ffournal of Autoimmunity (1992) 5, 83-91

Alkyllysophospholipid Prevents Induction of Experimental Allergic Encephalomyelitis

A n d r e a s K l e i n - F r a n k e a n d P a u l G. M u n d e r Max-Planck-lnstitut fiir Immunbiologie, Freiburg, Germany (Received 9 April 1991 and accepted 18 September 1991 ) Alkyllysophospholipids are synthetic analogues o f n a t u r a l phospholipids possessing a high i m m u n o m o d u l a t i n g a n d a n t i t u m o r a l capacity. E x p e r i m e n t a l a u t o i m m u n e encephalomyelitis is a m o d e l disease for multiple sclerosis which can be i n d u c e d by injecting rats with m y e l i n basic protein, M B P . The effect o f one alkyllysophospholipid, ET-18-OCH3, on the course of e x p e r i m e n t a l a u t o i m m u n e encephalomyelitis was investigated. It was f o u n d that a n i m a l s t r e a t e d with E T - 1 8 - O C H 3 showed only weak signs o f disease. M B P specific T-cell lines were co-cultivated with E T - 1 8 - O C H 3. The c o m p o u n d s u p p r e s s e d T-cell p r o l i f e r a t i o n m a r k e d l y , suggesting t h a t this m i g h t be its m o d e o f action in vivo. Since E T - I 8 - O C H 3 has only low toxicity in m a n , it could be of interest to p e r f o r m f u r t h e r studies on its effects on a u t o i m m u n e , d e m y e l i n a t i n g disease.

Introduction Phospholipids are basic c o m p o n e n t s of biological membranes. T h e y are continuously metabolized and a major intermediate p r o d u c t of phospholipid metabolism is 2-1ysophosphatidylcholine (2-LPC). Its concentration was found to be elevated in m e m b r a n e s of macrophages u p o n activation [1]. Alkyllysophospholipids ( A L P ) are synthetic analogs of naturally occurring 2 - L P C , with an ether b o n d in position 1 instead of the ester bond. T h i s modification renders the molecule more resistant to metabolization than its natural counterpart [2]. A L P are potent i m m u n o m o d u l a t o r s with a strong antitumoral activity preventing g r o w t h and metastasis of n u m e r o u s experimental tumors [3, 4]. T h i s antitumoral activity is due to the following p r o p e r ties: (a) A L P activate macrophages to cytotoxic effector cells [5], (b) they exert direct Correspondence to: Prof. P. G. Munder, Max-Planck-Institut fiir Immunbiologie, Stfibeweg51, 7800 Freiburg i. Br., Germany. 83 0896-8411/92/010083 + 09 $03.00/0

9 1992AcademicPress Limited

84

A n d r e a s K l e i n - F r a n k e and Paul G. M u n d e r

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and selective cytotoxic effects on malignant cells [6, 7], and (c) low doses of ALP have also been shown to induce differentiation of immature human leukemic cells [8]. The most effective ALP, racemic 1-octadecyl-2-methylglycero-3-phosphocholine (ET-18-OCH3, Figure 1), was used in clinical phase I studies in patients suffering from untreatable malignant disease [9] and is presently being tested in a multicenter phase I I study [ 10]. Two patients of a phase I study who suffered from both cancer and multiple sclerosis (MS) revealed not only a tumor response but also a dramatic improvement of the neurological symptoms (unpublished observation). This finding led us to investigate the influence of ET-18-OCH 3 on experimental allergic encephalomyelitis (EAE), which is generally considered to be a good animal model for the human disease. EAE is an experimental autoimmune disease of the central nervous system (CNS) white matter, characterized by a T-cell response to myelin basic protein (MBP). In susceptible animals, EAE can be induced by cutaneous injection of a CNS homogenate or MBP [ 11] mixed with complete Freund's adjuvant (CFA). The resulting disease is characterized by ascending paralysis and mononuclear cell inflammatory lesions of CNS white matter. EAE may also be passively induced by adoptive transfer ofsyngeneic, MBP-specific T cells which can be kept in culture as permanent cell lines [12]. EAE shares numerous clinical and pathological features with multiple sclerosis and is therefore regarded as a good experimental model for human demyelinating disease [13, 14]. Materials and methods

Animals

Female Lewis rats, between 8 and 12 weeks of age, were obtained from the breeding facilities of our institute. Induction of E A E

MBP was prepared from guinea pig CNS tissue according to the procedure of Eylar et al. [ 15]. Rats were immunized by injecting each of the four footpads with 0.05 ml of an emulsion of 100 ~tg MBP dissolved in 100 Ixl phosphate buffered saline (PBS) emulsified with 100 lal of mineral oil containing 200 Ixg Mycobacterium tuberculosis H 37 Ra (Difco, Detroit, MI, USA). Clinical assessment of disease

Animals were weighed daily and scored for signs of disease. A four grade scale was used: 1 = tail flaccidity, 2 = weakness of hind limbs, 3 = complete hind limb paresis

Alkyllysophospholipid in E A E

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and 4 = tetraparesis. Results are shown as mean of the maximum clinical score of each animal.

Phospholipids E T - 1 8 - O C H 3 (rac.l-octadecyl-2-methylglycero-3-phosphoholine), E T - 1 8 - O H (rac. 1-octadecyl-2-glycero-3-phosphocholine) and platelet activating factor (PAF, 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine)were purchased from Berchtold Inc., Bern, Switzerland. 2 - L P C (2-1ysophosphatidylcholine) was obtained from Serva Inc., Heidelberg, Germany.

Oral therapy Animals were treated daily with doses of between 0.5 and 10 mg. E T - 1 8 - O C H 3 was dissolved in 1 ml of full fat milk (0.5-10 mg) and administered by an intragastric canula. Control animals were treated with milk only.

Statistical analysis T h e Wilcoxon rank sum test was used for the evaluation of statistical significance of in vivo experiments.

MBP-specific T-cell lines Line L . M B P was established and maintained essentially according to the protocol of Ben-Nu et al. [ 12]. Briefly, lymph node cells of immunized Lewis rats were challenged in vitro with MBP. Responding lymphoblasts were isolated by density gradient centrifugation and cultured in interleukin-2 (IL2)-containing media. Supernatants of concanavalin-A (Con-A)-stimulated mouse spleen cells were used as a source of IL-2. Permanent T-cell lines were maintained by alternating restimulation (presentation of MB P by irradiated syngeneic thymus cells) and propagation in IL-2-containing media. Prior to experiments, the cells were tested for antigen-specificity by microculture proliferation test.

Proliferation assay by [3H]-thymidine incorporation I L- 2 dependent proliferation was measured by culturing 2,000 cells per round bottomed well in supplemented D M E M containing 10% horse serum and 15 % ConA supernatant. For assessment of antigen dependent proliferation, 104 MBP-specific T-cells per well were co-cultured with 106 irradiated (3,000 R) thymic presenter cells and stimulated with 0.2 mg M B P or 0.05 mg Con-A. Cultures were pulsed on day 3 with 0.2 mCi of [3H]-thymidine and were harvested 16 h later by a semi-automatic cell harvester. [3H]-thymidine incorporation was determined in a liquid scintillation counter.

86

A n d r e a s K l e i n - F r a n k e a n d P a u l G. M u n d e r T a b l e 1. Results of in vivo assays

Dose (mg/day) 0 0.5 0.5 1 1 2.5 2.5 5 5 10 I0

Treatment from day

EAE incidence

Clinical status

-0 5 0 5 0 5 0 5 0 5

7/7 4/4 4/4 7/7 7/7 7/7 5/7 3/4 3/4 3/5 3/5

3-4 3 2-3 2 1 2 1 0-I 0-1 0-1 0-1

(variance)

Duration (days)

Mortality

5.25 4.25 4.0 4.25 4.0 4.0 2.5 2.0 2.0 0.6 1.8

0/7 0/4 0/4 0/7 0/7 0/7 0/7 1/4" 0/4* 3/5* 4/5*

*Side effects= cachexia, apathy, dehydration. Table

2. Analysis of statistical significance by Wilcoxon

Rank sum test

Treatment I mg from day 0 I mg from day 5 2.5 mg from day 0 2.5 mg from day 5 2.5 mg from day 0 2.5 mg from day 5 1 mg from day 5 2.5 mg from day 5

Compared to

Statistical significance (%)

Control Control Control Control 1 mg from day 0 I mg from day 5 1 mg from day 0 2.5 mg from day 0

99.7 99.0 98.2 99.0 83.0 61.0 73.0 87.3

Results

Suppression of actively induced E A E with ET-18-OCH 3 Rats were treated daily with doses of between 0.5 and 10 m g (corresponding to 3-60 mg/kg). T r e a t m e n t c o m m e n c e d on the day of immunization, on day 5 postinjection (p.i.), or after the onset of disease (10 m g per day). T h e results are shown in Table 1. Doses between 0.5 and 2.5 m g E T - 1 8 - O C H 3 per day reduce the severity and duration of disease independent of the time of initiation of treatment. H i g h e r doses suppress clinical E A E almost completely but are toxic, leading to dehydration, apathy, cachexia and a high mortality. I n rats treated with 2.5 m g / d a y or less, no sideeffects were seen. T h e r a p y beginning after onset of clinical disease apparently did not influence the course of disease. Results o f the in vivo experiments were tested for significance by the Wilcoxon rank sum test (Table 2). T h e r a p e u t i c effects of 2 . 5 m g E T - 1 8 ~ 3 were

A l k y l l y s o p h o s p h o l i p i d in E A E

87

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Time (days) F i g u r e 2. Evolution of body weight o f treated a n d u n t r e a t e d animals. T h e average weight o f t h e animals o f each g r o u p is given in % of the initial weight. 9 = u n t r e a t e d r a t s , / x = i m m u n i z e d , b u t u n t r e a t e d rats, O = i m m u n i z e d rats receiving 1 m g E T - 1 8 - O C H 3 f r o m day 0, 9 = i m m u n i z e d rats treated with a daily dose of 10 m g from day 10. Seven rats/group.

significant at onset of treatment. There appeared to be dose-dependency but it was not significant. Onset of treatment on day 5 p.i. seemed to be more effective than on day 0 but the difference was not statistically significant. The onset of EAE is regularly accompanied by weight loss. Evolution of body weight of treated and untreated animals is compared in Figure 2. Rats receiving 10 mg ET-18-OCH3/day did not recover from the initial loss of weight after immunization. In untreated animals the onset of clinical EAE on day 10 is marked by a weight loss of 12% compared with only 6.5% in animals treated with 10 mg ET-18-OCH 3.

In vitro effects of E T - 1 8 - O C H 3 T-cell lines are cultured by alternating restimulation with MBP and IL-2dependent propagation. We tested for the effect of ET-18-OCH 3 on T-cell growth by [3H]-thymidine incorporation during both phases. T-blast proliferation during IL-2-dependent propagation was suppressed in a dose-dependent manner by adding ET-18-OCH 3 to the culture medium. A dose of 10 mg/ml resulted in almost complete suppression of T-cell growth. Other phospholipids (natural 2-LPC, PAF and ET-18-OH) did not affect cell proliferation (Figure 3).

88

A n d r e a s K l e i n - F r a n k e a n d P a u l G. M u n d e r

oo.

0 0.1

I 0.5

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Concentration (/.tg/ml) F i g u r e 3. Proliferation of L . M B P blasts d u r i n g I L - 2 d e p e n d e n t propagation in presence o f various concentrations of different phospholipids. Cells were cultured in t h e presence o f the p h o s p h o l i p i d for 48 h a n d t h e n [3H]-thymidine incorporation was m e a s u r e d a n d c o m p a r e d to that o f t h e control w i t h o u t a phospholipid added. [] = 2 - L P C , 9 = E T - 1 8 - O H , 9 = P A F , 9 = E T - 1 8 - O C H 3. Each point represents the m e a n of six different cultures. T h e e x p e r i m e n t was repeated four times with almost identical results.

D u r i n g restimulation by M B P the antiproliferative effect of E T - 1 8 - O C H 3 was far less marked. T-cell growth was inhibited only at 10 mg/ml (Figure 4). T h e same was true for Con-A-induced blast transformation. Discussion

T h e effect of the synthetic alkyllysophospholipid E T - 1 8 - O C H 3 on the course o f E A E was examined. E T - 1 8 - O C H 3 is an i m m u n o m o d u l a t i n g drug with strong antitumoral capacity. Clinical observations in two patients suffering from cancer and M S led us to investigate the drug's influence on EAE, a widely accepted model for M S . T h e experiments showed that E T - 1 8 - O C H 3 considerably diminished clinical signs of E A E provided it was given prior to the onset of clinical signs. After onset of clinical disease no therapeutic effect could be observed. T r e a t m e n t starting on day 5 p.i. appeared to be more effective than treatment beginning on the day o f immunization, though this was statistically not significant. T h e same effect has also been reported for cyclosphosphamide and some other immunosuppressive agents [ 16, 17]. F o r cyclosporine A and cyclophosphamide it has been reported that after withdrawal of the drug, animals developed clinical E A E [18, 19]. In contrast to animals treated with E T - 1 8 - O C H 3, clinical signs of E A E appeared faintly and for a shorter period but after the same asymptomatic lag phase as in control animals. Signs of disease were not observed after withdrawal of the drug.

Alkyllysophospholipid in E A E

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Time (h)

F i g u r e 4. Proliferation of L . M B P - b l a s t s d u r i n g restimulation with M B P in t h e presence o f different concentrations o f E T - 1 8 - O C H 3. [] = c o n t r o l , / ~ = 1 ~tg/ml, Q = 5 lig/ml, 9 = 10 ~tg/ml.

EAE is a T-cell dependent disease which can be adoptively transferred by T cells with a specificity for myelin basic protein, MBP. These cells can be kept as long term culture according to the protocol of Ben-Nun et al. [12] in the presence of IL-2 and with regular MBP restimulation. We established MBP-specific T-cell lines and assessed proliferation in the presence of various concentrations ET-18-OCH 3 by [3H]-thymidine incorporation. During IL-2-dependent propagation ET-18-OCH 3 strongly suppresses T-cell proliferation. Other more easily metabolizable phospholipids do not have this effect: natural LPC, which can be deacylated in position 1, and PAF and ET-18-OH, both of which can be acylated in position 2, fail to suppress T-cell proliferation. During restimulation with MBP or Con A, ET-18-OCH 3 inhibited T-cell proliferation only in high concentrations. This is probably a result of the presence of antigen presenting cells in the culture system which incorporate the drug into their cell membranes and withdraw it from the culture medium. The finding that ET-18-OCH 3 suppresses T-cell proliferation is in accordance with the finding of Andreesen et al. that the drug inhibits lectin induced transformation of human lymphocytes, whereas resting cells remain unaffected [20]. Little is known as yet as to the mode of action of ET-18-OCH 3. A L P are metabolized by an O-alkyl-cleaving enzyme. The belief that contrary to normal cells, malignant cells contain only low amounts of this enzyme and thus suffer from an accumulation of ALP [21] has recently been questioned [22]. ET-18-OCH 3 is reported to interfere with protein kinase C [23]. Since this enzyme plays an important role in the activation of lymphocytes, it might be possible that the ET-18-OCH3's effects on EAE can be explained by interference with protein kinase C.

90

Andreas Klein-Franke and Paul G. Munder

At present, therapy of M S is highly unsatisfactory, consisting only of drugs with uncertain therapeutic b u t strong adverse effects. E T - 1 8 - O C H 3 , unlike azathioprine and cyclosporin A, is not mutagenic [24], nor does it have critical side-effects such as bone m a r r o w depression or severe organ toxicity [25]. Since it passes t h r o u g h the blood-brain barrier and is highly suitable for long t e r m therapy [25], E T - 1 8 - O C H 3 m i g h t indeed be a p r o m i s i n g new a n t i - M S agent.

Acknowledgements

T h e authors wish to acknowledge the assistance of D r H a r t m u t Wekerle in establishing the animal model and the excellent technical assistance of M r K a r l - H e i n z Widmann. T h e work was s u p p o r t e d by a grant of the B u n d e s m i n i s t e r i u m f/ir F o r s c h u n g u n d Technologie.

References

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12. Ben-Nun, A.,H. Werkerle, andI. R. Cohen. 1981. The rapid isolation ofclonable antigen specific T lymphocyte lines capable of mediating autoimmune encephalomyelitis. Eur. J. Immunol. 11:195-199 13. Paterson, P. Y. 1978. The demyelinating diseases: clinical and experimental studies in animals and man. In Immunological Disease. M. Samter, D. W. Talmadge, K. F. Austen, J. H. Vaughan eds. Little Brown & Co., Boston. pp. 1400-1435 14. Lassmann, H. 1983. A comparative neuropathology of chronic EAE and MS. Neurology Series, Vol. 25. Springer, Berlin 15. Eylar, E. H., P. J. Kniskern, and J. J. Jackson. 1974. Myelin Basic Proteins. Meth. Enzymol. 32B: 323-341 16. Babington, R. G. and P. W. Wedeking. 1971. The influence of cinanserin and selected pharmacologic agents on EAE. J. Pharmacol. Exp. Ther. 177:454-460 17. Levine, S. and R. Sowinski. 1977. Suppression of the hypercute form of experimental allergic encephalomyelitis by drugs. Arch. Int. Pharmacodyn. 230:309 18. Elliot, G. A. 1973. Therapeutic effects of melengestrol acetate, cyclosphosphamide and palmitoyl cytarabne in established EAE of rats. Arch. Int. Pharmacol. Ther. 204:62-76 19. Bolton, C., G. Allsop, and M. L. Cuzner. 1982. The effect of cyclosporin A on the adoptive transfer of experimental allergic encephalomyelitis in the Lewis rat. Clin. Exp. Immunol. 47:127-132 20. Andreesen, R., M. Modolell, H. U. Weltzien, and P. G. Munder. 1979. Alkyllysophospholipid induced suppression of human lymphocyte response to mitogens and selective killing lymphoblasts. Immunbiol. 156:498-506 21. Modolell, A., R. Andreesen, W. Pahlke, U. Brugger, and P. G. Munder. 1979. Disturbance of phospholipid metabolism during the selective destruction of tumor cells induced by alkyl-lysophospholipids. Cancer Res. 39:4681-4686 22. Unger, L., H. J. Eibl, D. J. Kim et al. 1987. Sensitivity of leukemia cell lines to cytotoxic alkyllysophospholipids in relation to O-alkyl cleavage enzyme activities. J. Natl. Cancer Inst. 78:219-222 23. Oishi, K., B. Zheng, J. F. White, W. R. Vogler, and J. F. Kuo. 1988. Inhibition of Na, K-ATPase and Sodium Pump by anticancer ether lipids and protein kinase C inhibitors ET-18-OCH 3and BM 41.440. Biochem. Biophys. Res. Commun. 157:1000-1006 24. King, M. T., K. Eckhardt, E. Gocke, D. Wild, W. E. Berdel, and P. G. Munder. 1981. Failure to detect mutagenic effects of anti-tumor Aklyl-Lysophospholipids. Cancer Letters 12:217-222 25. Munder, P. G. and O. Westphal. 1990. Antitumoral and other biomedical activities of synthetic ether phospholipids. In Fifty Years Progress in Allergy. B. H. Waksman ed. Karger, Basel, pp. 206-235