Lysophospholipase Activation with Ascaris suum Extract

Abstract

In vivo lysophospholipase activity in mammals with nematode infections has been shown to be associated with increased tissue eosinophilia (Larsh et al., 1974, Exp. Parasitol. 36: 299306) and with the activation of lymphocytes (Goven and Moore, 1980, Z. Parasitenkd. 61: 265-269). An increase in numbers of Nippostrongylus brasiliensis results in increased lysophospholipase activity in the small intestine of rats (Goven, 1979, Int. J. Parasitol. 9: 193-198) but not in lung tissue infected with Angiostrongylus cantonensis (Laubach et al., 1978, J. Parasit. 64: 1145-1146). The mechanisms of lysophospholipase activation in leucocytes involved in the inflammatory reaction to nematode infections have not been characterized in vitro. This study explores the in vitro activation of cellular lysophospholipase. Peritoneal leucocytes were stimulated with varying amounts of Ascaris suum larval extract and assayed to determine if lysophospholipase activity was affected by cell contact with nematode substances. Ascaris suum larval extract was prepared by hatching infective eggs, collecting the live larvae by filtration through gauze, and homogenizing the larvae in Hanks' Balanced Salt Solution (HBSS) in a glass tissue grinder. The homogenate was frozen at -70 C, thawed and refrozen four times to disrupt larval tissues and cells. The mixture was then centrifuged at 7,000 g for 20 min and the supernatant fraction collected. The supematant fraction was sterilized using a Millipore filter and stored at -70 C. Total protein was determined using bovine serum albumin as a standard (Lowry et al., 1951, J. Biol. Chem. 193:265-275). One thousand A. suum eggs were orally administered to each of six 2to 3-mo-old mice (Harlan Sprague Dawley, Inc., Indianapolis, Indiana). Ten groups of mice were used, six mice per group. Mice, infected with A. suum eggs, were found to produce eosinophils in high numbers at 6 wk PI when challenged with A. suum extract, intraperitoneally. Uninfected mice produced few eosinophils. Peritoneal cells were harvested from infected mice following a single intraperitoneal injection of 10 jug of A. suum larval extract given to each mouse in 1 ml of HBSS followed 48 hr later by peritoneal lavage. The cells were washed three times with HBSS and resuspended to 2 x 106 cells/ml in HBSS. Leucocyte suspensions from each mouse contained one-half as eosinophils, one-fourth as neutrophils and one-fourth as mononuclear cells, differentiated by Wright staining. Total cell counts were determined using a hemacytometer and Turk's stain. Cell mixtures from each mouse (1 x 106 cells in 0.5 ml of HBSS) were incubated with 0.5-ml quantities of varying amounts (100, 10, 1, 1 x 10-1, 1 x 10-2, 1 x 10-3, 1 x 10-4, 1 x 10-5, and 1 x 10-6 mg protein) of A. suum larval extract at 37 C, aerobically, with 1.0% penicillinstreptomycin, for 48 hr. Cell viability was determined following incubation of the leucocyte mixtures using the trypan blue exclusion test. Significant differences between peritoneal cells incubated in the presence of various dilutions of A. suum extract were de-