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RESEARCH NOTE LOW MOLECULAR WEIGHT IMMUNOSUPPRESSORS SECRETED BY ADULT NEMATOSPIROIDES DUBIUS FERNANDO G. MONROY, COLIN DOBSON Department
St. Lucia, Queensland
13 May 1988; accepted 25 August 1988)
Abstract-MoNRou F. G., DOBSON C. and ADAMSJ. H. 1989. Low molecular weight immunosuppressors secreted by adult Nemutospiroides dubius. lnternationul Journal for Parasitology 19: 125-l 27. Adult Nemafospiroides dubius excretory-secretory products (ES) were collected from worms cultured in vitro, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) into four fractions (FI-IV), electroeluted and assessed for their ability to inhibit the proliferation of mouse lymphocytes stimulated by mitogens in vifro. The proliferation of mitogen- and ES-stimulated mouse spleen lymphocytes from normal and infected mice was inhibited by low mol. wt ES F-IV (<26,000). INDEX antigens.
THE longevity of Nematospiroides dubius in mice is associated with the depression of protective immunity. Adult worms suppress immune reactions in mice to both homologous and heterologous antigens and may be associated with soluble factors which circulate in mice and appear to enhance parasite survival (see Dobson & Cayzer, 1982; Behnke, Hannah & Pritchard, 1983; Losson, Lloyd & Soulsby, 1985). Losson et al. (1985) found low mol. wt components (< 14,000) in ES from larval N. dubius which depressed the proliferation of mouse lymphocytes in response to mitogens in vitro. Here we identified immunosuppressors in adult N. dubius ES which also depressed murine lymphocyte proliferation in vitro. We used methods similar to those used by O’Donnell, Dineen, Rothwell & Marshall (1985) to identify protective immunogens in Trichostrongylus colubriformis after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and on Western blots. The antigens used here were released from adult N. dubius which were incubated for 48 h in RPM1 1640 tissue culture medium plus 100 i.u. penicillin and 100 pg ml-’ streptomycin, 2 mM-L-glutamine and 25 mM-HEPES at 37°C under 5% CO,-95% air (Adams, Monroy, East & Dobson, 1987). ES, 100 p g protein, were resolved by SDS-PAGE in 10 separate runs in 1.5 mm thick, 10% acrylamide gels. The ES were then divided into four mol. wt fractions (FI-IV), by reference to prestained mol. wt standards (Fig. l), and recovered from the gel by electroelution over 37 h (Biorad model 422) using a 3500 mol. wt cut-off membrane, at a constant current of 10 mA/glass tube in 25 ma-Tris, 192 mr+glycine, 0.1% SDS, pH 8.3 buffer. Eluted fractions were electrodialysed (Biorad model 422) against three changes of electroelution 125
buffer containing 0.01% SDS for 24 h at a constant current of 10 mA/glass tube. Spleen lymphocytes from uninfected and infected mice were treated with mitogens and ES in vitro (Bailey, Lloyd, Martin & Soulsby, 1984; Losson etal., 1985). Either 1 pg of phytohaemagglutinin (PHA), 5 pg of Concanavalin A (Con A), or 50 fig of lipopolysaccharide (LPS) was added to 2.5 X 10h viable spleen lymphocytes in each of 96 wells on microtitre plates and incubated for either 3 or 5 days at 37°C in 7% COz. Control cultures contained medium, and either ES or electroeluted media from SDS-PAGE alone. Cultures were pulsed with 37 kBq tritiated thymidine (Amersham Australia Pty. Ltd) and harvested after 24 h in an automated cell harvester. The amount of radioactivity associated with cell DNA was measured in a liquid scintillation spectrometer (Beckman Instruments). Results were expressed as the mean number of counts min-’ (c.p.m.) f S.D. from triplicate cultures. Twenty microlitres of N. dubius ES and their fractions (10 pug ml-‘) were assayed for their ability to transform lymphocytes from infected and uninfected mice compared with cultures of cells from the same mice treated with 20 p 1 of mitogens and with culture media as controls. Cells from infected mice were also used to monitor immunosuppression by ES and ES fractions in cultures stimulated by mitogens and ES. Lymphocytes from uninfected mice neither proliferated nor died when cultured in vitro with different concentrations of N. dubius ES. Indeed murine lymphocytes retained > 90% viability 72 h after treatment with up to 50 pg ml-’ ES in a trypan blue exclusion assay. The optimal concentration of ES protein which induced infected mouse lymphocytes to
F. G. MONROY, C. DOSSONand J. H. ADAMS
116 * 84 * 58 L 48L+ESi
FIG. 3. Inhibition of proliferation of lymphocytes (L) from
mice infected for 11 days with 100 N. dubius L3 by Nematospiroides dubius ES and its FI-IV fractions. FI-IV
were incubated in the presence of 10 fig ml-’ ES protein.
FIG. 1. SDS-PAGE of Nematospiroides dubius ES in 10% acrylamide gel stained with Coomassie blue. Arrows show wheregels were cut to electroelutefractions (FI-IV).
FIG. 2. Effect of adult Nematospiroides dubius ES on mitogen induced proliferation of spleen lymphocytes (L) from uninfected (H) mice. Mitogen-stimulated lymphocytes from infected (I) mice were included for comparison. I-IV, fractions electroeluted from ES; ES* represents the mean of the response to whoholeIv. dub&s ES and to whole ES electroeluted from SDS-PAGE gels.
FIG. 4. Profile of low molecufar weight ES from female and maie ~~~tosp~ro~des dub&s separated in 12.5% acrylamide gel by SDS-PAGE.
transform was 10 pg ml-‘. The mitogenic effects of PHA, Con A and LPS on splenic lymphocytes from uninfected and infected mice were depressed by ES compared with the effect of these mitogens on cells treated with culture fluid. Spleen lymphocytes from infected mice responded more strongly to mitogens than did cells from uninfected controls which suggested that they were activated by the infection. Lymphocyte transformation by mitogens was depressed by ES and by all ES fractions but particularly by ES, F-IV (mol. wt < 26,000) (Fig. 2). When lymphocytes from infected mice were stimulated with 10 p g ml-’ ES, their transformation was depressed by the addition of ES F-IV (83%) (Fig. 3). F-IV contained at least five polypeptides (mol. wt < 26,000 to 4000) which showed qualitative and quantitative different protein profiles in male and female parasites (Fig. 4). ES immunosuppressors were not deactivated by electroelution, by 0.1% SDS nor by boiling for 2 min. ES was not toxic to lymphocytes nor did ES and F-IV contain amounts of glycoprotein which could bind the lectins and abrogate their proliferative effect on the lymphocytes. N. dubius evades host immune responses and survives during primary infections because it reduces the effectiveness of cellular reactions engendered (Cross, 1960; Dehlawi, Wakelin & Behnke, 1987; Schmitz, Possart-Schmitz, Gehrung, Stauffer, Mossmann & Fischer, 1982). Pritchard, Ali & Behnke (1984) also suggested that N. dub& facilitates its own survival by generating suppressor T cells which inhibit rejection of the worm from the murine intestine. Our results show that ES from adult N. dubius contained components among at least five low mol. wt molecules, among which some appear to be gender specific and which appear to suppress both T and B lymphocytes in the early stages of proliferation similar to those reported by Losson et al. (1985) for larvae recovered 5-6 days after infection. Acknowledgements-This work was supported by a grant from the Australian Research Grant Scheme. We thank Dr Iain J. East for his interest.
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