Induction of mast cell accumulation, histamine release and skin edema by N49 phospholipase A2

Abstract

Background: It has been recognized that phospholipase A2 (PLA2) is a crucial component of snake venom, which contributes greatly to snake venom induced inflammation in man. However, the mechanisms through which N49 PLA2 provoke inflammation remain unclear. Recently, a N49 PLA2, TM-N49 from Protobothrops mucrosquamatus crude venom was characterized in our laboratory. Since the purification procedure developed is able to supply us with relatively large quantity of highly purified TM-N49, we investigated the ability of TM-N49 in induction of inflammation.

Results: The results showed that TM-N49 provoked a dose dependent increase in microvascular leakage in the skin of rats. The potency of TM-N49 in induction of skin edema appeared similar potency of bradykinin and histamine. Pretreatment of rats with compound 48/80 diminished TM-N49 induced skin reaction and reduced mast cell numbers in rats. Ginkgolide B and cyproheptadine, but not terfenadine and quinacrine, inhibited TM-N49 elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, TM-N49 was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting TM-N49 elicited histamine release. TM-N49 induced mast cell accumulation in the peritoneum of mice, which was inhibited by co-injection of ginkgolide B, cyproheptadine and terfenadine. Intravenous injection of monoclonal antibodies against CD18, ICAM-1 and CD11a also blocked TM-N49 induced mast cell accumulation.

Conclusion: TM-N49 is a potent stimulus for skin edema, mast cell activation and accumulation.

Figure 1

Effect of TM-N49 on rat dermal microvascular leakage. Various doses of TM-N49 were injected into the skin of rat for 20 min. Also shown are the responses to BSA, bradykinin and histamine alone at a dose of 5 μg and normal saline control. Skin reaction represented the area of Evan's blue extravasation. Data are displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values other than outliers (O) (defined as those which are more than 1.5 box lengths from the median) for 6 animals in each group. * P < 0.05 compared with the response to the diluent only control animals.

Figure 2

Influence of compound 48/80 on rat dermal microvascular leakage induced by TM-N49 and histamine (A), and on mast cell numbers in the peritoneum of rats (B). Rats were intra-peritoneally injected with compound 48/80 at a dose of 0.6 mg·kg^-1 for 6 h, twice a day for 3 days, and doubled dose in day 4 before TM-N49 (5 μg) or histamine (5 μg) being administrated for 20 min. Skin reaction represented the area of Evan's blue extravasation. Data are displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values other than outliers (O) (defined as those which are more than 1.5 box lengths from the median) for 6 animals in each group. In (A), * P < 0.05 compared with the corresponding response to untreated animals and in (B), * P < 0.05 compared with the response to untreated animals.

Figure 3

Influence of anti-inflammatory drugs on rat dermal microvascular leakage induced by TM-N49 (5 μg), bradykinin (5 μg), PAF (5 μg) and histamine (5 μg). Ginkgolide (GG, 5 μg), cyproheptadine (CH, 5 μg), terfenadine (TF, 5 μg) or quinacrine (QC, 5 μg) were co-injected with TM-N49, bradykinin or histamine, respectively for 20 min, whereas PAF was only co-injected with GG. Skin reaction represented the area of Evan's blue extravasation. Data are displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values other than outliers (O) (defined as those which are more than 1.5 box lengths from the median) for 6 animals in each group. * P < 0.05 compared with the response to the corresponding uninhibited control animals.

Figure 4

Effects of TM-N49 on histamine release from colon, lung and tonsil mast cells in the presence or absence of exogenous calcium and magnesium. The values shown are mean ± SE for four to five separate experiments. Various concentrations of TM-N49 were incubated with cells for 15 min before termination of the reactions. * P < 0.05 compared with the response to the corresponding buffer alone group, ^†P < 0.05 compared with the response to the corresponding group in the absence of calcium and magnesium.

Figure 5

Time course for histamine release from colon, lung and tonsil mast cells induced by TM-N49 (30 μg/ml), anti-IgE antibody (10 μg/ml) and calcium ionophore (1 μg/ml). The values shown are mean ± SE of the percentage of maximum histamine release, which equals (actual net histamine release/maximum net histamine release induced by the testing compound) × 100%, for four to five separate experiments.

Figure 6

Effect of TM-N49 (TM) on mast cell numbers in mouse peritoneum. Various doses of TM-N49 were injected into the peritoneum of mice for 10 min, 2 h, 6 h or 16 h. Also shown are the responses to BSA and normal saline control. Data are displayed as a boxplot, which indicates the median, interquartile range, the largest and smallest values other than outliers (O) (defined as those which are more than 1.5 box lengths from the median) for 6 animals in each group. * P < 0.05 compared with the response to the corresponding diluent only control animals.