA 2 puri�ed from the same snake, can activate mast cells and induce skin edema [24]. Both TM-N49 and promutoxin are devoid of catalytic activity and are thought to contribute to Protobothrops mucrosquamatus venom toxicity [5,8]. Moreover, both TM-N49 and promutoxin are potent stimuli for induction of neutrophil, lymphocyte, macrophage, and eosinophil accumulation in the mouse peritoneum [25]. ese observations suggested that the two novel subgroups of group II PLA 2 may contribute to the in�ammatory process caused by snake-venom poisoning. However, the ability of R49 PLA 2 on induction of mast-cell accumulation has not yet been reported. In the present study, we investigated the mechanisms of promutoxin-induced mast-cell accumulation.

Puri�cation of Promutoxin.
Promutoxin was isolated from Protobothrops mucrosquamatus crude venom following the procedures described previously [8]. Brie�y, the lyophilized venom (1.5 g) was dissolved in 20 mL of 0.05 M sodium phosphate buffer (pH 5.8) before being loaded on an SP-Sephadex C-25 column. e absorbed proteins were eluted with a linear gradient (0-0.8 M NaCl). Peak 7 was collected and loaded on Superdex 75 column in 25 mM sodium phosphate buffer (pH 5.8 with 0.15 M NaCl). e main peak was collected, and then loaded on a reversephase C 18 high-performance liquid chromatography (HPLC) (Waters Corporation, Milford, MA, USA). e main peak fraction, representing the puri�ed promutoxin, was pooled, lyophilized, and stored at -20 ∘ C.
Protein concentration was determined by using a Coomassie Plus assay kit with BSA as standard. e PLA 2 activity was routinely assayed by a titration method using egg yolk as substrate [26] and by a colorimetric assay using L-phosphatidylcholine as substrate [27]. Honey-bee PLA 2 was employed as positive control.

Induction of Mast-Cell Accumulation.
Various doses of promutoxin, BSA or normal saline were injected in 0.5 mL volumes into the peritoneum of male BALB/c mice, whose abdominal skin was swabbed with 70% ethanol, a group of 6 mice for each dose. is model was adapted from that described by omas and colleagues [28], which complied with the European Community guidelines for use of experimental animals. At 10 min, 2 h, 6 h, or 16 h following injection, animals were sacri�ced by inhalation of carbon dioxide, and their peritoneal lavages were collected following a standardized procedure with 5 mL normal saline. Aer centrifugation at 500 g for 10 min at 4 ∘ C, supernatants were collected and stored at −40 ∘ C until use, and cells were resuspended in 1 mL of MEM. e total cell numbers were determined by enumerating them with an Improved Neubauer haemocytometer aer being stained with 0.1% trypan blue. For the differential cell counting, cytocentrifuge preparations were made, air dried, and stained with modi�ed Wright's stain. Differential cell counts were performed for a minimum of 500 cells. e results were expressed as absolute numbers of each cell type per mouse peritoneum.
For the experiments investigating mast-cell-migration mechanisms, groups of mice were pretreated intravenously (tail vein injection) with monoclonal antibodies against the adhesion molecules L-selectin, CD11a, CD18, and ICAM-1 (all at a dose of 1 mg⋅kg −1 ) [29][30][31], respectively, for 30 min before intraperitoneal injection of 5 g of promutoxin. Control animals received an equivalent dose of the corresponding normal rat or hamster IgG isotype control. At 6 h following injection, the mice were sacri�ced and their peritoneal lavages were processed as described above.

Statistical
Analysis. Data are shown as mean ± SE for the number of experiments indicated, and ANOVA followed by Tukey's tests were used for statistical comparison of the data. In all analyses, was taken as statistically signi�cant.

Puri�cation and Characteri�ation of Promutoxin.
Approximately 25 mg of promutoxin was obtained from 1.5 g Protobothrops mucrosquamatus crude venom following the procedures described above. e purity of the PLA 2 was at least 98% as assessed by SDS-PAGE, HPLC, and mass spectrometry analysis [24].

Induction of Mast-Cell Accumulation by Promutoxin.
As early as 10 min following injection, 5 g of promutoxin was able to induce signi�cant mast-cell accumulation in the peritoneum of mice. e mast-cell accumulation appeared to last for 16 h (Figure 1). Approximately, up to 6-fold increase in mast-cell numbers was achieved at 16 h following injection (Figure 1).

Discussion
It is found for the �rst time that promutoxin, a novel member of a minor subgroup (R49 PLA 2 ) of snake-venom group II PLA 2 s, can induce mast-cell accumulation. e observation supports our previous �nding that N49 PLA 2 , another minor subgroup of snake-venom group II PLA 2 s [23] can induce mast-cell accumulation. Obviously, promutoxin does not induce mast-cell accumulation in a concentrationdependent manner [24]. We previously found that promutoxin could activate mast-cells. e reduction of mast-cell T 1: e in�uence of anti-in�ammatory compounds on promutoxin-(5 g) induced mast-cell accumulation in mouse peritoneum.

Compound injected
Number number induced by promutoxin in higher doses may be due to the activation of accumulated mast-cells by promutoxin. Ginkgolide B, a PAF antagonist, inhibited more than 35.9% promutoxin-induced mast-cell migration, indicating that PAF is likely involved in mast-cell migration by promutoxin. is result appears to agree with a previous work which found that PAF may contribute to mast-cell migration induced by TM-N49 [23]. Inhibition of 71.3 and 92.6% promutoxin-induced mast-cell accumulation by cyproheptadine, a histamine/5-HT antagonist, and terfenadine a selective histamine-H 1 -receptor antagonist, implies that histamine is likely to be involved in the above event through its H 1 receptor. Indeed, we have found previously that promutoxin can activate mast cells to release histamine [24] and anticipate herein that released histamine then elicits mast-cell migration. e fact that terfenadine inhibited promutoxin-provoked mast-cell recruitment to a greater extent than cyproheptadine further implies the selectivity of histamine receptor involved. It was found that snake-venom promutoxin could induce mast-cell accumulation even at 10 min following injection, but the number of lymphocyte, macrophage, eosinophil, and neutrophil migration was not altered at 10 min following injection of promutoxin [25]. It seemed the accumulation of neutrophils, lymphocyte, macrophage, and eosinophil induced by promutoxin could be a secondary event, in which accumulated mast cells are activated by promutoxin and release their chemoattractant factors such as serine proteinases [36,37], histamine, and PAF to recruit in�ammatory cells.
ICAM-1 appears to be a key adhesion molecule of promutoxin-induced mast-cell accumulation as an antibody against ICAM-1 blocked more than 76.7% of the in�uence of promutoxin on the cell migration. ICAM-1 involved in lymphocyte [38], macrophage [39], eosinophil [40,41] and mast-cell accumulation [23] has been reported previously, which may support our current observations. An antibody against CD11a (LFA-1) blocked more than 53.8% promutoxin-induced mast-cell accumulation, indicating that CD11a plays an important role in the migration of mast cells, which is consistent with previous reports that TM-N49-induced mast-cell accumulation is mediated by CD18, CD11a, and ICAM-1 [23]. As expected, antibody against CD18 blocked more than 87.2% promutoxin-induced mastcell accumulation in the present study. ough L-selectin is involved in the neutrophil and eosinophil accumulation provoked by promutoxin, it seemed that L-selectin is not involved in the promutoxin-induced mast-cell accumulation.
Promutoxin, as a novel member of minor subgroup of PLA 2 , is an enzymatically inactive enzyme. It induced mastcell accumulation via a PAF, and histamine H 1 receptordependent mechanism, and through a CD11a/CD18-and ICAM-1-associated adhesion pathway. Accumulated mast cells may contribute to snake-venom wound.