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The magnitude and kinetics of delayed-type hypersensitivity responses in mice vaccinated with irradiated cercariae of Schistosoma mansoni E. C. RATCLIFFE and R. A. WILSON Department of Biology, University of York, York YO1 5DD (Received 20 September 1990; revised 18 January 1991 ; accepted 18 January 1991) S U M MARY
A footpad assay was used to measure the D T H of mice to soluble worm antigens (SWAP), and to living day 7 lung schistosomula, following vaccination and challenge infections with Schistosoma mansoni. D T H to SWAP was first observed on day 10, and reached its maximum on day 17 post-vaccination. Treatment of mice with anti-CD4 antibody on the 3 days prior to footpad challenge completely abrogated this response. Reactivity to living parasites was of a lower order than that to SWAP; it also peaked earlier, on day 10 post-vaccination. By day 35, responsiveness to both sets of antigens had declined almost to control levels. There was no correlation between the level of D T H to living schistosomula, at any time, and the degree of resistance subsequently developed. Percutaneous challenge of vaccinated mice was followed by a resurgence of reactivity to SWAP. This secondary response occurred more rapidly than the primary response, peaking on day 7 postchallenge, and was of a similar magnitude. We were unable to detect a similar recall of D T H to living schistosomula, possibly because the assay was insufficiently sensitive. We conclude that the intensity and kinetics of D T H responsiveness are crucial features of the irradiated vaccine model, and suggest that further investigation of cell-mediated immune reactions, particularly those occurring in the lungs, is vital to a better understanding of events underlying the development and expression of immunity. Key words: Schistosoma mansoni, vaccination, delayed-type hypersensitivity.
INTRODUCTION
Vaccination of C57B1/6 mice with radiationattenuated cercariae of Schistosoma mansoni induces high levels of resistance against challenge (James, Labine & Sher, 1981). Although the underlying mechanisms are incompletely understood, evidence from a variety of sources has implicated cellmediated immunity. Studies of inbred mouse strains (James & Sher, 1983) revealed an association between protection and the ability to mount a delayedtype hypersensitivity (DTH) response upon footpad challenge with soluble parasite antigens. Additionally, Ch'ang & Colley (1986), showed that cutaneous sensitivity was manifested by vaccinated mice upon challenge with irradiated cercariae. This response was exemplified by an increase in thickness of the ear challenge-site, and was mediated by T-cells of the Lyt-1 phenotype. Both the inductor and effector mechanisms of immunity have since been shown in ablation studies to depend upon T-lymphocytes of the CD4 + subset (Phillips et al. 1987; Vignali et al. 1989). In our experimental system, the resistance mechanism operates during the lung phase of challenge parasite migration (Wilson, Coulson & Dixon, 1986). After vaccination, there is intense and persistent mononuclear cell infiltration into the pulmonary Parasitology (1991), 103, 65 75 Printed in Great Britain
airways (Aitken, Coulson & Wilson, 1988; Menson, Coulson & Wilson, 1989). It is possible that this represents an 'arming' of the lungs, so that a challenge infection can be rapidly eliminated. It is envisaged that secondary exposure to parasites results in a D T H reaction in the lung, with the migrating schistosomulum as the target for the immune response. Ultrastructural evidence for this was provided by Crabtree & Wilson (1986). The initiation of a D T H response depends upon the recruitment from the bloodstream of specifically sensitized T-cells. Contact with antigen causes these lymphocytes to produce cytokines, some of which are able to attract non-specifically, and activate, mononuclear cells from the circulation. It is macrophages recruited in this way which give rise to swelling by causing non-specific tissue destruction, either directly, or by releasing inflammatory cytokines (reviewed by Liew, 1984; Greene, Schatten & Bromberg, 1984). Measurement of D T H at a peripheral site such as the footpad or the ear provides a means of quantifying the degree of sensitization to a given antigen. The size of the swelling reflects in part the number of reactive cells in the circulation, a fact supported by adoptive transfer experiments, in which a positive association between the number of cells transferred and the size of lesion generated has been demonstrated (Turk, 1980).
E. C. Ratcliffe and R. A. Wilson
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Although it would be ideal to examine antischistosome responses in the lung, this organ is relatively inaccessible to functional assays of cellular immunity. In the present study we have therefore used the footpad-swelling test to probe for DTH to soluble worm antigens, and to living day 7 lung schistosomula, the putative target for the immune response in vivo. This has enabled us to investigate the dynamics of cell-mediated immunity, during the vaccinating and challenge infections, in relation to resistance in this model.
after which they were stored at 4 °C until use the following day. Living lung-stage schistosomula were prepared by the method of Wilson & Coulson (1986). Briefly, day 7 parasites were recovered from the lungs of heavily infected donor mice by mincing and incubation. After 3-4 h at 37 °C, emergent schistosomula were separated from lung fragments by filtration through 400-mesh stainless-steel gauze, pelleted by low-speed centrifugation and handcounted into the desired aliquots. Fixation, where required, was carried out as described above.
MATERIALS AND METHODS
Isotopic labelling and autoradiography
Mice and parasites C57B1/6 strain mice, age- and sex-matched within each experiment, were used throughout this study. A Puerto Rican strain of Schistosoma mansoni is routinely maintained by passage through albino Biomphalaria glabrata snails and LACA mice in our laboratory. Vaccination and percutaneous challenge Anaesthetized mice were vaccinated percutaneously via the abdomen with 500 cercariae, attenuated with 20 Krad gamma irradiation from a 60Co source at the Department of Radiobiology, Cookridge Hospital, Leeds. Where percutaneous challenge was necessary, groups of vaccinated (V) and control (C) mice were exposed via the tail to 200 normal cercariae, 5 weeks post-vaccination. For assay of protection, adult worms were recovered by portal perfusion 35 days post-challenge, and resistance was calculated according to the formula: C-V
Preparation of soluble worm antigens (SWAP) The method used was adapted from that of James (1981). Adult worms, recovered by perfusion from mice infected 7—8 weeks previously with 200 cercariae, were homogenized in a small volume of Hanks' Balanced Salt Solution (HBSS). The homogenate was centrifuged (100000 £; 4 °C;1 h), and the supernatant quenched in liquid nitrogen for storage at —80 °C. Protein concentration was determined by the Lowry method (Lowry et al. 1951). Preparation of living and fixed schistosomula Mechanically transformed 3h schistosomula were prepared by the technique of Ramalho-Pinto et al. (1974). At the end of the 3h culture period, the schistosomula were fixed by transfer to 1 % paraformaldehyde in 0-1 M sodium phosphate buffer,
Techniques for parasite labelling, preparation of mouse tissues and autoradiographic tracking of schistosomula were as previously described (Wilson & Coulson, 1986; Wilson et al. 1986), with the following modifications. Parasites labelled with [75Se]methionine were recovered from the lungs of donor mice 7 days after infection as described above. Schistosomula were hand-counted into aliquots of 100 parasites and used for footpad challenge of naive recipients. Footpad skin, popliteal, inguinal and axillary lymph nodes, lungs and liver were removed and compressed for autoradiography. Prepared organs were exposed to X-ray films in contact with intensifying screens at — 80 °C for 4 weeks, to maximize detection efnciencv. Measurement of DTH to SWAP Mice were anaesthetized, and their right hind footpads measured with a dial gauge micrometer, (Mitotoyo, Japan), as described by Miller & Jenkins (1986). Aliquots containing 100/
The magnitude and kinetics of delayed-type hypersensitivity responses in mice vaccinated with irradiated cercariae of Schistosoma mansoni E. C. RATCLIFFE and R. A. WILSON Department of Biology, University of York, York YO1 5DD (Received 20 September 1990; revised 18 January 1991 ; accepted 18 January 1991) S U M MARY
A footpad assay was used to measure the D T H of mice to soluble worm antigens (SWAP), and to living day 7 lung schistosomula, following vaccination and challenge infections with Schistosoma mansoni. D T H to SWAP was first observed on day 10, and reached its maximum on day 17 post-vaccination. Treatment of mice with anti-CD4 antibody on the 3 days prior to footpad challenge completely abrogated this response. Reactivity to living parasites was of a lower order than that to SWAP; it also peaked earlier, on day 10 post-vaccination. By day 35, responsiveness to both sets of antigens had declined almost to control levels. There was no correlation between the level of D T H to living schistosomula, at any time, and the degree of resistance subsequently developed. Percutaneous challenge of vaccinated mice was followed by a resurgence of reactivity to SWAP. This secondary response occurred more rapidly than the primary response, peaking on day 7 postchallenge, and was of a similar magnitude. We were unable to detect a similar recall of D T H to living schistosomula, possibly because the assay was insufficiently sensitive. We conclude that the intensity and kinetics of D T H responsiveness are crucial features of the irradiated vaccine model, and suggest that further investigation of cell-mediated immune reactions, particularly those occurring in the lungs, is vital to a better understanding of events underlying the development and expression of immunity. Key words: Schistosoma mansoni, vaccination, delayed-type hypersensitivity.
INTRODUCTION
Vaccination of C57B1/6 mice with radiationattenuated cercariae of Schistosoma mansoni induces high levels of resistance against challenge (James, Labine & Sher, 1981). Although the underlying mechanisms are incompletely understood, evidence from a variety of sources has implicated cellmediated immunity. Studies of inbred mouse strains (James & Sher, 1983) revealed an association between protection and the ability to mount a delayedtype hypersensitivity (DTH) response upon footpad challenge with soluble parasite antigens. Additionally, Ch'ang & Colley (1986), showed that cutaneous sensitivity was manifested by vaccinated mice upon challenge with irradiated cercariae. This response was exemplified by an increase in thickness of the ear challenge-site, and was mediated by T-cells of the Lyt-1 phenotype. Both the inductor and effector mechanisms of immunity have since been shown in ablation studies to depend upon T-lymphocytes of the CD4 + subset (Phillips et al. 1987; Vignali et al. 1989). In our experimental system, the resistance mechanism operates during the lung phase of challenge parasite migration (Wilson, Coulson & Dixon, 1986). After vaccination, there is intense and persistent mononuclear cell infiltration into the pulmonary Parasitology (1991), 103, 65 75 Printed in Great Britain
airways (Aitken, Coulson & Wilson, 1988; Menson, Coulson & Wilson, 1989). It is possible that this represents an 'arming' of the lungs, so that a challenge infection can be rapidly eliminated. It is envisaged that secondary exposure to parasites results in a D T H reaction in the lung, with the migrating schistosomulum as the target for the immune response. Ultrastructural evidence for this was provided by Crabtree & Wilson (1986). The initiation of a D T H response depends upon the recruitment from the bloodstream of specifically sensitized T-cells. Contact with antigen causes these lymphocytes to produce cytokines, some of which are able to attract non-specifically, and activate, mononuclear cells from the circulation. It is macrophages recruited in this way which give rise to swelling by causing non-specific tissue destruction, either directly, or by releasing inflammatory cytokines (reviewed by Liew, 1984; Greene, Schatten & Bromberg, 1984). Measurement of D T H at a peripheral site such as the footpad or the ear provides a means of quantifying the degree of sensitization to a given antigen. The size of the swelling reflects in part the number of reactive cells in the circulation, a fact supported by adoptive transfer experiments, in which a positive association between the number of cells transferred and the size of lesion generated has been demonstrated (Turk, 1980).
E. C. Ratcliffe and R. A. Wilson
66
Although it would be ideal to examine antischistosome responses in the lung, this organ is relatively inaccessible to functional assays of cellular immunity. In the present study we have therefore used the footpad-swelling test to probe for DTH to soluble worm antigens, and to living day 7 lung schistosomula, the putative target for the immune response in vivo. This has enabled us to investigate the dynamics of cell-mediated immunity, during the vaccinating and challenge infections, in relation to resistance in this model.
after which they were stored at 4 °C until use the following day. Living lung-stage schistosomula were prepared by the method of Wilson & Coulson (1986). Briefly, day 7 parasites were recovered from the lungs of heavily infected donor mice by mincing and incubation. After 3-4 h at 37 °C, emergent schistosomula were separated from lung fragments by filtration through 400-mesh stainless-steel gauze, pelleted by low-speed centrifugation and handcounted into the desired aliquots. Fixation, where required, was carried out as described above.
MATERIALS AND METHODS
Isotopic labelling and autoradiography
Mice and parasites C57B1/6 strain mice, age- and sex-matched within each experiment, were used throughout this study. A Puerto Rican strain of Schistosoma mansoni is routinely maintained by passage through albino Biomphalaria glabrata snails and LACA mice in our laboratory. Vaccination and percutaneous challenge Anaesthetized mice were vaccinated percutaneously via the abdomen with 500 cercariae, attenuated with 20 Krad gamma irradiation from a 60Co source at the Department of Radiobiology, Cookridge Hospital, Leeds. Where percutaneous challenge was necessary, groups of vaccinated (V) and control (C) mice were exposed via the tail to 200 normal cercariae, 5 weeks post-vaccination. For assay of protection, adult worms were recovered by portal perfusion 35 days post-challenge, and resistance was calculated according to the formula: C-V
Preparation of soluble worm antigens (SWAP) The method used was adapted from that of James (1981). Adult worms, recovered by perfusion from mice infected 7—8 weeks previously with 200 cercariae, were homogenized in a small volume of Hanks' Balanced Salt Solution (HBSS). The homogenate was centrifuged (100000 £; 4 °C;1 h), and the supernatant quenched in liquid nitrogen for storage at —80 °C. Protein concentration was determined by the Lowry method (Lowry et al. 1951). Preparation of living and fixed schistosomula Mechanically transformed 3h schistosomula were prepared by the technique of Ramalho-Pinto et al. (1974). At the end of the 3h culture period, the schistosomula were fixed by transfer to 1 % paraformaldehyde in 0-1 M sodium phosphate buffer,
Techniques for parasite labelling, preparation of mouse tissues and autoradiographic tracking of schistosomula were as previously described (Wilson & Coulson, 1986; Wilson et al. 1986), with the following modifications. Parasites labelled with [75Se]methionine were recovered from the lungs of donor mice 7 days after infection as described above. Schistosomula were hand-counted into aliquots of 100 parasites and used for footpad challenge of naive recipients. Footpad skin, popliteal, inguinal and axillary lymph nodes, lungs and liver were removed and compressed for autoradiography. Prepared organs were exposed to X-ray films in contact with intensifying screens at — 80 °C for 4 weeks, to maximize detection efnciencv. Measurement of DTH to SWAP Mice were anaesthetized, and their right hind footpads measured with a dial gauge micrometer, (Mitotoyo, Japan), as described by Miller & Jenkins (1986). Aliquots containing 100/
