Agents Actions, 35 (1992)
0065-4299/92/020079-06$1.50+ 0.20/0 9 1992 Birkh/iuserVerlag,Basel
The efficacy of Cyclosporin A, FK-506 and Prednisolone to modify the adoptive transfer of Experimental Allergic Encephalomyelitis (EAE) C. Bolton PharmacologyGroup, Schoolof Pharmacy& Pharmacology,Universityof Bath, ClavertonDown, Bath BA2 7AY
Abstract
The in vitro potency of the immunosuppressants Cyclosporin A (CsA), FK-506 and Prednisolone was assessed using the adoptive transfer model of EAE in the Lewis rat. Co-culture of encephalitogen-sensitised splenic leukocytes with Prednisolone did not inhibit the transfer of disease to naive histocompatible recipients despite significant suppression of neuroantigen-stimulated leukocyte proliferation by the drug. The addition of CsA (100 nM) to cultures inhibited the induction of adoptive EAE but a lower dose of the agent (10 nM) did not prevent the development of clinico-histopathological signs of disease. FK-506 (1 nM) was 100 times more effective than CsA at suppressing adoptive EAE thus emphasising the usefulness of the model in determining the relative efficacy of compounds to modify cell-dependent autoimmune disease.
Introduction
The initial observation by Paterson [1] that the autoimmune disease Experimental Allergic Encephalomyelitis (EAE) could be induced by the transfer of lymphocytes from actively-sensitised rats to naive histocompatible recipients confirmed the condition to be, principally, an immune cellmediated phenomenon. Subsequent studies revealed the specific requirement of antigen-presenting macrophages and T-cell subsets for the successful mediation of EAE [2, 3, 4]. Furthermore, work by Panitch and McFarlin [5] and Richert et al. [6] demonstrated that adoptivelytransferred lymphocytes, when pre-incubated with either the polyclonal mitogen Concanavalin A or the neuroantigen myelin basic protein (MBP), had an improved capacity to generate disease. EAE has become welt established as an animal counterpart for the human central nervous system
(CNS) disease Multiple Sclerosis (MS) [7] and has contributed to a clearer understanding of the mechanisms ~which may underlie the etiology of demyelination [8]. In addition, pharmacological studies utilising both the active and adoptive models of EAE have provided useful information on the mechanisms by which steroidal and nonsteroidal immunomodulatory drugs may act and be of potential value in the treatment of MS [911]. Indeed, the rationale for instigating clinical trials with the immunosuppressive compound Cyclosporin A (CsA) originated from extensive work investigating the efficacy of the drug in various models of EAE [12, 13]. Recently the novel macrolide antibiotic FK-506 was shown to prevent or significantly modify the course of actively-induced EAE in the Lewis rat [14] and additional studies demonstrated the drug to be effective in other experimental models of autoimmunity [15-18]. The present study exam-
Agents Actions, 35 (1992)
80 ines the effects of FK-506 on the adoptive transfer of EAE and determines the potency of the drug relative to CsA and the steroidal compound Prednisolone.
with I/~ Ci 3H-thymidine per well (specific activity 5.0 Ci/mmol) 24 hr prior to harvesting and radionucleotide uptake was quantitated using an LKB-Wallae Liquid Scintillation Counter.
Materials and methods
Assessment of neurological EAE
Animals
The body weight and neurological status of each animal was recorded daily. The day on which clinical signs of EAE developed in each rat was noted and symptoms were graded as follows: 1. flaccid tail; 2. hind limb hypotonia; 3. partial hind limb paralysis; 4. complete hind limb paralysis.
Male Lewis rats, weighing 200-225 g, were obtained from Bantin and Kingman Ltd, Hull, UK, and maintained on Labsure CRM rat diet and water ad libitum.
Induction of active EAE Animals received, in each hind footpad, 0.2 ml of a water-in-oil emulsion containing 50 ~g of solubilised MBP, prepared by the method of Dunkley and Carnegie [19] and an equal volume of Freund's in complete adjuvant containing 5mg/ml Mycobacterium tuberculosis H37Ra (Difco, Surrey, UK).
Assessment of histological EAE Cervical spinal cords were removed from animals, following complete loss of symptoms, and fixed in 10% formal saline. Sections, 10/a thick, were cut, stained in haematoxylin-eosin, coded to prevent identification and the presence or absence of perivascular lesions was determined by light microscopy.
Induction of adoptive EAE The adoptive transfer of disease was performed as previously described by Bolton e t al. [9]. Briefly, spleens were aseptically removed from rats showing paralytic signs of EAE, 13 days post-inoculation, and pooled single cell suspensions were prepared in RPMI 1640 medium containing 5% heatinactivated, mycoplasma-screened foetal calf serum, 2 m M glutamine, 2 x 1 0 - S M 2-mercaptoethanol, 100 units penicillin and 100 gg streptomycin/ml-1 (Gibco Ltd, Uxbridge, UK). Viable leukocytes, as assessed by trypan blue exclusion, at a concentration of 2 x 106 cells/ml- 1 were cultured in the presence of 1 gg/ml-a MBP for 72 hrs at 37~ in a humidified atmosphere of 5% CO2. Harvested cells were washed thoroughly in Hank's balanced salt solution and 2 - 4 x 107 viable leukocytes were injected, via the tail vein, into each histocompatible recipient.
In vitro determination of MBP-induced proliferation At the initiation of culture 200 gl aliquots were removed from cell preparations, dispensed in groups of five in round-bottom microculture plates and incubated as above. Cells were pulsed
Drugs Prednisolone (Sigma, UK), CsA (Sandoz Ltd, Switzerland) and FK-506 (Fujisawa Pharmaceutical Co. Ltd., Japan) were solubilised in absolute alcohol, diluted to the required concentrations in complete medium and added, simultaneously with MBP, to preparations at the intiation of culture.
Statistical analysis Students t-test was used to assess differences between control and drug treatments. Results
Drug effects on the adoptive transfer of EAE to naive recipients Splenic leukocytes cultured in the presence of MBP and injected into recipient rats induced neurological and histological signs of EAE whereas cells incubated without the encephalitogen did not transfer disease (Table 1). The addition of 1 gm Prednisolone to MBP-stimulated cultures proved to be cytotoxic as assessed by the trypan blue exclusion test (data not shown). A lower concentra-
Agents Actions, 35 (1992)
81
50
t~ v W (3 Z
40
40
30
30
30
20
20
20
10
10
0
7 0 12
-10
-10
-20
-20
-30
-30
-40 J
-40
I O
------'4 ,
,
,
13 ~ - ~ 2
w t~ >m -10 b -20
8
Figure 1
b
DAY NUMt3FR POST-CELL TRANSFER
Average body weight change (g) of recipient rats relative to original body weight (represented as zero) at time of cell transfer. Recipients received cells cultured in the absence A: o--o; or presence, A, B and C: o - - o ; of MBP (1 pg/ml) plus Prednisolone: A (100 nM): n - - n ; or CsA: B (100 nM): n - - ~ , (10 riM) m--m; or FK-506: C (100 riM): o--u, (1 riM) m--m, (0.1 riM) a - - z . a: Average day of onset of neurological EAE. b: Average day of loss of all symptoms.
Table 1
The influence of Prednisolone, CsA and FK-506 on the adoptive transfer of EAE. MBP present (+)/Drug (riM) absent (--)
-
+ + + + + + +
-
-Prednisolone (100) CsA (100) CsA (10) FK-506 (100) FK-506 (1) FK-506 (0.1)
a Pooled data from 4 experiments.
No. animals with neurological symptoms/total
Average max neurological score
No. animals with cervical spinal lesions/ total
0/6
0.0
0/6
29/29 a 5/5 0/6 6/6 0/5 0/5 5/5
2.9 2.2 0.0 3.2 0,0 0,0 3.2
29/29 5/5 0/6 6/6 0/5 0/5 5/5
82
Agents Actions, 35 (1992)
tlon of the steroid (100 nM) did not affect cell viability but failed to inhibit the development of adoptive EAE. In contrast, a comparable dose of CsA (100 nM) prevented the cellular transfer of clinico-histopathological signs of disease. However, all recipients receiving donor leukocytes previously co-cultured with MBP and 10 n M CsA showed characteristic symptoms of EAE. The onset of neurological EAE and the appearance of CNS infiltrates were completely suppressed by incubation of spleen ceils with 1 n M FK-506, a concentration 100-fold less than the minimum effective dose established for CsA. Body weight fluctuations in recipient rats
The average body weight of all groups of rats receiving splenic leukocytes incubated with MBP alone began to decrease 2 - 3 days before the onset of EAE and continued to decline with the development of disease (Fig. 1 A, B, C). Body weights increased after 7 days post-cell transfer preceding the complete loss of symptoms of EAE by 2 - 3 days and despite the persistence of CNS lesions. Comparable weight changes and disease activity occurred in animals injected with cells previously cocultured with encephalitogen and either Prednisolone (100nM), CsA (10nM) or FK-506 (0.1 nM). Rats receiving cells maintained in vitro in the absence of MBP and animals treated with Table 2 Drug-induced inhibition of aH-thymidine uptake by MBP-stimulated splenic leukocytes from EAE-diseased rats ~
Drug (nM)
Prednisolone (100) CsA (100) CsA (10) FK-506 (100) FK-506 (1) FK-506 (0.1)
% Inhibition of 3H-thymidine uptake b 72 43 39 48 44 --25
Degree of significance (p < ) c 0.001 0.01 0.05 0.001 0.02 NSD
a The percentage of viable cells recorded in drug-treated triplicate cultures was comparable to values obtained from preparations containing MBP alone (80-85% of cells viable after 72 hrs incubation). b Mean counts/min_ SEM recorded for control cultures from 4 experiments (cells + MBP) = 16 483 -I-4642. ~ Mean no. of counts/min recorded from drug-treated cultures compared to values obtained from MBP-stimulated cells (minus background counts from cultures containing vehicle only).
cells exposed to antigen plus high doses of CsA (100nM) or FK-506 (100nM and 1 nM) increased their body weight and remained healthy for the duration of the experiments. The effects of in vitro drug treatment on the proliferative response of splenic leukocytes to M B P
Significant inhibition of radionucleotide uptake into antigen-stimulated cells was achieved by the addition of 100nM Prednisolone to cultures (Table 2). Although significant, approximately 30% less inhibition of 3H-thymidine incorporation was recorded when 100 nM and 10 nM CsA was added to cells. Similarly, high doses of FK-506 were effective in preventing cellular responses to MBP but 0.1 n M of the drug did not curtail eneephalitogen-directed proliferation. Discussion
Several studies have established that the immunomodulatory drugs, Prednisolone, CsA and FK-506 suppress either actively or adoptively-induced EAE in the Lewis rat [9, 14, 20]. The current investigation has extended the earlier observation by assessing, in vitro, the relative potency of these compounds in the adoptive transfer model of EAE. Exposure of MBP-conditioned leukocytes to a non-cytotoxic concentration of the steroid Prednisolone did not prevent the transfer of disease and associated body weight loss in recipients despite the anti-proliferative effects of the drug in vitro. However, a comparable dose of CsA did inhibit the cellular induction of EAE but a lower concentration of the drug although significantly inhibiting radionucleotide uptake by encephalitogen-sensitised cells, failed to curtail the subsequent transfer of disease. These observations highlight the disparity between the significant in vitro effects of a compound and the consequence of introducing drug-treated encephalitogen-sensitised cells into naive recipients. Indeed, differences between the significant suppressive effects of drugs on immunologically-primed leukocytes in vitro and the subsequent ability of these cells to retain diseaseinducing activity has been previously documented by Abreu [21] who showed that selected doses of rat interferon could markedly affect thymidine uptake into MBP-sensitised lymph node cells but
83
Agents Actions, 35 (1992)
were unable to prevent the manifestation of adoptively-induced clinical and histological signs of EAE. The current work also suggests that the percentage of cells which remain insensitive to the controlling influence of Prednisolone and CsA in vitro are primarily responsible for disease induction. Furthermore, it would appear that inhibition of activelyinduced EAE by the immunosuppressants may be effected through mechanisms additional to the drugs influence on antigen-directed immune cell activation. Indirect support for this proposal is provided by the studies of Desai and Barton [11] who described the differential effectiveness of Dexamethasone and CsA in the active and passive models of rat EAE. Work presented herein has also shown FK-506, a neutral macrolide isolated from the fermentation broth of a soil organism Streptomyces tsukubaensis, to be 100 times more effective in suppressing the adoptive transfer of EAE compared to CsA. The difference in potency between the two immunosuppressants is in agreement with data obtained from in vitro studies using murine and human lymphocyte populations [22-24]. However, the previously reported EDs0 value of 0.1 n M proved to be ineffective [25] when employed to suppress adoptive EAE or inhibit thymidine uptake by MBP-conditioned leul~ocytes. Nevertheless, the results imply that the two higher doses of FK-506 inhibited specifically the cells responsible for disease induction even though more than 50% of leukocytes remained beyond the anti-mitogenic influence of the drug. CsA and FK-506 are structurally unrelated but have several mechanistic similarities which may account, in part, for their ability to modify the induction of adoptive EAE in the Lewis rat. Of particular relevance to the current study is the finding that both compounds directly inhibit interleukin 2 (IL-2) production in activated T-cells, by down-regulating IL-2 gene transcription [26]. Subsequent work by Dumont et al. [27] revealed that the suppressive effects of CsA and FK-506 on T-cell proliferation could be reversed by the addition of recombinant IL-2 to cultures. Bower et al. [28] have recently shown that IL-2 is a prerequisite for the successful adoptive transfer of EAE and that 1L-2-depleted cultures were ineffective at mediating disease. However, the reported capacity to induce EAE was restored by in vitro sup-
plementation with recombinant IL-2. Consequently, the ability of CsA and FK-506 to profoundly affect IL-2 production may offer one mechanism through which the drugs may act to prevent the development of adoptive disease. In conclusion, this study has determined the relative efficacy of three established immunosuppressants to modify adoptively induced EAE in the Lewis rat. The investigation also emphasises the usefulness of the model to confirm observations of drug effects in vitro and may serve to assess, more specifically, the pharmacological actions of immunoregulatory compounds.
Acknowledgements The financial support of the Multiple Sclerosis Society of Great Britain and Northern Ireland and the technical assistance provided by Mrs L. Moore is gratefully acknowledged. Received 15 April 1991; accepted by I. Ahnfelt-Ronne 18 June 1991
References [1] P. Y. Paterson, Transfer of allergic encephalomyelitis in rats by means of lymph node cells. J. exp. Med. 111, 119-135 (1960). [2] A. Ben-Nun, H. Wekerle and I. R. Cohen, The rapid isolation of clonable antigen-specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. Immunol. 11, 195-199 (1981), [3] R. H. Swanborg, Autoimmune effector cells V: a monoclonal antibody specific for rat helper T lymphocytes inhibits adoptive transfer of autoimmune encephalomyelitis. J. Immunol. 130, 1503-1505 (1983). [4] A. M. Carbone, H. Ovadia and P. Y. Paterson, Role of macrophage-myelin basic protein interaction in the induction of experimental allergic encephalomyelitis in Lewis rats. J. Immunol. 131, 1263-1267 (1983). [5] H. S. Panitch and D. E. McFarlin, Experimental allergic encephalomyelitis: enhancement of cell-mediated transfer by concanavalin A. J. Immunol. 119, 1134-1137 (1977). [6] J. R. Richert, B. E Driscoll, M. Kies and E, C. Alvord, Adoptive transfer of experimental allergic encepohalomyelitis: Incubation of rat spleen cells with specific antigen. J. Immunol. 122, 494-496 (1979). [7] P. Y. Paterson, Autoimmune diseases of myelin. Prog. Clin. Biol. Res. 49, 19-36 (1980). [8] B. G. W. Arnason, Re&vance of experimental allergic encephalomyelitis to multiple slcerosis. Neuro]. Clin. 1, 765782 (1983). [9] C. Bolton, G. Atlsopp and M. L. Cuzner, The effect of cyclasporin A on the adoptive transfer of experimental allergic encephalomyelitis in the Lewis rat. Clin. exp. Immunol. 47, 127-132 (1982). [10] M. E. Westarp, H. Wekerle, A. Ben-Nun, I. R. Cohen, M. L. Vohl and H. Przuntek, T lymphocyte line-mediated experimental allergic encephalomyelitis - a pharmacologic modelfor
84 testing of immunosuppressive agents for the treatment of central nervous system disease. J. Pharmacol. Exp. Ther. 242, 614-620 (1987). [11] S. Desai and R. Barton, Pharmacological comparison of active and passive experimental allergic encephalomyelitis in the rat. Ag. Act. 27, 351-355 (1989). [12] C. BoRon, J. E Borel, M. L. Cuzner, A. N. Davison and A. M. Turner, Autoimmunity: Cyclosporin A therapy in Experimental Allergic Encephalomyelitis. In Cyclosporin A. (Ed. D. J. G. White) pp. 135-152, Oxford, UK, 1982. [13] P. Rudge, J. C. Koetsier, J. Mertin, J. O. Mispelblom Beyer, H. K. Van Walbeck, R. Clifford Jones, J. Harrison, K. Robinson, B. Mellein, T. Poole, J. C. J. M. Stokvis and P. Timonen, Randomised double blind controlled trial of cyclosporin in multiple sclerosis. J. Neurol. Neurosurg. Psych. 52, 559-565 (1989). [14] N. Inamura, M. Hashimoto, K. Nakahara, Y. Nakajima, M. Nishio, H. Aoki, I. Yamaguchi and M. Kohsaka, Immunosuppressive effect of FK-506 on experimental allergic encephalomyelitis in rats. Int. J. Immunopharmacol. 10, 991995 (1988). [15] H. Kawashima, Y. Fujino and M. Mochizuki, Effects of a new immunosuppressive agent, FK-506, on experimental autoimmune uveoretinitis in rats. Invest. Opthalmol. Vis. Sci. 29, 1265-1271 (1988). [16] N. Inamura, M. Hashimoto, K. Nakahara, H. Aoki, I. Yamaguchi and M. Kohsaka, Immunosuppressive effect of FK506 on collagen-induced arthritis in rats. Clin. Immunol. Immunopathol. 46, 82-90 (1988). [17] K. Takagishi, M. Yamamoto, A. Nishimura, G. Yamasaki, N. Kanazawa, T. Hotokebuchi and Kaibara, Effect of FK506 on collagen arthritis in mice. Transplant Proc. 21, 10561058 (1989). [18] K. Takabayashi, T. Koike, K. Kurasawa, R. Matsumura, T. Sato, H. Tomioka, I. Ito, T. Yoshiki and S. Yoshida, Effect of FK-506, a novel immunosuppressive drug on murine systemic lupus erythematasus. Clin. Immunol. Immunopathol. 46, 82-90 (1989). [19] P. R. Dunkley and P. Carnegi, Isolation of myelin basic proteins. In Research Methods in Neurochemistry Vol. 2,
Agents Actions, 35 (1992) (Eds. N. M. and R. Rodnight) pp. 219-245, Plenum Press, N.Y. 1974. [20] S. Levine and R. Sowinski, Suppression of the hyperacute form of experimental allergic encephalomyelitis by drugs. Arch. Int. Pharmacodyn. 230, 309-318 (1977). [21] S. L. Abreu, Interferon in experimental autoimmune encephalomyelitis (EAE) : Effects o f interferon on the antigenenhanced adoptive transfer of EAE. Int. Archs. Allergy Appl. Immun. 76, 302-307 (1985). [22] S. Sawada, G. Suzuki, Y. Kawase and F. Takaku, Novel immunosuppressive agent, FK-506. In vitro effects on the cloned T-cell activation. J. Immunol. 139, 1797 - 1803 (1987). [23] J. Woo, M. Stephen and A. W. Thompson, Spleen lymphocyte populations and expression of activation markers in rats treated with the potent new immunosuppressive agent FK-506. Immunology, 65, 153-155 (1988). [24] J. E. Kay, S. E. Doe and C. R. Benzie, The mechanism of action of the immunosuppressive drug FK-506. Cell. Immunol. 124, 175-181 (1989). [25] T. Kino, H. Hatanaka, S. Miyata, N. Inamura, M. Nishiyama, T. Yajima, T. Goto, M. Okuhara, M. Kohsaka, H. Aoki and T. Ochiai, FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J. Antibiot. (Tokyo) 40, 1256-1265 (1987). [26] M. J. Tocci, D. A. Matkovich, K. A. Collier, P. Kwot, E Dumont, S. Lin, S. Degudicibus, J. J. Siekierka, J. Chin and N. I. Hutchinson, The immunosuppressant FK-506 selectively inhibits expression of early T cell activation genes. J. Immunol. 143, 718-726 (1989). [27] E J. Dumont, M. J. Stanch, S. L. Koprak, M. R. Melinon and N. H. Sigal, Distinct mechanisms of suppression of murine T cell activation by the related macrolides FK-506 and rapamycin, J. Immunol. 144, 2151-258 (1990). [28] H. G. A. Bouwer, G. N. Dietsch and D. J. Hinrichs, Adoptive transfer of experimental allergic encephalomyelitis: conditions influencing memory and effector cell development. Cell Immunol. 131, 219-231 (1990).
0065-4299/92/020079-06$1.50+ 0.20/0 9 1992 Birkh/iuserVerlag,Basel
The efficacy of Cyclosporin A, FK-506 and Prednisolone to modify the adoptive transfer of Experimental Allergic Encephalomyelitis (EAE) C. Bolton PharmacologyGroup, Schoolof Pharmacy& Pharmacology,Universityof Bath, ClavertonDown, Bath BA2 7AY
Abstract
The in vitro potency of the immunosuppressants Cyclosporin A (CsA), FK-506 and Prednisolone was assessed using the adoptive transfer model of EAE in the Lewis rat. Co-culture of encephalitogen-sensitised splenic leukocytes with Prednisolone did not inhibit the transfer of disease to naive histocompatible recipients despite significant suppression of neuroantigen-stimulated leukocyte proliferation by the drug. The addition of CsA (100 nM) to cultures inhibited the induction of adoptive EAE but a lower dose of the agent (10 nM) did not prevent the development of clinico-histopathological signs of disease. FK-506 (1 nM) was 100 times more effective than CsA at suppressing adoptive EAE thus emphasising the usefulness of the model in determining the relative efficacy of compounds to modify cell-dependent autoimmune disease.
Introduction
The initial observation by Paterson [1] that the autoimmune disease Experimental Allergic Encephalomyelitis (EAE) could be induced by the transfer of lymphocytes from actively-sensitised rats to naive histocompatible recipients confirmed the condition to be, principally, an immune cellmediated phenomenon. Subsequent studies revealed the specific requirement of antigen-presenting macrophages and T-cell subsets for the successful mediation of EAE [2, 3, 4]. Furthermore, work by Panitch and McFarlin [5] and Richert et al. [6] demonstrated that adoptivelytransferred lymphocytes, when pre-incubated with either the polyclonal mitogen Concanavalin A or the neuroantigen myelin basic protein (MBP), had an improved capacity to generate disease. EAE has become welt established as an animal counterpart for the human central nervous system
(CNS) disease Multiple Sclerosis (MS) [7] and has contributed to a clearer understanding of the mechanisms ~which may underlie the etiology of demyelination [8]. In addition, pharmacological studies utilising both the active and adoptive models of EAE have provided useful information on the mechanisms by which steroidal and nonsteroidal immunomodulatory drugs may act and be of potential value in the treatment of MS [911]. Indeed, the rationale for instigating clinical trials with the immunosuppressive compound Cyclosporin A (CsA) originated from extensive work investigating the efficacy of the drug in various models of EAE [12, 13]. Recently the novel macrolide antibiotic FK-506 was shown to prevent or significantly modify the course of actively-induced EAE in the Lewis rat [14] and additional studies demonstrated the drug to be effective in other experimental models of autoimmunity [15-18]. The present study exam-
Agents Actions, 35 (1992)
80 ines the effects of FK-506 on the adoptive transfer of EAE and determines the potency of the drug relative to CsA and the steroidal compound Prednisolone.
with I/~ Ci 3H-thymidine per well (specific activity 5.0 Ci/mmol) 24 hr prior to harvesting and radionucleotide uptake was quantitated using an LKB-Wallae Liquid Scintillation Counter.
Materials and methods
Assessment of neurological EAE
Animals
The body weight and neurological status of each animal was recorded daily. The day on which clinical signs of EAE developed in each rat was noted and symptoms were graded as follows: 1. flaccid tail; 2. hind limb hypotonia; 3. partial hind limb paralysis; 4. complete hind limb paralysis.
Male Lewis rats, weighing 200-225 g, were obtained from Bantin and Kingman Ltd, Hull, UK, and maintained on Labsure CRM rat diet and water ad libitum.
Induction of active EAE Animals received, in each hind footpad, 0.2 ml of a water-in-oil emulsion containing 50 ~g of solubilised MBP, prepared by the method of Dunkley and Carnegie [19] and an equal volume of Freund's in complete adjuvant containing 5mg/ml Mycobacterium tuberculosis H37Ra (Difco, Surrey, UK).
Assessment of histological EAE Cervical spinal cords were removed from animals, following complete loss of symptoms, and fixed in 10% formal saline. Sections, 10/a thick, were cut, stained in haematoxylin-eosin, coded to prevent identification and the presence or absence of perivascular lesions was determined by light microscopy.
Induction of adoptive EAE The adoptive transfer of disease was performed as previously described by Bolton e t al. [9]. Briefly, spleens were aseptically removed from rats showing paralytic signs of EAE, 13 days post-inoculation, and pooled single cell suspensions were prepared in RPMI 1640 medium containing 5% heatinactivated, mycoplasma-screened foetal calf serum, 2 m M glutamine, 2 x 1 0 - S M 2-mercaptoethanol, 100 units penicillin and 100 gg streptomycin/ml-1 (Gibco Ltd, Uxbridge, UK). Viable leukocytes, as assessed by trypan blue exclusion, at a concentration of 2 x 106 cells/ml- 1 were cultured in the presence of 1 gg/ml-a MBP for 72 hrs at 37~ in a humidified atmosphere of 5% CO2. Harvested cells were washed thoroughly in Hank's balanced salt solution and 2 - 4 x 107 viable leukocytes were injected, via the tail vein, into each histocompatible recipient.
In vitro determination of MBP-induced proliferation At the initiation of culture 200 gl aliquots were removed from cell preparations, dispensed in groups of five in round-bottom microculture plates and incubated as above. Cells were pulsed
Drugs Prednisolone (Sigma, UK), CsA (Sandoz Ltd, Switzerland) and FK-506 (Fujisawa Pharmaceutical Co. Ltd., Japan) were solubilised in absolute alcohol, diluted to the required concentrations in complete medium and added, simultaneously with MBP, to preparations at the intiation of culture.
Statistical analysis Students t-test was used to assess differences between control and drug treatments. Results
Drug effects on the adoptive transfer of EAE to naive recipients Splenic leukocytes cultured in the presence of MBP and injected into recipient rats induced neurological and histological signs of EAE whereas cells incubated without the encephalitogen did not transfer disease (Table 1). The addition of 1 gm Prednisolone to MBP-stimulated cultures proved to be cytotoxic as assessed by the trypan blue exclusion test (data not shown). A lower concentra-
Agents Actions, 35 (1992)
81
50
t~ v W (3 Z
40
40
30
30
30
20
20
20
10
10
0
7 0 12
-10
-10
-20
-20
-30
-30
-40 J
-40
I O
------'4 ,
,
,
13 ~ - ~ 2
w t~ >m -10 b -20
8
Figure 1
b
DAY NUMt3FR POST-CELL TRANSFER
Average body weight change (g) of recipient rats relative to original body weight (represented as zero) at time of cell transfer. Recipients received cells cultured in the absence A: o--o; or presence, A, B and C: o - - o ; of MBP (1 pg/ml) plus Prednisolone: A (100 nM): n - - n ; or CsA: B (100 nM): n - - ~ , (10 riM) m--m; or FK-506: C (100 riM): o--u, (1 riM) m--m, (0.1 riM) a - - z . a: Average day of onset of neurological EAE. b: Average day of loss of all symptoms.
Table 1
The influence of Prednisolone, CsA and FK-506 on the adoptive transfer of EAE. MBP present (+)/Drug (riM) absent (--)
-
+ + + + + + +
-
-Prednisolone (100) CsA (100) CsA (10) FK-506 (100) FK-506 (1) FK-506 (0.1)
a Pooled data from 4 experiments.
No. animals with neurological symptoms/total
Average max neurological score
No. animals with cervical spinal lesions/ total
0/6
0.0
0/6
29/29 a 5/5 0/6 6/6 0/5 0/5 5/5
2.9 2.2 0.0 3.2 0,0 0,0 3.2
29/29 5/5 0/6 6/6 0/5 0/5 5/5
82
Agents Actions, 35 (1992)
tlon of the steroid (100 nM) did not affect cell viability but failed to inhibit the development of adoptive EAE. In contrast, a comparable dose of CsA (100 nM) prevented the cellular transfer of clinico-histopathological signs of disease. However, all recipients receiving donor leukocytes previously co-cultured with MBP and 10 n M CsA showed characteristic symptoms of EAE. The onset of neurological EAE and the appearance of CNS infiltrates were completely suppressed by incubation of spleen ceils with 1 n M FK-506, a concentration 100-fold less than the minimum effective dose established for CsA. Body weight fluctuations in recipient rats
The average body weight of all groups of rats receiving splenic leukocytes incubated with MBP alone began to decrease 2 - 3 days before the onset of EAE and continued to decline with the development of disease (Fig. 1 A, B, C). Body weights increased after 7 days post-cell transfer preceding the complete loss of symptoms of EAE by 2 - 3 days and despite the persistence of CNS lesions. Comparable weight changes and disease activity occurred in animals injected with cells previously cocultured with encephalitogen and either Prednisolone (100nM), CsA (10nM) or FK-506 (0.1 nM). Rats receiving cells maintained in vitro in the absence of MBP and animals treated with Table 2 Drug-induced inhibition of aH-thymidine uptake by MBP-stimulated splenic leukocytes from EAE-diseased rats ~
Drug (nM)
Prednisolone (100) CsA (100) CsA (10) FK-506 (100) FK-506 (1) FK-506 (0.1)
% Inhibition of 3H-thymidine uptake b 72 43 39 48 44 --25
Degree of significance (p < ) c 0.001 0.01 0.05 0.001 0.02 NSD
a The percentage of viable cells recorded in drug-treated triplicate cultures was comparable to values obtained from preparations containing MBP alone (80-85% of cells viable after 72 hrs incubation). b Mean counts/min_ SEM recorded for control cultures from 4 experiments (cells + MBP) = 16 483 -I-4642. ~ Mean no. of counts/min recorded from drug-treated cultures compared to values obtained from MBP-stimulated cells (minus background counts from cultures containing vehicle only).
cells exposed to antigen plus high doses of CsA (100nM) or FK-506 (100nM and 1 nM) increased their body weight and remained healthy for the duration of the experiments. The effects of in vitro drug treatment on the proliferative response of splenic leukocytes to M B P
Significant inhibition of radionucleotide uptake into antigen-stimulated cells was achieved by the addition of 100nM Prednisolone to cultures (Table 2). Although significant, approximately 30% less inhibition of 3H-thymidine incorporation was recorded when 100 nM and 10 nM CsA was added to cells. Similarly, high doses of FK-506 were effective in preventing cellular responses to MBP but 0.1 n M of the drug did not curtail eneephalitogen-directed proliferation. Discussion
Several studies have established that the immunomodulatory drugs, Prednisolone, CsA and FK-506 suppress either actively or adoptively-induced EAE in the Lewis rat [9, 14, 20]. The current investigation has extended the earlier observation by assessing, in vitro, the relative potency of these compounds in the adoptive transfer model of EAE. Exposure of MBP-conditioned leukocytes to a non-cytotoxic concentration of the steroid Prednisolone did not prevent the transfer of disease and associated body weight loss in recipients despite the anti-proliferative effects of the drug in vitro. However, a comparable dose of CsA did inhibit the cellular induction of EAE but a lower concentration of the drug although significantly inhibiting radionucleotide uptake by encephalitogen-sensitised cells, failed to curtail the subsequent transfer of disease. These observations highlight the disparity between the significant in vitro effects of a compound and the consequence of introducing drug-treated encephalitogen-sensitised cells into naive recipients. Indeed, differences between the significant suppressive effects of drugs on immunologically-primed leukocytes in vitro and the subsequent ability of these cells to retain diseaseinducing activity has been previously documented by Abreu [21] who showed that selected doses of rat interferon could markedly affect thymidine uptake into MBP-sensitised lymph node cells but
83
Agents Actions, 35 (1992)
were unable to prevent the manifestation of adoptively-induced clinical and histological signs of EAE. The current work also suggests that the percentage of cells which remain insensitive to the controlling influence of Prednisolone and CsA in vitro are primarily responsible for disease induction. Furthermore, it would appear that inhibition of activelyinduced EAE by the immunosuppressants may be effected through mechanisms additional to the drugs influence on antigen-directed immune cell activation. Indirect support for this proposal is provided by the studies of Desai and Barton [11] who described the differential effectiveness of Dexamethasone and CsA in the active and passive models of rat EAE. Work presented herein has also shown FK-506, a neutral macrolide isolated from the fermentation broth of a soil organism Streptomyces tsukubaensis, to be 100 times more effective in suppressing the adoptive transfer of EAE compared to CsA. The difference in potency between the two immunosuppressants is in agreement with data obtained from in vitro studies using murine and human lymphocyte populations [22-24]. However, the previously reported EDs0 value of 0.1 n M proved to be ineffective [25] when employed to suppress adoptive EAE or inhibit thymidine uptake by MBP-conditioned leul~ocytes. Nevertheless, the results imply that the two higher doses of FK-506 inhibited specifically the cells responsible for disease induction even though more than 50% of leukocytes remained beyond the anti-mitogenic influence of the drug. CsA and FK-506 are structurally unrelated but have several mechanistic similarities which may account, in part, for their ability to modify the induction of adoptive EAE in the Lewis rat. Of particular relevance to the current study is the finding that both compounds directly inhibit interleukin 2 (IL-2) production in activated T-cells, by down-regulating IL-2 gene transcription [26]. Subsequent work by Dumont et al. [27] revealed that the suppressive effects of CsA and FK-506 on T-cell proliferation could be reversed by the addition of recombinant IL-2 to cultures. Bower et al. [28] have recently shown that IL-2 is a prerequisite for the successful adoptive transfer of EAE and that 1L-2-depleted cultures were ineffective at mediating disease. However, the reported capacity to induce EAE was restored by in vitro sup-
plementation with recombinant IL-2. Consequently, the ability of CsA and FK-506 to profoundly affect IL-2 production may offer one mechanism through which the drugs may act to prevent the development of adoptive disease. In conclusion, this study has determined the relative efficacy of three established immunosuppressants to modify adoptively induced EAE in the Lewis rat. The investigation also emphasises the usefulness of the model to confirm observations of drug effects in vitro and may serve to assess, more specifically, the pharmacological actions of immunoregulatory compounds.
Acknowledgements The financial support of the Multiple Sclerosis Society of Great Britain and Northern Ireland and the technical assistance provided by Mrs L. Moore is gratefully acknowledged. Received 15 April 1991; accepted by I. Ahnfelt-Ronne 18 June 1991
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