Metacercariae of P. westermani were collected from naturally infected freshwater crayfish, Cambaroides similis, at an endemic area in Korea. Excretory-secretory product (ESP) of P. westermani metacercariae was prepared by transferring 5,000 newly excysted metacercariae into 5 ml physiological saline and incubating at 37℃ in a 5% CO₂ incubator for 12 hr. The incubation medium was dialyzed against distilled water and centrifuged at 12,000 rpm for 30 min. The resulting supernatant were lyophilized and diluted with an appropriate medium to the desired concentration immediately before use. The amounts of proteins in the ESP were measured using the bicinchoninic acid protein assay kit (Pierce, IL, USA).

Granulocytes were isolated from the peripheral blood of normal individuals. Granulocytes were isolated by using a gradient percoll solution. Briefly, venous blood anticoagulated with 50 U/ml heparin was diluted with PIPES buffer (25 mM PIPES, 50 mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose, pH 7.4) at a 1:1 ratio. Diluted blood was overlaid on an isotonic Histopaque solution (density, 1.083 g/ml) (Sigma) and centrifuged at 1,000 g for 30 min at 4℃. The supernatant and mononuclear cells at the interface were carefully removed, and erythrocytes in the sediment were lysed by two cycles of hypotonic water lysis. Isolated granulocytes were suspended in the reaction medium. Freshly isolated granulocytes were stimulated by incubating them in a 96-wells plate coated with human IgG (Sigma) in the absence or presence of ESP of PwNEM. Briefly, 96-well tissue culture plates (Nunc, Denmark) were coated with human IgG diluted in PBS at concentrations from 0 to 100 µg/ml in the absence or presence (2, 6, 20 µg) of the ESP for 2 hr at 37℃. After incubation, the wells were aspirated and then washed twice with PBS. Two hundred microliters of cell suspension (2.5 × 10⁵ cell/ml) in HBSS with 10 mM HEPES, 0.03% gelatin and 100 µM cytochrome c (Sigma) were dispensed in the wells coated with IgG in the absence or presence of the ESP. As a positive control, 200 µl of 1 ng/ml PMA in HBSS with 10 mM HEPES were added to the cell suspension dispensed in the wells not coated with IgG in the absence of the ESP. Immediately after the addition of the cells, the absorbance in each well was measured at 550 nm with a micoplate autoreader (Thermomax; Molecular Devices, USA), followed by repeated readings. Between absorbance measurement, the plate was incubated at 37℃. Each reaction was conducted in duplicates and superoxide anion generation was calculated with an extinction coefficient of 21.1 × 10^-3 M/cm for reduced cytochrome c at 550 nm, and was expressed as nmoles of cytochrome c reduction/1 × 10⁵ cells. Data are presented as mean±SE from the number of experiments indicated. Statistical significance of the differences between the two groups was assessed with paired Student's t-test.

To investigate whether the ESP of PwNEM could influence the effector function of granulocytes stimulated with IgG, the effect of ESP on superoxide production of granulocyte stimulated with immobilized IgG was tested in vitro. As shown in Fig. 1, granulocytes released superoxide in response to immobolized IgG alone in a time-dependent manner. The kinetics of superoxide production of granulocytes stimulated with three different concentrations (10, 30, 100 µg/ml) of IgG was nearly identical, although the amount of superoxide produced in response to the immobilized IgG was concentration-dependent. Granulocytes, stimulated by 10 µg/ml IgG, produced superoxide which was detectable after 90 min and gradually increased thereafter. Superoxide production of granulocytes stimulated by 30 and 100 µg/ml IgG was detectable after 60 and 45 min, respectively. In contrast, when granulocytes were incubated in non-coated plates, little or no production of superoxide was found. These IgG-induced superoxide production of granulocytes were strongly inhibited when the cells were incubated in the wells coated with IgG in the presence of the ESP (6 or 20 µg) (inhibition range; 73.6-89.6%, p<0.01) (Table 1). However, the inhibitory effect of the lowest amount (2 µg) of the ESP was minimal (IgG 30 µg/ml, 21.3% inhibition; IgG 100 µg/ml, 23% inhibition) (Table 1).

This study shows that the ESP of PwNEM inhibit superoxide production of granulocytes stimulated with immobilized IgG in a dose-dependent manner. In addition, the ESP degraded IgG in vitro in a dose-dependent manner (data not shown). This result suggests that PwNEM secrete some biological molecules that interfere with IgG-stimulated effector functions. Chung et al. (1997) reported that the ESP obtained from 20 newly live excysted metacercariae or purified cysteine proteases from the ESP in vitro cleaved the heavy chain of IgG within 2 hr, and that the secreted metacercarial cysteine proteases of P. westermani revealed stronger activity in cleaving IgG than those of the juveniles and adult worms. Therefore, the inhibitory effect of the ESP on IgG-induced superoxide production of granulocytes is likely due to degradation of IgG induced by cysteine proteolytic activity in the ESP. In addition, it is reported that the cathepsin L proteases secreted by Fasciola hepatica in vitro prevented antibody-mediated eosinophil attachment to newly excysted juvenile (Carmona et al., 1993). Thus, all together, these findings suggest that proteolytic enzyme in the ESP produced by helminth worm is a crucial molecule for evading cell-mediated ADCC against the worms, and that higher amount and activity of proteolytic enzyme in the ESP secreted by younger worms is required for survival of the worms and successful migration to their final destination in parasitic infections.