- Email: [email protected]
Neuritogenic activity of peripheral nerve myelin proteins in Lewis rats
Neuroscience Letters, 19 (1980) 353-358
353
© Elsevier/North-Holland Scientific Publishers Ltd.
NEURITOGENIC ACTIVITY OF PERIPHERAL NERVE MYELIN PROTEINS IN LEWIS RATS
MASARU SUZUKI, KUNIO KITAMURA, KEIICHI UYEMURA, YOSHIHIRO OGAWA, YOSHIHIRO ISHIHARA and HARUO MATSUYAMA
Department of Physiology, Saitama Medical School, lruma-gun, Saitama, 350-04 (Japan) and
(Y. 0., Y.L and ll.M.) Department of Neuropathology, Tokyo Metropolitan Institute for Neurosciences, Fuchu-shi, Tokyo, 183 (Japan) (Received May 2nd, 1980) (Revised version received June 13th, 1980) (Accepted June 18th, 1980)
SUMMARY
The neuritogenic activity of human peripheral nerve myelin proteins in Lewis rats was examined. Experimental allergic neuritis (EAN) was induced by human myelin fraction in all Lewis rats examined. Purified human P2 protein induced mild but definite EAN. None of purified P0 protein, total myelin lipids, gangliosides and cerebrosides induced EAN. Addition of total myelin lipids or gangliosides to the P2 protein did not enhance the neuritogenicity of the P2 protein. Bovine P2 protein, which was inactive in rabbits and guinea pigs, showed considerable neuritogenicity in Lewis rats. We postulate that induction of EAN depends both on the species of host animal and the source of antigen.
Experimental allergic neuritis (EAN), first described by Waksman and Adams [15], is generally considered a model of the Guillain-Barr~ syndrome, a human demyelinating disease of the peripheral nervous system (PNS). The antigen to induce EAN, however, is not yet well understood, while the basic protein has been commonly accepted as the responsible antigen of experimental allergic encephalomyelitis (EAE) in the central nervous system. We have purified myelin proteins in bovine peripheral nerve and reported that purified bovine P2 protein [31, a basic protein (molecular weight 14,859 [9]) in peripheral nerve myelin, was inactive in guinea pigs and rabbits, while bovine
354
peripheral nerve myelin, as a whole, was able to induce severe EAN consistently [12-14]. Recently, the Lewis rat was reported to be more susceptible to EAN when injected with bovine P2 protein [6] or peripheral nerve myelin fractions of a number of species [11]. In this paper, we focused our attention on the neuritogenic activity of myelin components of human peripheral nerve in Lewis rats. Lewis rats of either sex (original source Charles River, Japan), 10-15 weeks of age and weighing 250-300 g (female) or 450-500 g (male) each, were maintained under controlled humidity, temperature and lighting conditions. The inoculum consisted of one part (250 t~l) of antigen solution and one part of Freund's complete adjuvant (Difco, MI). The mixture was given intradermally in both hind foot pads of the Lewis rats. The animals were weighed and observed daily for clinical signs of EAN and EAE. Symptoms developed usually between 14 and 20 days after inoculation. Animals were sacrificed either when neurological symptoms developed or at 30 days after inoculation. Brain, brain stem, spinal cord, Gasserian ganglia, dorsal ganglia, spinal roots and sciatic nerves of the rats were removed and fixed in 10% formalin. All tissues were embedded in paraffin, stained with Luxol fast blue and hematoxylin-eosin and examined for lesions. H u m a n spinal roots were obtained at autopsy within several hours after death. The tissue was homogenized in 0.85 M sucrose (pH 7.0) with a Teflon-glass homogenizer. The myelin fraction was prepared by the method of density gradient ultracentrifugation with 0.32 M and 0.85 M sucrose as reported previously [12]. The acid extract and residue of the myelin fraction were obtained by treatment with 0.03 M HC1 (pH 2.0). The P2 protein was purified from the acid extract by Sephadex G75 column chromatography [7]. The acid-extract residue was delipidated, solubilized with 10°70 sodium dodecyl sulfate (SDS) in 0.1 M phosphate buffer (pH 7.2) and applied on a Sephadex G-200 column for purification of the P0 protein [8] (a glycoprotein of molecular weight 28,000 in PNS myelin). Bovine P2 protein was also purified from bovine spinal roots by the same procedures. The 2mercaptoethanol (2-ME)-treated P2 protein was prepared by incubation of the bovine P2 protein in 0.9°70 NaC1 with 1°70 2-ME at 45°C for 1 h. For preparation of lipids, myelin fractions were treated with 20 vol. of c h l o r o f o r m - m e t h a n o l (CM) (2:1, v/v). Large amount of insoluble residue was present. Total lipids were obtained by evaporating the CM extract to dryness on a rotary flash evaporator. The CM extract of myelin was also partitioned by addition of 0.2 vol. of 0.88°7o KCI as described by Folch et al. [4]. The lower phase was washed once with the theoretical upper phase and evaporated. The combined upper phase was also evaporated and dissolved in a small volume of chloroform. Each lipid fraction was applied on a Unisil column and eluted with stepwise increasing concentration of methanol in chloroform by the method of Eto et al. [2]. From the upper phase fraction, gangliosides were eluted with c h l o r o f o r m - m e t h a n o l 4 : 6 and 2 : 8 (v/v); from the lower phase, cerebrosides were obtained with c h l o r o f o r m - m e t h a n o l 9 : 1 (v/v). Phosphatidylserine was purchased from Sigma Chemical Co.
355 TABLE 1 ANTIGENIC ACTIVITY TO INDUCE EXPERIMENTAL ALLERGIC NEURITIS IN LEWIS RATS Antigen
Number of animals tested
Number with histological lesions* in PNS -
+
in CNS + +
-
+
+ +
Human PNS myelin Myelin (500 ~g protein) P2 protein (500 #g) Gangliosides P2 + gangliosides P2 + total lipids P0 protein (500 #g) Cerebrosides P0 + cerebrosides
5 8 7 8 4 3 2 3
0 4 7 5 2 3 2 3
0 3 0 2 0 0 0 0
5 1 0 1 2 0 0 0
5 5 7 8 4 3 2 3
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
Bovine PNS myelin P2 protein (200 #g) P2 (2-ME-treated) P2 + phosphatidylserine P2 + total lipids
4 4 4 3
1 2 3 3
0 1 1 0
3 1 0 0
4 4 4 3
0 0 0 0
0 0 0 0
*Severity of lesions is classified as: - , no lesion; +, cell infiltration; + +, demyelination. In T a b l e I, n e u r i t o g e n i c activities o f myelin c o m p o n e n t s o f h u m a n a n d b o v i n e p e r i p h e r a l nerve in Lewis rats are s u m m a r i z e d . I n j e c t i o n o f h u m a n p e r i p h e r a l nerve myelin f r a c t i o n (500/~g p r o t e i n / a n i m a l ) with F r e u n d ' s c o m p l e t e a d j u v a n t p r o d u c e d clinical s y m p t o m s a n d h i s t o l o g i c a l lesions o f d e m y e l i n a t i o n in p e r i p h e r a l n e r v o u s system, b u t n o t in central n e r v o u s system, in all 5 Lewis rats. P u r i f i e d h u m a n P2 p r o t e i n (500 /~g/animal) i n d u c e d less severe E A N in 4 o f 8 rats. T h r e e o f t h e m s h o w e d mild clinical s y m p t o m s a n d histological cell i n f i l t r a t i o n w i t h o u t d e m y e l i n a t i o n , a n d in o n e case, as s h o w n in Fig. 1, a p p a r e n t d e m y e l i n a t i o n was observed. H i s t o l o g i c a l e x a m i n a t i o n o f the a n i m a l s injected with p u r i f i e d h u m a n P0 p r o t e i n (500 /~g/animal), a m a j o r g l y c o p r o t e i n in p e r i p h e r a l nerve myelin, revealed no r e c o g n i z a b l e lesions in the p e r i p h e r a l a n d central n e r v o u s system. N e i t h e r the g a n g l i o s i d e s (30 /zg/animal) n o r the c e r e b r o s i d e s (1 m g / a n i m a l ) o b t a i n e d f r o m h u m a n p e r i p h e r a l nerve m y e l i n were active in causing E A N w h e n injected into Lewis rats. T h e rats injected with t h e m i x t u r e o f the P2 p r o t e i n a n d g a n g l i o s i d e s or t o t a l lipids o f h u m a n p e r i p h e r a l nerve myelin s h o w e d m i l d signs o f E A N , j u s t as t h o s e injected with the P 2 p r o t e i n alone. T h e g a n g l i o s i d e s o r t o t a l lipids d i d n o t e n h a n c e the a n t i g e n i c activity o f the P2 p r o t e i n . T h e m i x t u r e o f h u m a n P0 p r o t e i n a n d c e r e b r o s i d e s s h o w e d n o lesion in a n y n e r v o u s system o f the rats. O n the o t h e r h a n d , b o v i n e P2 p r o t e i n ( 2 0 0 / ~ g / a n i m a l ) p r o d u c e d severe h i s t o l o g i c a l lesions with
356
iiiiiiiiiiiIIIII,C?CI L!IIIIIiiiii ii Fig. 1. Demyelination of the spinal root of the Lewis rat injected with human P2 protein (500/~g). Luxol fast-blue and hematoxylin-eosin staining, x 320 (reduced by 20°7o in production).
demyelination in peripheral nervous system in 3 of 4 rats, without lesions in the central nervous system. In the presence of total lipids or phosphatidylserine, bovine P2 protein did not enhance but reduced its neuritogenicity. The 2-ME-treated P2 protein, i.e. after cleavage of the intramolecular disulfide bonds by 2mercaptoethanol (2-ME), showed EAN lesion in 2 of 4 rats; one with cell infiltration and one with demyelination. From the present results, together with our previous reports, we can draw the following conclusions. (1) Injection of bovine peripheral nerve myelin induced typical EAN in rabbits and guinea pigs [12, 13]. The myelin fraction of human peripheral nerve also induced EAN without EAE in Lewis rats. These results agreed with the previous report of Smith et al. [11] that the myelin of human peripheral nerve induced EAN in Lewis rats. (2) Purified bovine P2 protein showed definite EAN antigenicity in Lewis rats while it was inactive in rabbits and guinea pigs. This supports Kadlubowsky and Hughes's report [6] that bovine P2 protein induced EAN in Lewis rats. Purified human P2 protein also induced definite EAN, but to a lesser extent in half of the Lewis rats injected. We have reported that human P2 protein had similar but not identical properties to bovine P2 protein [14] as follows: (a) human P2 protein cross-reacted immunologically with anti-bovine P2 protein antibody; (b) electrophoretic mobilities of the two proteins were similar but significantly different at pH 4.5. These differences of the two proteins may reflect the differences in their neuritogenicity in Lewis rats. (3) Neither gangliosides nor cerebrosides, total lipids and purified P0 protein from human peripheral nerve myelin had neuritogenic activity in Lewis rats. As purified bovine P0 protein was reported to have no activity in inducing EAN in guinea pigs [14], it is not probable that the P0 protein is a responsible antigen for EAN. However, there are some possibilities that the neuritogenicity of the P0 protein may be lost during preparation procedures such as the treatment with a high concentration of the SDS solution. (4) The mixture of human P2 protein and gangliosides or total lipids
357
induced EAN at a similar rate and severity to the P2 protein alone. Addition of phosphatidylserine or total lipids to bovine P2 protein did not enhance but rather reduced the neuritogenic activity of bovine P2 protein. Nagai et al. [10] reported that bovine P2 protein exhibited full neuritogenic activity in rabbits when combined with gangliosides from the same source. Ishaque et al. [5] reported that Lewis rats, injected with rabbit P2 protein (200 gg) along with acidic lipids (1 mg), developed severe clinical signs of EAN, while rabbit P2 protein (200 t~g) alone was inactive in rats. Curtis et al. [1] also confirmed that purified rabbit P2 protein alone did not induce EAN in Lewis rats. Therefore, we postulate that induction of EAN depends both on the species of host animal and the source of antigen. Recently, we reported the complete amino acid sequence of bovine P2 protein elsewhere [9]. Studies on the antigenic determinants of bovine P2 protein in Lewis rats are in progress in our laboratory. REFERENCES 1 2 3 4 5 6 7 8
9 10
11 12 13
Curtis, B.M., Forno, L.S. and Smith, M.E., Reactivation of neuritogenic activity of P2 protein from rabbit PNS myelin, Brain Res., 175 (1979) 387-391. Eto, T., Ichikawa, Y., Nishimura, K., Ando, S. and Yamakawa, T., Chemistry of lipid of the posthemolytic residue or stroma of erythrocytes, J. Biochem., 69 (1968) 205-213. Eylar, E.H., Uyemura, K., Brostoff, S.W., Kitamura, K., Ishaque, A. and Greenfield, S., Proposed nomenclature for PNS myelin proteins, Neurochem. Res., 4 (1979) 289-293. Folch, J., Lees, M. and Sloane-Stanley, G.H.S., Simple method for the isolation and purification of total lipids from animal tissue, J. biol. Chem., 226 (1957) 289-293. lshaque, A., Hofmann, T. and Eylar, E.H., The structure and disease-inducing property of the P2 protein of peripheral nerve myelin, Fed. Proc., 38 (1979) 514 (abstract). Kadlubowsky, M. and Hughes, R.A.C., Identification of the neuritogen for experimental allergic neuritis, Nature (Lond.), 277 (1979) 140-141. Kitamura, K., Yamanaka, T. and Uyemura, K., On basic proteins in bovine peripheral nerve myelin, Biochim. biophys. Acta (Amst.), 379 (1975) 582-591. Kitamura, K., Suzuki, M. and Uyemura, K., Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane, Biochim. biophys. Acta (Amst.), 445 (1976) 806-816. Kitamura, K., Suzuki, M., Suzuki, A. and Uyemura, K., The complete amino acid sequence of the P2 protein in bovine peripheral nerve myelin, FEBS Lett., in press. Nagai, Y., Uchida, T., Takada, S. and Ikuta, F., Restoration of activity for induction of experimental allergic neuritis by a combination of myelin basic protein P2 and gangliosides from peripheral nerve, Neurosci. Lett., 8 (1978) 247-254. Smith, M.E., Forno, L.S. and Hofmann, W.W., Experimental allergic neuritis in the Lewis rat, J. Neuropath. exp. Neurol., 38 (1979) 377-391. Uyemura, K., Tobari, C., Hirano, S. and Tsukada, Y., Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord, J. Neurochem., 19 (1972) 2607-2614. Uyemura, K., Kitamura, K., Ogawa, Y. and Matsuyama, H., Studies on the antigenic protein to induce experimental allergic neuritis. In H. Shiraki, T. Yonezawa and Y. Kuroiwa (Eds.), The Aetiology and Pathogenesis of the Demyelinating Disease, Japan Science Press, Tokyo, 1976, pp. 181-192.
358 14 Uyemura, K., Suzuki, M. and Kitamura, K., Studies on myelin proteins in human peripheral nerve, Advanc. exp. Med. Biol., 100 (1978) 95-115. 15 Waksman, B.H. and Adams, R.D., Allergic neuritis: an experimental disease of rabbits induced by the injection of peripheral nervous tissue and adjuvants, J. exp. Med., 103 (1955) 213-235.
353
© Elsevier/North-Holland Scientific Publishers Ltd.
NEURITOGENIC ACTIVITY OF PERIPHERAL NERVE MYELIN PROTEINS IN LEWIS RATS
MASARU SUZUKI, KUNIO KITAMURA, KEIICHI UYEMURA, YOSHIHIRO OGAWA, YOSHIHIRO ISHIHARA and HARUO MATSUYAMA
Department of Physiology, Saitama Medical School, lruma-gun, Saitama, 350-04 (Japan) and
(Y. 0., Y.L and ll.M.) Department of Neuropathology, Tokyo Metropolitan Institute for Neurosciences, Fuchu-shi, Tokyo, 183 (Japan) (Received May 2nd, 1980) (Revised version received June 13th, 1980) (Accepted June 18th, 1980)
SUMMARY
The neuritogenic activity of human peripheral nerve myelin proteins in Lewis rats was examined. Experimental allergic neuritis (EAN) was induced by human myelin fraction in all Lewis rats examined. Purified human P2 protein induced mild but definite EAN. None of purified P0 protein, total myelin lipids, gangliosides and cerebrosides induced EAN. Addition of total myelin lipids or gangliosides to the P2 protein did not enhance the neuritogenicity of the P2 protein. Bovine P2 protein, which was inactive in rabbits and guinea pigs, showed considerable neuritogenicity in Lewis rats. We postulate that induction of EAN depends both on the species of host animal and the source of antigen.
Experimental allergic neuritis (EAN), first described by Waksman and Adams [15], is generally considered a model of the Guillain-Barr~ syndrome, a human demyelinating disease of the peripheral nervous system (PNS). The antigen to induce EAN, however, is not yet well understood, while the basic protein has been commonly accepted as the responsible antigen of experimental allergic encephalomyelitis (EAE) in the central nervous system. We have purified myelin proteins in bovine peripheral nerve and reported that purified bovine P2 protein [31, a basic protein (molecular weight 14,859 [9]) in peripheral nerve myelin, was inactive in guinea pigs and rabbits, while bovine
354
peripheral nerve myelin, as a whole, was able to induce severe EAN consistently [12-14]. Recently, the Lewis rat was reported to be more susceptible to EAN when injected with bovine P2 protein [6] or peripheral nerve myelin fractions of a number of species [11]. In this paper, we focused our attention on the neuritogenic activity of myelin components of human peripheral nerve in Lewis rats. Lewis rats of either sex (original source Charles River, Japan), 10-15 weeks of age and weighing 250-300 g (female) or 450-500 g (male) each, were maintained under controlled humidity, temperature and lighting conditions. The inoculum consisted of one part (250 t~l) of antigen solution and one part of Freund's complete adjuvant (Difco, MI). The mixture was given intradermally in both hind foot pads of the Lewis rats. The animals were weighed and observed daily for clinical signs of EAN and EAE. Symptoms developed usually between 14 and 20 days after inoculation. Animals were sacrificed either when neurological symptoms developed or at 30 days after inoculation. Brain, brain stem, spinal cord, Gasserian ganglia, dorsal ganglia, spinal roots and sciatic nerves of the rats were removed and fixed in 10% formalin. All tissues were embedded in paraffin, stained with Luxol fast blue and hematoxylin-eosin and examined for lesions. H u m a n spinal roots were obtained at autopsy within several hours after death. The tissue was homogenized in 0.85 M sucrose (pH 7.0) with a Teflon-glass homogenizer. The myelin fraction was prepared by the method of density gradient ultracentrifugation with 0.32 M and 0.85 M sucrose as reported previously [12]. The acid extract and residue of the myelin fraction were obtained by treatment with 0.03 M HC1 (pH 2.0). The P2 protein was purified from the acid extract by Sephadex G75 column chromatography [7]. The acid-extract residue was delipidated, solubilized with 10°70 sodium dodecyl sulfate (SDS) in 0.1 M phosphate buffer (pH 7.2) and applied on a Sephadex G-200 column for purification of the P0 protein [8] (a glycoprotein of molecular weight 28,000 in PNS myelin). Bovine P2 protein was also purified from bovine spinal roots by the same procedures. The 2mercaptoethanol (2-ME)-treated P2 protein was prepared by incubation of the bovine P2 protein in 0.9°70 NaC1 with 1°70 2-ME at 45°C for 1 h. For preparation of lipids, myelin fractions were treated with 20 vol. of c h l o r o f o r m - m e t h a n o l (CM) (2:1, v/v). Large amount of insoluble residue was present. Total lipids were obtained by evaporating the CM extract to dryness on a rotary flash evaporator. The CM extract of myelin was also partitioned by addition of 0.2 vol. of 0.88°7o KCI as described by Folch et al. [4]. The lower phase was washed once with the theoretical upper phase and evaporated. The combined upper phase was also evaporated and dissolved in a small volume of chloroform. Each lipid fraction was applied on a Unisil column and eluted with stepwise increasing concentration of methanol in chloroform by the method of Eto et al. [2]. From the upper phase fraction, gangliosides were eluted with c h l o r o f o r m - m e t h a n o l 4 : 6 and 2 : 8 (v/v); from the lower phase, cerebrosides were obtained with c h l o r o f o r m - m e t h a n o l 9 : 1 (v/v). Phosphatidylserine was purchased from Sigma Chemical Co.
355 TABLE 1 ANTIGENIC ACTIVITY TO INDUCE EXPERIMENTAL ALLERGIC NEURITIS IN LEWIS RATS Antigen
Number of animals tested
Number with histological lesions* in PNS -
+
in CNS + +
-
+
+ +
Human PNS myelin Myelin (500 ~g protein) P2 protein (500 #g) Gangliosides P2 + gangliosides P2 + total lipids P0 protein (500 #g) Cerebrosides P0 + cerebrosides
5 8 7 8 4 3 2 3
0 4 7 5 2 3 2 3
0 3 0 2 0 0 0 0
5 1 0 1 2 0 0 0
5 5 7 8 4 3 2 3
0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0
Bovine PNS myelin P2 protein (200 #g) P2 (2-ME-treated) P2 + phosphatidylserine P2 + total lipids
4 4 4 3
1 2 3 3
0 1 1 0
3 1 0 0
4 4 4 3
0 0 0 0
0 0 0 0
*Severity of lesions is classified as: - , no lesion; +, cell infiltration; + +, demyelination. In T a b l e I, n e u r i t o g e n i c activities o f myelin c o m p o n e n t s o f h u m a n a n d b o v i n e p e r i p h e r a l nerve in Lewis rats are s u m m a r i z e d . I n j e c t i o n o f h u m a n p e r i p h e r a l nerve myelin f r a c t i o n (500/~g p r o t e i n / a n i m a l ) with F r e u n d ' s c o m p l e t e a d j u v a n t p r o d u c e d clinical s y m p t o m s a n d h i s t o l o g i c a l lesions o f d e m y e l i n a t i o n in p e r i p h e r a l n e r v o u s system, b u t n o t in central n e r v o u s system, in all 5 Lewis rats. P u r i f i e d h u m a n P2 p r o t e i n (500 /~g/animal) i n d u c e d less severe E A N in 4 o f 8 rats. T h r e e o f t h e m s h o w e d mild clinical s y m p t o m s a n d histological cell i n f i l t r a t i o n w i t h o u t d e m y e l i n a t i o n , a n d in o n e case, as s h o w n in Fig. 1, a p p a r e n t d e m y e l i n a t i o n was observed. H i s t o l o g i c a l e x a m i n a t i o n o f the a n i m a l s injected with p u r i f i e d h u m a n P0 p r o t e i n (500 /~g/animal), a m a j o r g l y c o p r o t e i n in p e r i p h e r a l nerve myelin, revealed no r e c o g n i z a b l e lesions in the p e r i p h e r a l a n d central n e r v o u s system. N e i t h e r the g a n g l i o s i d e s (30 /zg/animal) n o r the c e r e b r o s i d e s (1 m g / a n i m a l ) o b t a i n e d f r o m h u m a n p e r i p h e r a l nerve m y e l i n were active in causing E A N w h e n injected into Lewis rats. T h e rats injected with t h e m i x t u r e o f the P2 p r o t e i n a n d g a n g l i o s i d e s or t o t a l lipids o f h u m a n p e r i p h e r a l nerve myelin s h o w e d m i l d signs o f E A N , j u s t as t h o s e injected with the P 2 p r o t e i n alone. T h e g a n g l i o s i d e s o r t o t a l lipids d i d n o t e n h a n c e the a n t i g e n i c activity o f the P2 p r o t e i n . T h e m i x t u r e o f h u m a n P0 p r o t e i n a n d c e r e b r o s i d e s s h o w e d n o lesion in a n y n e r v o u s system o f the rats. O n the o t h e r h a n d , b o v i n e P2 p r o t e i n ( 2 0 0 / ~ g / a n i m a l ) p r o d u c e d severe h i s t o l o g i c a l lesions with
356
iiiiiiiiiiiIIIII,C?CI L!IIIIIiiiii ii Fig. 1. Demyelination of the spinal root of the Lewis rat injected with human P2 protein (500/~g). Luxol fast-blue and hematoxylin-eosin staining, x 320 (reduced by 20°7o in production).
demyelination in peripheral nervous system in 3 of 4 rats, without lesions in the central nervous system. In the presence of total lipids or phosphatidylserine, bovine P2 protein did not enhance but reduced its neuritogenicity. The 2-ME-treated P2 protein, i.e. after cleavage of the intramolecular disulfide bonds by 2mercaptoethanol (2-ME), showed EAN lesion in 2 of 4 rats; one with cell infiltration and one with demyelination. From the present results, together with our previous reports, we can draw the following conclusions. (1) Injection of bovine peripheral nerve myelin induced typical EAN in rabbits and guinea pigs [12, 13]. The myelin fraction of human peripheral nerve also induced EAN without EAE in Lewis rats. These results agreed with the previous report of Smith et al. [11] that the myelin of human peripheral nerve induced EAN in Lewis rats. (2) Purified bovine P2 protein showed definite EAN antigenicity in Lewis rats while it was inactive in rabbits and guinea pigs. This supports Kadlubowsky and Hughes's report [6] that bovine P2 protein induced EAN in Lewis rats. Purified human P2 protein also induced definite EAN, but to a lesser extent in half of the Lewis rats injected. We have reported that human P2 protein had similar but not identical properties to bovine P2 protein [14] as follows: (a) human P2 protein cross-reacted immunologically with anti-bovine P2 protein antibody; (b) electrophoretic mobilities of the two proteins were similar but significantly different at pH 4.5. These differences of the two proteins may reflect the differences in their neuritogenicity in Lewis rats. (3) Neither gangliosides nor cerebrosides, total lipids and purified P0 protein from human peripheral nerve myelin had neuritogenic activity in Lewis rats. As purified bovine P0 protein was reported to have no activity in inducing EAN in guinea pigs [14], it is not probable that the P0 protein is a responsible antigen for EAN. However, there are some possibilities that the neuritogenicity of the P0 protein may be lost during preparation procedures such as the treatment with a high concentration of the SDS solution. (4) The mixture of human P2 protein and gangliosides or total lipids
357
induced EAN at a similar rate and severity to the P2 protein alone. Addition of phosphatidylserine or total lipids to bovine P2 protein did not enhance but rather reduced the neuritogenic activity of bovine P2 protein. Nagai et al. [10] reported that bovine P2 protein exhibited full neuritogenic activity in rabbits when combined with gangliosides from the same source. Ishaque et al. [5] reported that Lewis rats, injected with rabbit P2 protein (200 gg) along with acidic lipids (1 mg), developed severe clinical signs of EAN, while rabbit P2 protein (200 t~g) alone was inactive in rats. Curtis et al. [1] also confirmed that purified rabbit P2 protein alone did not induce EAN in Lewis rats. Therefore, we postulate that induction of EAN depends both on the species of host animal and the source of antigen. Recently, we reported the complete amino acid sequence of bovine P2 protein elsewhere [9]. Studies on the antigenic determinants of bovine P2 protein in Lewis rats are in progress in our laboratory. REFERENCES 1 2 3 4 5 6 7 8
9 10
11 12 13
Curtis, B.M., Forno, L.S. and Smith, M.E., Reactivation of neuritogenic activity of P2 protein from rabbit PNS myelin, Brain Res., 175 (1979) 387-391. Eto, T., Ichikawa, Y., Nishimura, K., Ando, S. and Yamakawa, T., Chemistry of lipid of the posthemolytic residue or stroma of erythrocytes, J. Biochem., 69 (1968) 205-213. Eylar, E.H., Uyemura, K., Brostoff, S.W., Kitamura, K., Ishaque, A. and Greenfield, S., Proposed nomenclature for PNS myelin proteins, Neurochem. Res., 4 (1979) 289-293. Folch, J., Lees, M. and Sloane-Stanley, G.H.S., Simple method for the isolation and purification of total lipids from animal tissue, J. biol. Chem., 226 (1957) 289-293. lshaque, A., Hofmann, T. and Eylar, E.H., The structure and disease-inducing property of the P2 protein of peripheral nerve myelin, Fed. Proc., 38 (1979) 514 (abstract). Kadlubowsky, M. and Hughes, R.A.C., Identification of the neuritogen for experimental allergic neuritis, Nature (Lond.), 277 (1979) 140-141. Kitamura, K., Yamanaka, T. and Uyemura, K., On basic proteins in bovine peripheral nerve myelin, Biochim. biophys. Acta (Amst.), 379 (1975) 582-591. Kitamura, K., Suzuki, M. and Uyemura, K., Purification and partial characterization of two glycoproteins in bovine peripheral nerve myelin membrane, Biochim. biophys. Acta (Amst.), 445 (1976) 806-816. Kitamura, K., Suzuki, M., Suzuki, A. and Uyemura, K., The complete amino acid sequence of the P2 protein in bovine peripheral nerve myelin, FEBS Lett., in press. Nagai, Y., Uchida, T., Takada, S. and Ikuta, F., Restoration of activity for induction of experimental allergic neuritis by a combination of myelin basic protein P2 and gangliosides from peripheral nerve, Neurosci. Lett., 8 (1978) 247-254. Smith, M.E., Forno, L.S. and Hofmann, W.W., Experimental allergic neuritis in the Lewis rat, J. Neuropath. exp. Neurol., 38 (1979) 377-391. Uyemura, K., Tobari, C., Hirano, S. and Tsukada, Y., Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord, J. Neurochem., 19 (1972) 2607-2614. Uyemura, K., Kitamura, K., Ogawa, Y. and Matsuyama, H., Studies on the antigenic protein to induce experimental allergic neuritis. In H. Shiraki, T. Yonezawa and Y. Kuroiwa (Eds.), The Aetiology and Pathogenesis of the Demyelinating Disease, Japan Science Press, Tokyo, 1976, pp. 181-192.
358 14 Uyemura, K., Suzuki, M. and Kitamura, K., Studies on myelin proteins in human peripheral nerve, Advanc. exp. Med. Biol., 100 (1978) 95-115. 15 Waksman, B.H. and Adams, R.D., Allergic neuritis: an experimental disease of rabbits induced by the injection of peripheral nervous tissue and adjuvants, J. exp. Med., 103 (1955) 213-235.