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Immunophenotypic analysis of blood and spleen lymphocyte subsets in rats protected against schistosomiasis by cyclosporin A
Immunology Letters, 17 (1988) 169-172 Elsevier IML 01002
Immunophenotypic analysis of blood and spleen lymphocyte subsets in rats protected against schistosomiasis by cyclosporin A A. W. T h o m s o n ~ and L. H. Chappell 2 llmmunopathology Laboratory, Department of Pathology and 2Department of Zoology, University of Aberdeen, Aberdeen, U.K. (Received 7 September 1987; revision received 15 October 1987; accepted 19 October 1987)
1. Summary
The antischistosomal property of a short course of cyclosporin A (CsA), administered at the time of infection was demonstrated in the rat. In comparison with vehicle-treated controls, no schistosomes were recovered from CsA-treated animals, 18-19 days post infection. Flow cytometric analysis of blood and spleen lymphocyte populations from rats protected by CsA revealed no significant changes in functional T lymphocyte subsets, B cells or Ia-positive cells. Moreover, there were no significant differences in eosinophil numbers between the two experimental groups. These data add credence to the view that the unexplained antischistosomal property of cyclosporins is not mediated immunologically.
hypersensitivity (DTH) in rats [5] and guinea pigs [6], induction of syngeneic graft-versus-host disease in rats [7] and prevention of high dose antigeninduced tolerance in mice [8-10]. In most instances, these latter effects of CsA have been attributed to impairment of a regulatory (suppressor) cell subset. CsA exhibits antiparasite activity in rodents against a variety of, but not all species examined [11]. Several groups have shown that the drug exerts potent prophylactic and therapeutic activity against Schistosomiasis mansoni [12-14], although to date, no explanation for these observations has been found. In view of the paucity of information concerning the immunological status of animals exhibiting CsA-induced resistance to S. mansoni, we have conducted an analysis of circulating and splenic lymphocyte subsets in rats given a short course of CsA at the time of infection with S. mansoni.
2. Introduction
The immunosuppressive activities of cyclosporin A (CsA), first described by Borel et al. [1], are well recognized and have been extensively reviewed [2-4]. In addition, however, "paradoxical" effects of CsA on cell-mediated immunity have been described. These include, under well-defined experimental conditions, potentiation of delayed-type
3. Materials and Methods
3.1. Animals Groups of 6 male Sprague-Dawley rats (170220 g) purchased from Harlan Olac Ltd, Shaw's Farm, Blackthorn, Bicester, Oxon, UK, were used. They were maintained in a light- and temperatureregulated environment and received Oxoid rat and mouse breeding diet with tap water ad libitum.
Key words: S. mansoni; Rats; Cyclosporin A; Lymphocyte subsets
Correspondence to: Dr. A. W. Thomson, Immunopathology Laboratory, Department of Pathology, University of Aberdeen, Foresterhill, Aberdeen, AB9 2ZD, U.K.
3.2. Cyclosporin A Cyclosporin A was provided in powder form by Sandoz Ltd, Basle, Switzerland and dissolved initially in one part anhydrous ethanol, to which was ad-
0165-2478 / 88 / $ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
169
ded nine parts olive oil (Boots PLC, Nottingham, UK) to give a concentration of 25 mg/ml. The solution was stored in the dark for no more than 3 days and administered subcutaneously (20 mg/kg/day) on days - 1, 0 and + 1, in relation to infection. Controis received drug vehicle alone. 3.3. Infection and recovery of S. mansoni Rats were infected with 250 cercariae of S. mansoni by the abdominal skin penetration route under Sagatal (Nembutal, May and Baker) anaesthesia. Worms were recovered by aortic perfusion with citrate saline (4 °C) at 18-19 days post infection. Addition of 1070 saponin to the perfusate facilitated counting of the juvenile schistosomes obtained. 3.4. Haematological examination Rats were bled under Fluothane (Halothane, ICI) anaesthesia from the cleaned tail tips into capillary containers (Sarstedt) with EDTA (1 mg/ml) as anticoagulant. Blood was diluted 1:500 in Isoton, then analysed, within 1 h of collection, by a Coulter Haemo W counter to provide a total WBC count. Films were prepared, then stained with Wright's stain and a differential WBC count undertaken on 200 cells. 3.5. Isolation of mononuclear cells Blood obtained by cardiac puncture was collected into acid citrate dextrose, then diluted two-fold in RPMI-1640 (Gibco, Paisley, Renfrewshire, Scotland, UK). Mononuclear cells (MNC) were then isolated by centrifugation (400×g; 20 min at room temperature) (RT) over Ficoll/Isopaque (specific gravity 1.077 g/I). Cells removed from the interface were washed three times (350 g) in RPMI-1640, counted using an improved Neubauer haemocytometer and finally resuspended (107/ml) in RPMI-1640, containing 1007o heat-inactivated foetal bovine serum (Gibco). Spleens were finely chopped in RPMI-1640 and the cells expressed through 80-mesh stainless steel gauze. Debris was allowed to settle for 10 min at room temperature, before removal of the supernatant cell suspension. Eosinophils were estimated using Discombe's solution, as detailed previously [15]. MNC were then isolated as described above. 3.6. Immunostaining and flow cytometry An indirect immunofluorescence procedure was 170
employed to demonstrate cell phenotypes, using the appropriate mouse anti-rat monoclonal antibodies (Serotec Ltd, Bicester, Oxon, UK) at the following final working dilutions: OX-19 (pan T), 1:1000; W3/25 (T helper cells, macrophages), 1:1000; OX-8 (T cytotoxic/suppressor cells, natural killer cells), 1:800; OX-12 (K light chains) 1:1000; OX-6 (Ia antigen), 1:200. The secondary antibody employed, at 1:50, was FITC-conjugated goat anti-mouse Ig (Becton Dickinson), which was preincubated for 45 min at 4°C in 0.05 M Tris/HCl, pH 7.6, containing 5°70 normal rat serum. Fifty /zl of cell suspension (107 cells/ml) were mixed with an equal volume of primary antibody for 1 h at RT. After two washes in phosphate-buffered saline (PBS), the cells were resuspended in 100/A of secondary antibody (at a final dilution of 1:50) and incubated for 30 min at 4°C. After two further washes, the cells were suspended in 1 ml PBS and maintained on ice until analysis. Controls consisted of omission of the primary layer mouse monoclonals (PBS control) and replacement o f the primary antibodies by normal mouse immunoglobulins. The stained cells were analysed with an "Epics C" flow cytometer (Coulter Electronics, Luton, UK) as described elsewhere [16]. Ten thousand cells were analysed for each sample. 3.7. Statistics Results were expressed as means _+ SD. The significances of differences between means were determined using the Student's t-test for independent samples.
4. Results
4.1. Influence of CsA on infection The mean number of schistosomes recovered following aortic perfusion of control rats was 30.4 +_ 28.4 (individual numbers, 18, 11, 35, 78, 10). In contrast, no worms were detected in perfusates of any of the CsA-treated animals. 4.2. Analysis of cells in blood Table 1 shows the absolute numbers of leucocyte populations, lymphocyte subsets and M H C class II(Ia)-positive cells in blood of control and CsA-
Table 1 Leukocyte populations in blood of S. mansoni-infected rats. Treatment (No.)
Control (5) CsA (6)
Cell No. (x 109/1) WBC
L
M
N
E
15.6_+3.7 12.7_+2.4
12.1+_3.8 7.6_+2.5*
2.3_+1.0 2.8-+1.1
1.8+_0.4 1.9-+0.6
0.1 +0.1 0.2+0.2
OX-8
OX-12
OX-6
4.6-+1.3 3.3+_0.6
3.7+1.1 3.7-+0.7
3.7+_1.1 2.9+-0.5
Lymphocyte subsets (x 109/1) OX-19 Control (5) CsA (6)
8.8+2.5 4.8+0.9**
W3/25 8.4+2.4 5.2_+0.9
Results are means + 1 SD. WBC, white blood cells; L, lymphocytes; M, monocytes; N, neutrophils; E, eosinophils. * p<0.05; **, p<0.01 compared with control values. treated rats. The only statistically significant effects o f the drug were a reduction in absolute lymphocyte numbers and in OX-19 + cells. In other respects, cell populations in both groups remained within normal limits, as detailed previously for rats of this strain [17]. 4.3. Analysis of cells in spleen In spleen, neither total WBC nor eosinophil numbers were affected by CsA administration (Table 2). Furthermore, no influence of CsA on the incidence of cells expressing the various phenotypic markers studied was detected.
5. Discussion These data confirm the potent anti-schistosomal effect in rodents of short courses of non-toxic doses o f CsA, administered around the time of infection. Moreover, they provide evidence for the first time that this remarkable and unexplained phenomenon is not associated with significant changes in eosinophil numbers, lymphocyte subsets (apart from some reduction in OX-19 + cells), or the expression of an activation marker (Ia antigen) on mononuclear cells in blood or spleen of animals exhibiting resistance to primary infection. In contrast, we have shown that in mice, the augmentation by CsA of delayed hypersensitivity reactions to otherwise tolerogenic doses of a non-parasite antigen (xenogeneic erythrocytes) is accompanied by signifi-
cant increases in splenic T helper cells (L3T4 +) and in the T helper/suppressor cell ratio [16]. Indeed, we have found that the reduction in parasite numbers in CsA-treated mice is associated with concomitant decreases in the size of T-dependent hepatic granulomata in response to schistosome eggs [14]. There are however, clear differences between rats and mice with respect to S. mansoni infections. In the rat, worms only survive about 3 wk and never reach maturity, whereas in mice, the parasites mature to produce eggs and there is evidence o f concomitant immunity. We are not aware of any published information on lymphocyte subsets within blood, spleen or hepatic granulomata of CsA-treated, S. mansoniinfected mice. The present findings add credence to the view that the antischistosomal activity of CsA may not be mediated immunologically. Thus Bout et al. [13] found that the drug was also effective in congenitally athymic hosts and we have also reported than nonimmunosuppressive cyclosporin derivatives exhibit antischistosomal activity in vivo [18]. CsA (10 or 20/xg/ml) has no effect on survival o f schistosomules or adult worms in vitro [14]. Whilst it is conceivable that CsA could delay migration o f worms from the lungs to the hepatic circulation, we have failed to recover schistosomes by hepatic perfusion of mice 47, 62 and 98 days after a short course of CsA (5 daily doses o f 50 m g / k g s.c.) at the time of infection. No information currently exists on the site of parasite attrition in CsA-treated animals. Such data, together with further information concerning 171
Table 2 Leukocyte populations in spleens of S. mansoni-infected rats. Treatment (No.)
Cell No. ( x 107) WBC
Control CsA
(5) (6)
E
63.4_+ 7.9 56.3_+ 8.7
0.79+ 0.66 0.91_+ 0.56
Lymphocyte subsets (o70)
Control CsA
(5) (6)
OX-19
W3/25
OX-8
OX-12
OX-6
47.2+ 5.8 46.3 _+10.8
35.9_+11.0 33.5 _+ 13.7
33.2_+20.5 32.1 _+16.2
40.4_+ 4.7 44.9 + 15.9
40.9_+ 4.0 41.6+_ 14.0
Results are means _+ 1 SD. WBC, white blood cells; E, eosinophils.
the influence of CsA on host and parasite physiology, are clearly required to elucidate this biologically significant phenomenon, with possible human therapeutic implications.
Acknowledgements CsA was kindly donated by Professor J. E Borel, Sandoz, Basle, Switzerland. We thank Miss Anne Johnston, Mr. James Milton and Mrs. Janet Walker for skilled technical assistance, Dr. R. J. L. Davidson for haematological analyses and Dr. H. E Sewell for advice on flow cytometry. The financial support from University of Aberdeen Pooled Medical Endowment Funds is gratefully acknowledged.
References [1] Borel, J. E, Feurer, C., Gubler, H. U. and Stahelin, H. (1976) Immunology 32, 1017-1024. [2] Thomson, A. W. (1983) Aust. J. Exp. Biol. Med. Sci. 61, 147 - 172.
[3] Shevach, E. M. (1985) Ann. Rev. Immunol. 3, 397-423. [4] Borel, J. E, Ed. (1986) Ciclosporin. Karger, Basel.
172
[5] Kaibara, N., Hotokebuchi, T., Takagishi, K. and Katsuki, I. (1983) 3. Exp. Med. 158, 2007-2015. [6] Thomson, A. W., Moon, D. K., Inoue, Y., Geczy, C. L. and Nelson, D. S. (1983) Immunology 48, 301-308. [7] Hess, A. D., Vogelsang, G. B., Heyd, J. and Beschorner, W. E. (1987) Transplant Proc. 19, 2683-2686. [8] Altmann, D. M. and Blyth, W. A. (1985) Clin. Exp. Immunol. 59, 17-22. [9] Braida, M. and Knop, J. (1986) Immunology 59, 503-507. [10] Webster, L. M. and Thomson, A. W. (1987) Immunology 60, 409- 414. [11] Thomson, A.W., Smith, S. W. G. and Chappell, L.H. (1986) Parasitol. Today 2, 288-290. pell, L. H. (1986) Parasitol Today 2" 288-290. [12] Bueding, E., Hawkins, J. and Cha, Y. N. (1981) Agents Actions 11, 380-383. [13] Bout, D., Deslee, D. and Capron, A. (1986) Infect. lmmun. 52, 823- 827. [14] Smith, S. W. G., Chappell, L. H., Thomson, A. W., McGowan, A. P. and Simpson, J. G. (1987) Int. Archs. Allergy Appl. Immunol., in press. [15] Thomson, A. W., Milton, J. I., Aldridge, R. D., Davidson, R. J. L. and Simpson, J. G. (1986) Scand. J. Immunol. 24, 163-170. [16] Webster, L. M. and Thomson, A. W. (1987) Clin. Exp. Immunol., in press. [17] Thomson, A. W., Mathie, I. H. and Sewell, H. E (1987) Immunology 60, 383-388. [18] Chappell, L. H., Thomson, A. W., Barker, G. C. and Smith, S. W. G. (1987) Antimicrob. Agents Chemother., in press.
Immunophenotypic analysis of blood and spleen lymphocyte subsets in rats protected against schistosomiasis by cyclosporin A A. W. T h o m s o n ~ and L. H. Chappell 2 llmmunopathology Laboratory, Department of Pathology and 2Department of Zoology, University of Aberdeen, Aberdeen, U.K. (Received 7 September 1987; revision received 15 October 1987; accepted 19 October 1987)
1. Summary
The antischistosomal property of a short course of cyclosporin A (CsA), administered at the time of infection was demonstrated in the rat. In comparison with vehicle-treated controls, no schistosomes were recovered from CsA-treated animals, 18-19 days post infection. Flow cytometric analysis of blood and spleen lymphocyte populations from rats protected by CsA revealed no significant changes in functional T lymphocyte subsets, B cells or Ia-positive cells. Moreover, there were no significant differences in eosinophil numbers between the two experimental groups. These data add credence to the view that the unexplained antischistosomal property of cyclosporins is not mediated immunologically.
hypersensitivity (DTH) in rats [5] and guinea pigs [6], induction of syngeneic graft-versus-host disease in rats [7] and prevention of high dose antigeninduced tolerance in mice [8-10]. In most instances, these latter effects of CsA have been attributed to impairment of a regulatory (suppressor) cell subset. CsA exhibits antiparasite activity in rodents against a variety of, but not all species examined [11]. Several groups have shown that the drug exerts potent prophylactic and therapeutic activity against Schistosomiasis mansoni [12-14], although to date, no explanation for these observations has been found. In view of the paucity of information concerning the immunological status of animals exhibiting CsA-induced resistance to S. mansoni, we have conducted an analysis of circulating and splenic lymphocyte subsets in rats given a short course of CsA at the time of infection with S. mansoni.
2. Introduction
The immunosuppressive activities of cyclosporin A (CsA), first described by Borel et al. [1], are well recognized and have been extensively reviewed [2-4]. In addition, however, "paradoxical" effects of CsA on cell-mediated immunity have been described. These include, under well-defined experimental conditions, potentiation of delayed-type
3. Materials and Methods
3.1. Animals Groups of 6 male Sprague-Dawley rats (170220 g) purchased from Harlan Olac Ltd, Shaw's Farm, Blackthorn, Bicester, Oxon, UK, were used. They were maintained in a light- and temperatureregulated environment and received Oxoid rat and mouse breeding diet with tap water ad libitum.
Key words: S. mansoni; Rats; Cyclosporin A; Lymphocyte subsets
Correspondence to: Dr. A. W. Thomson, Immunopathology Laboratory, Department of Pathology, University of Aberdeen, Foresterhill, Aberdeen, AB9 2ZD, U.K.
3.2. Cyclosporin A Cyclosporin A was provided in powder form by Sandoz Ltd, Basle, Switzerland and dissolved initially in one part anhydrous ethanol, to which was ad-
0165-2478 / 88 / $ 3.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
169
ded nine parts olive oil (Boots PLC, Nottingham, UK) to give a concentration of 25 mg/ml. The solution was stored in the dark for no more than 3 days and administered subcutaneously (20 mg/kg/day) on days - 1, 0 and + 1, in relation to infection. Controis received drug vehicle alone. 3.3. Infection and recovery of S. mansoni Rats were infected with 250 cercariae of S. mansoni by the abdominal skin penetration route under Sagatal (Nembutal, May and Baker) anaesthesia. Worms were recovered by aortic perfusion with citrate saline (4 °C) at 18-19 days post infection. Addition of 1070 saponin to the perfusate facilitated counting of the juvenile schistosomes obtained. 3.4. Haematological examination Rats were bled under Fluothane (Halothane, ICI) anaesthesia from the cleaned tail tips into capillary containers (Sarstedt) with EDTA (1 mg/ml) as anticoagulant. Blood was diluted 1:500 in Isoton, then analysed, within 1 h of collection, by a Coulter Haemo W counter to provide a total WBC count. Films were prepared, then stained with Wright's stain and a differential WBC count undertaken on 200 cells. 3.5. Isolation of mononuclear cells Blood obtained by cardiac puncture was collected into acid citrate dextrose, then diluted two-fold in RPMI-1640 (Gibco, Paisley, Renfrewshire, Scotland, UK). Mononuclear cells (MNC) were then isolated by centrifugation (400×g; 20 min at room temperature) (RT) over Ficoll/Isopaque (specific gravity 1.077 g/I). Cells removed from the interface were washed three times (350 g) in RPMI-1640, counted using an improved Neubauer haemocytometer and finally resuspended (107/ml) in RPMI-1640, containing 1007o heat-inactivated foetal bovine serum (Gibco). Spleens were finely chopped in RPMI-1640 and the cells expressed through 80-mesh stainless steel gauze. Debris was allowed to settle for 10 min at room temperature, before removal of the supernatant cell suspension. Eosinophils were estimated using Discombe's solution, as detailed previously [15]. MNC were then isolated as described above. 3.6. Immunostaining and flow cytometry An indirect immunofluorescence procedure was 170
employed to demonstrate cell phenotypes, using the appropriate mouse anti-rat monoclonal antibodies (Serotec Ltd, Bicester, Oxon, UK) at the following final working dilutions: OX-19 (pan T), 1:1000; W3/25 (T helper cells, macrophages), 1:1000; OX-8 (T cytotoxic/suppressor cells, natural killer cells), 1:800; OX-12 (K light chains) 1:1000; OX-6 (Ia antigen), 1:200. The secondary antibody employed, at 1:50, was FITC-conjugated goat anti-mouse Ig (Becton Dickinson), which was preincubated for 45 min at 4°C in 0.05 M Tris/HCl, pH 7.6, containing 5°70 normal rat serum. Fifty /zl of cell suspension (107 cells/ml) were mixed with an equal volume of primary antibody for 1 h at RT. After two washes in phosphate-buffered saline (PBS), the cells were resuspended in 100/A of secondary antibody (at a final dilution of 1:50) and incubated for 30 min at 4°C. After two further washes, the cells were suspended in 1 ml PBS and maintained on ice until analysis. Controls consisted of omission of the primary layer mouse monoclonals (PBS control) and replacement o f the primary antibodies by normal mouse immunoglobulins. The stained cells were analysed with an "Epics C" flow cytometer (Coulter Electronics, Luton, UK) as described elsewhere [16]. Ten thousand cells were analysed for each sample. 3.7. Statistics Results were expressed as means _+ SD. The significances of differences between means were determined using the Student's t-test for independent samples.
4. Results
4.1. Influence of CsA on infection The mean number of schistosomes recovered following aortic perfusion of control rats was 30.4 +_ 28.4 (individual numbers, 18, 11, 35, 78, 10). In contrast, no worms were detected in perfusates of any of the CsA-treated animals. 4.2. Analysis of cells in blood Table 1 shows the absolute numbers of leucocyte populations, lymphocyte subsets and M H C class II(Ia)-positive cells in blood of control and CsA-
Table 1 Leukocyte populations in blood of S. mansoni-infected rats. Treatment (No.)
Control (5) CsA (6)
Cell No. (x 109/1) WBC
L
M
N
E
15.6_+3.7 12.7_+2.4
12.1+_3.8 7.6_+2.5*
2.3_+1.0 2.8-+1.1
1.8+_0.4 1.9-+0.6
0.1 +0.1 0.2+0.2
OX-8
OX-12
OX-6
4.6-+1.3 3.3+_0.6
3.7+1.1 3.7-+0.7
3.7+_1.1 2.9+-0.5
Lymphocyte subsets (x 109/1) OX-19 Control (5) CsA (6)
8.8+2.5 4.8+0.9**
W3/25 8.4+2.4 5.2_+0.9
Results are means + 1 SD. WBC, white blood cells; L, lymphocytes; M, monocytes; N, neutrophils; E, eosinophils. * p<0.05; **, p<0.01 compared with control values. treated rats. The only statistically significant effects o f the drug were a reduction in absolute lymphocyte numbers and in OX-19 + cells. In other respects, cell populations in both groups remained within normal limits, as detailed previously for rats of this strain [17]. 4.3. Analysis of cells in spleen In spleen, neither total WBC nor eosinophil numbers were affected by CsA administration (Table 2). Furthermore, no influence of CsA on the incidence of cells expressing the various phenotypic markers studied was detected.
5. Discussion These data confirm the potent anti-schistosomal effect in rodents of short courses of non-toxic doses o f CsA, administered around the time of infection. Moreover, they provide evidence for the first time that this remarkable and unexplained phenomenon is not associated with significant changes in eosinophil numbers, lymphocyte subsets (apart from some reduction in OX-19 + cells), or the expression of an activation marker (Ia antigen) on mononuclear cells in blood or spleen of animals exhibiting resistance to primary infection. In contrast, we have shown that in mice, the augmentation by CsA of delayed hypersensitivity reactions to otherwise tolerogenic doses of a non-parasite antigen (xenogeneic erythrocytes) is accompanied by signifi-
cant increases in splenic T helper cells (L3T4 +) and in the T helper/suppressor cell ratio [16]. Indeed, we have found that the reduction in parasite numbers in CsA-treated mice is associated with concomitant decreases in the size of T-dependent hepatic granulomata in response to schistosome eggs [14]. There are however, clear differences between rats and mice with respect to S. mansoni infections. In the rat, worms only survive about 3 wk and never reach maturity, whereas in mice, the parasites mature to produce eggs and there is evidence o f concomitant immunity. We are not aware of any published information on lymphocyte subsets within blood, spleen or hepatic granulomata of CsA-treated, S. mansoniinfected mice. The present findings add credence to the view that the antischistosomal activity of CsA may not be mediated immunologically. Thus Bout et al. [13] found that the drug was also effective in congenitally athymic hosts and we have also reported than nonimmunosuppressive cyclosporin derivatives exhibit antischistosomal activity in vivo [18]. CsA (10 or 20/xg/ml) has no effect on survival o f schistosomules or adult worms in vitro [14]. Whilst it is conceivable that CsA could delay migration o f worms from the lungs to the hepatic circulation, we have failed to recover schistosomes by hepatic perfusion of mice 47, 62 and 98 days after a short course of CsA (5 daily doses o f 50 m g / k g s.c.) at the time of infection. No information currently exists on the site of parasite attrition in CsA-treated animals. Such data, together with further information concerning 171
Table 2 Leukocyte populations in spleens of S. mansoni-infected rats. Treatment (No.)
Cell No. ( x 107) WBC
Control CsA
(5) (6)
E
63.4_+ 7.9 56.3_+ 8.7
0.79+ 0.66 0.91_+ 0.56
Lymphocyte subsets (o70)
Control CsA
(5) (6)
OX-19
W3/25
OX-8
OX-12
OX-6
47.2+ 5.8 46.3 _+10.8
35.9_+11.0 33.5 _+ 13.7
33.2_+20.5 32.1 _+16.2
40.4_+ 4.7 44.9 + 15.9
40.9_+ 4.0 41.6+_ 14.0
Results are means _+ 1 SD. WBC, white blood cells; E, eosinophils.
the influence of CsA on host and parasite physiology, are clearly required to elucidate this biologically significant phenomenon, with possible human therapeutic implications.
Acknowledgements CsA was kindly donated by Professor J. E Borel, Sandoz, Basle, Switzerland. We thank Miss Anne Johnston, Mr. James Milton and Mrs. Janet Walker for skilled technical assistance, Dr. R. J. L. Davidson for haematological analyses and Dr. H. E Sewell for advice on flow cytometry. The financial support from University of Aberdeen Pooled Medical Endowment Funds is gratefully acknowledged.
References [1] Borel, J. E, Feurer, C., Gubler, H. U. and Stahelin, H. (1976) Immunology 32, 1017-1024. [2] Thomson, A. W. (1983) Aust. J. Exp. Biol. Med. Sci. 61, 147 - 172.
[3] Shevach, E. M. (1985) Ann. Rev. Immunol. 3, 397-423. [4] Borel, J. E, Ed. (1986) Ciclosporin. Karger, Basel.
172
[5] Kaibara, N., Hotokebuchi, T., Takagishi, K. and Katsuki, I. (1983) 3. Exp. Med. 158, 2007-2015. [6] Thomson, A. W., Moon, D. K., Inoue, Y., Geczy, C. L. and Nelson, D. S. (1983) Immunology 48, 301-308. [7] Hess, A. D., Vogelsang, G. B., Heyd, J. and Beschorner, W. E. (1987) Transplant Proc. 19, 2683-2686. [8] Altmann, D. M. and Blyth, W. A. (1985) Clin. Exp. Immunol. 59, 17-22. [9] Braida, M. and Knop, J. (1986) Immunology 59, 503-507. [10] Webster, L. M. and Thomson, A. W. (1987) Immunology 60, 409- 414. [11] Thomson, A.W., Smith, S. W. G. and Chappell, L.H. (1986) Parasitol. Today 2, 288-290. pell, L. H. (1986) Parasitol Today 2" 288-290. [12] Bueding, E., Hawkins, J. and Cha, Y. N. (1981) Agents Actions 11, 380-383. [13] Bout, D., Deslee, D. and Capron, A. (1986) Infect. lmmun. 52, 823- 827. [14] Smith, S. W. G., Chappell, L. H., Thomson, A. W., McGowan, A. P. and Simpson, J. G. (1987) Int. Archs. Allergy Appl. Immunol., in press. [15] Thomson, A. W., Milton, J. I., Aldridge, R. D., Davidson, R. J. L. and Simpson, J. G. (1986) Scand. J. Immunol. 24, 163-170. [16] Webster, L. M. and Thomson, A. W. (1987) Clin. Exp. Immunol., in press. [17] Thomson, A. W., Mathie, I. H. and Sewell, H. E (1987) Immunology 60, 383-388. [18] Chappell, L. H., Thomson, A. W., Barker, G. C. and Smith, S. W. G. (1987) Antimicrob. Agents Chemother., in press.