- Email: [email protected]
Experimental allergic encephalomyelitis— properties of an iodinated encephalitogenic polypeptide
Immunochendstry. Pergamon Press 1967. Vol. 4, pp. 273-281. Printed in Great Britain COMMUNICATION
TO THE
EDITORS
Experimental allergic encephalomyelitis--Properties of an iodinated encephalitogenic polypeptide* (Received 3 March 1967) Abstract~A bovine encephalitogenic polypeptide fraction (BEP) from ox spinal cord was iodinated with 195I and 1~I by the technique of Hunter and Greenwood.~ Iodinated BEP retained its electrophoretic and chromatographic properties and encephalitogenic activity. Hydrolysis of 1~I BEP showed that tyrosine and histidine were labelled. Delayed hypersensitivity reactions with BEP used as antigen performed on guinea pigs immunized with BEP trace labelled with 1~5I were positive. However the incidence of delayed hypersensitivity reactions on animals immunized with heavily iodinated la6I BEP was low, in spite of their normal encephalitogenic response. Foetal calf serum, the serum of adult animals and man and human cerebrospinal fluid appeared to contain a proteinase or peptidase capable of digesting BEP. Proteolysis of supposedly sequestered autoantigens of myelin may be a normal means of preventing them from reaching the immunological system. INTRODUCTION EXPERIMENTAL allergic encephalomyelitis (EAE) can be produced by injecting a bovine encephalitogenic polypeptide fraction (BEP) with Freund's complete adjuvant (FCA) into guinea pigs. BEP is isolated from an acid extract of an acetone dried powder of bovine spinal cord. There are three polypeptides in BEP all of relatively low molecular weight (3500). These polypeptides are thought to be derived from a basic protein of myelin by the action of an acid proteinase during the extraction with acid. tl~ The encephalomyelitis produced by immunization with BEP is preceded by cutaneous delayed hypersensitivity to BEP but no humoral antibody to BEP has been detected by conventional methods, t2~ We labelled BEP with radioactive iodine to aid the study of its immunological properties. This paper describes the iodination of BEP and the effects of iodination on the encephalitogenic and immunological activity of BEP. In addition we studied the effect of normal serum and cerebrospinal fluid on iodinated BEP.
MATERIALS AND METHODS Carnegie and Lumsden 'a~ isolated a group of polypeptides from bovine spinal cord and subsequently a more highly purified fraction was isolated tl~ as follows. A mixture of basic proteins and polypeptides was obtained by extracting an acetone dried powder of bovine spinal cord with HCI. This extract was neutralized and applied to carboxymethyl-Sephadex G25. The retained fraction was eluted with HCI and the proteins and polypeptides precipitated with 90 per cent acetone. This 'basic fraction' was passed repeatedly through a column of Sephadex G50, using formic acid : acetic acid : water (1 : 4 : 45) as solvent, to isolate the protein and polypeptide fractions. Bovine encephalitogenic polypeptide (BEP) was obtained as an apparently homogeneous fraction with an elution volume equivalent * Publication No. 1131 from The Walter and Eliza Hall Institute of Medical Research. 273
274
C o m m u n i c a t i o n to the Editors
to a polypeptide with a molecular weight of 3500. Amino acid analysis showed a high content of lysine, arginine, histidine, serine and glycine. On electrophoresis in polyacrylamide gel it was possible to observe three zones with only minor differences in mobility, the mobility with reference to cytochrome C being 1.85-2.05. ta~ The tyrosine content of BEP is 0.21/~-mole per mg and the histidine content 0.44 v-mole per mg. tl~ Iodination of BEP. Labelling with 1~5I was performed by the technique of Hunter and Greenwood. tS~ In a typical experiment 20/~g of BEP in 20/~1 of 1 M phosphate buffered saline (pH 7.3) was mixed with 2 mc (30/~1) of carrier-free radioactive sodium iodide (Radiochemical Centre, Amersham, England). Five microlitres of chloramine T, 10 mg/ml in water were added and allowed to react for 3 min. To stop the iodination 5/~1 of sodium metabisulfite, 24 mg/ml in water, were added. The la5I BEP was separated from inorganic iodide by chromatography of the mixture on a small column (6 cm × 0.6 cm) of Sephadex G25 fine (Pharmacia, Uppsala, Sweden). The column was washed several times with phosphate buffered saline (pH 7.3) and treated with 50/~g of BEP to minimize adsorption of 1~5I BEP onto Sephadex and glassware. The column was washed with 3 to 6 volumes of 0.1 N HC1 prior to fractionating the iodination mixture. The 125I BEP was eluted with 0.1 N HC1 and collected in tubes numbered 4 to 7 (fraction size 0.2 ml) and immediately buffered to pH 7.3 with a 1 M solution of sodium phosphate buffered saline (Fig. 1). The radioactivity in each fraction was determined using a sodium iodide scintillation crystal (Type No. 883A, Ekco Electronics Ltd., England). Other samples of low radioactivity were counted in the well of a sodium iodide scintillation crystal counter with an efficiency of about 60 per cent (Type No. 664A, Ekco Electronics Ltd., England). Electrophoresis of BEP on polyacrylamide gel was as described. ~4} Peptide maps were made from tryptic digests of BEP mixed with a trace amount of t25I BEP as described by Carnegie, Bencina and Lamoureux. tl~ The digests were examined by electrophoresis and chromatography. A typical experiment is described in Fig. 6. The digests were examined also by a peptide mapping technique whereby the sample is separated first on a micro-column of Sephadex G25 in phenol-acetic acid-water (1 : 1 : 1, w/v/v) followed by paper chromatography with n-butanol : pyridine : acetic acid : water (30 : 30 : 6 : 24 by volume) as solvent. 161 The papers were examined using the reagents described by Easley. c7~ Chromatograms were placed in contact with Kodak X-ray film for 3 to 24 hr to obtain autoradiographs. The encephalitogenic activity of BEP and the iodinated preparations of BEP were tested by immunizing guinea pigs as described by Lamoureux et al. ~2~ in doses shown in Table 1. Guinea pigs which develop EAE become incontinent and show loss of righting reflexes, hind limb paresis or hind limb paralysis; however guinea pigs which did not develop hind limb paresis or paralysis were not scored as having 'clinical' EAE. The brain and upper spinal cord of all animals were examined microscopically for our histological criteria of EAE which were perivascular aggregates of mononuclear cells in the brain substance in several separate fields. Cutaneous sensitivity to encephalitogen was tested 9 to 10 days after immunization, using 10 to 25 #g of BEP. The cutaneous injection site was examined at 3 hr
Experimental Allergic Encephalomyelitis
275
for an immediate reaction and at 24 and 48 hr for delayed reactions, as described by Lamoureux et aL <2~ Humoral antibody. Pooled sera were tested by immunodiffusion in agar geF s~ and passive cutaneous anaphylaxis (PCA)(9~ using BEP as antigen. For immunodiffusion we used small petri dishes containing 4 ml of melted Noble agar (Difco, Detroit, Michigan, U.S.A.) at a concentration of 0.3, 0.5 and 0.75 per cent and the gels were buffered at pH 4.8 with 0.2 M sodium acetate, at pH 6.0, 6.5 and 7.3 with 0.2 M sodium phosphate and at pH 8.6 with 0.05 M veronal buffer. EXPERIMENTAL RESULTS
Iodination of BEP. Iodination of BEP with 125I occurred readily. The total recovery of radioactivity was at least 85 per cent after chromatography on Sephadex G25 with 0.1 N HC1 as eluent (Fig. 1). In every case all the radioactivity which was recovered was attached to BEP and no peak of free 125I was detected. The specific activity of 125I BEP in eight iodination experiments ranged from 10 to 180 ~c per tzg of BEP. Because of the low molecular weight of BEP, preparations even of high specific activity had only a small proportion of molecules labelled.
o 1500
--
¢0
"
1
\%. I
10
~5
2O
Frocli0n number
FIG. 1. Chromatography of BEP on Sephadex G25 after iodination with x~5I (for conditions, see text). Fraction size 0"2 rnl; column size 6 cm ×0"6 era; eluent 0.1 N HCI. Fractions between the cross lines were pooled to give 125I BEP.
BEP was more heavily labelled with a mixture of 127I and a trace amount of 125I. Two preparations were obtained, one with a ratio of 0.3 atoms of iodine per mole of tyrosine in BEP (1271 BEPa) and the other with a ratio of 1.68 atoms of iodine per mole of tyrosine (187I BEPb). In the latter experiment a distinct peak of free iodide was obtained when the mixture was fractionated after iodination (Fig. 2). Chemical properties of iodinated BEP. Chromatography of 1251 BEP on Sephadex G50 three weeks after iodination showed that 75-80 per cent of the radioactivity
Communication to the Editors
276
was still bound to BEP and the peak of radioactivity coincided with the peak of the non-iodinated BEP (Fig. 3); a second peak in the salt region contained radioactivity. Electrophoresis on polyacrylamide gel of lzsI BEP three weeks after labelling showed that the mobility of 125I BEP relative to cytochrome C was unchanged, the I000 --
g
\
,-o~/
I
2O
Fraction
number
FIO. 2. Separation of iodinated BEP (600 ~g) from iodide. In this experiment a ratio of three atoms of iodide 18¢I per mole of tyrosine in BEP was used in the iodination. A trace of lasI was added to the iodide used for labelling. T h e fLrst peak is 125I BEP and the second peak is excess iodide. For conditions of chromatography, see Fig. 1.
--
3000C
LI~
O-O6O
Ila5 B E P
E --~040
2000C
N >,
1 0 IO00C
--
-
).O~n
(~
Fraction number
FIG. 3. Chromatography of a mixture of BEP (2-4 vg) and 1~51 BEP (0-08 ~g, 70 ~ / ~ g ) on Sephadex G50 (10g) in formic acid:acetlc a c i d : w a t e r (1]: 4 : 45). Radioactivity shown by ( ), optical density at 280 m~ (. . . . ).
l
FIG. 4. Electrophoresis on polyacrylamide gel of BEP (2), BEP mixed with 125I BEP (3) and mixture of basic protein and polypeptides (4). Cytochrome C (1) was used as reference. T h e gel contained 15 per cent polyacrylamide in formic acid : acetic acid : water (1 : 4 : 45) and 4 M urea p H 2"5.
FIG. 5. Autoradiography of the polyacrylamide gel shown in Fig. 4. Conditions and samples as in Fig. 4. (Facing p. 276)
Experimental Allergic Encephalomyelitis
277
Mc-value being 1.90 to 2.05 (Fig. 4); an autoradiograph of this particular gel showed only one radioactive spot corresponding to BEP (Fig. 5). t2¢I BEPa and 12¢I BEPb also had unchanged Mc-values. t~sI BEP was digested for 5 hr at 37°C with pronase at pH 7.5 and the digest examined by paper chromatography in butanol : acetic acid : water (5 : 1 : 5). '1°~ Iodine was found to be present as monoiodo-tyrosine, monoiodo-histidine and diiodo-tyrosine. When 125I BEP was left for 3 months at pH 2 the spot corresponding to monoiodo-histidine was greatly diminished and a spot of free iodide was evident. We concluded that iodination with a ratio of up to 1.68 atoms of iodine per mole of tyrosine in BEP did not significantly alter the chromatographic or electrophoretic properties of BEP and that most of the iodine remained bound to the BEP molecules for at least 3 weeks. Peptide mapping oftryptic digest of125I BEP. Peptide mapping was performed on tryptic digest of BEP together with a trace of 12~I BEP as a preliminary step to a study of the encephalitogenic determinant. The position of the peptides which stained with ninhydrin and spots which contained radioactivity are shown in Fig. 6. The spots of radioactivity did not coincide with any ninhydrin positive spots; the radioactive spots had a slightly higher Rf than the spots which contained tyrosine. These radioactive spots possibly correspond to iodotyrosine spots; if so the presence of the iodine in the peptide molecules would have caused an increase in the Rf-value in a manner similar to the change caused in the Rf of free tyrosine when it is iodinated. ~m Peptide mapping by the alternative procedure wherein the
© -r a
o O
a, T t3
5
X
5
I0
cm
ELECTROPHORE51S
FIG. 6. Tryptic digest of BEP (400 ~,g) mixed with trace of t~sI BEP (3 ~ , specific activity 35/~c/~g). Electrophoresis in pyridine acetate p H 6"5, 40 min at 50 V/cm. Chromatography in n-butanol : pyridine : acetic acid : water (30 : 20 : 6 : 24). Ninhydrin positive spots are shown by O. Spots which stained positive for tyrosine are shown by ~ and those which were radio° active by • (strong) and ~ (weak).
278
Communication
to the Editors
peptides were first separated according to molecular size and then by chromatography t6~ showed that the main spot of radioactivity migrated in Sephadex G25 as a peptide composed of five amino acids. We concluded that in the tryptic digest x85I was attached to relatively small peptides which were neutral or only slightly cationic at pH 6.5 and no radioactivity was associated with the strongly basic peptides. Immunological activity of iodinated BEP. The preparations studied were BEP, 125I BEP with a specific activity of 35/~c per ~g of BEP and the more heavily labelled preparations 127I BEPa and 1~7I BEPb which are described above. All preparations produced EAE in guinea pigs (Table 1). With the iodinated preparations it is possible that the encephalitogenic activity could be accounted for by small amounts of unlabelled BEP, since 50 ng of BEP are able to produce EAE. c2~ None of the preparations induced precipitating antibody to BEP as measured by immunodiffusion. Pooled sera from guinea pigs immunized with 127I BEPa gave positive PCA reactions with BEP, although sera from guinea pigs immunized with unlabelled BEP did not. Guinea pigs immunized with BEP, x25I BEP and 1~'7I BEPa developed typical cutaneous delayed hypersensitivity to BEP (Table 1). However delayed hypersensitivity reactions in animals immunized with heavily iodinated BEP (x27I BEPb) were less decisive than those obtained with guinea pigs immunized with other preparations, since only two of seven animals gave positive reactions (Table 1), yet five had lesions of EAE. We can at present offer no explanation for these apparent differences. TABLE 1. (DHS)
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE), DELAYED HYPERSENSITIVITY AND CIRCULATING ANTIBODY IN GUINEA PIGS AFTER IMMUNIZATION W I T H BEP AND IODINATED BEP IN FREUND'S COMPLETE ADJUVANT
Fractions injected (dose)
EAE
Incidence of Time of neurological onset disorder (days) BEP (50 ~g) 9/11 12-18 12sI BEP 35 p.c//zg 6/10 14--19 (2-5 t,g) lZ7I BEPa (40-50 t'g) 5/6 12-18 1~7I BEPb (40-50 tLg) 4/7 14-18
DHS to BEP Incidence of histological lesions 10/11 5/10 6/6 5/7
Incidence of positive reactions 8/11
Antibody* to BEP IDt
PCA++
-
-
6/10
-
:k
4/5
-
+
2/7t
--
:k
* Pooled sera. t ID --immunodiffusion; ++Weak reactions. PCA--passive cutaneous anaphylaxis.
Degradation of 1~5I BEP by normal serum and cerebrospinalfluid. Chromatography on Sephadex G50 was used to study the effect of normal serum on 1251 BEP. Fifty to 1000 ng of l~sI BEP were incubated for 30 rain at room temperature with 0.2 ml of adult guinea pigs serum; chromatography of the mixture on Sephadex G50 revealed a small peak of radioactivity in the protein region, a peak of 125I
Experimental Allergic Encephalomyelitis
279
6oc 2
w
3
4
30'
I
--
o
I Time,
I
2
0'
hr
FIG. 7. Chromatography of a mixture of 1~5I BEP mixed with normal guinea pig serum and applied immediately to the column (0') and after 30 min incubation (30'). Column was prepared from 10 g Sephadex G50 with 0.1 M Tris buffer pH 7"5. Flow rate 60 ml per hour, radioactivity monitored in a Nuclear Chicago flow counter. Peaks occurred where protein 1, 300(0-4000 molecular weight polypeptides 2, iodide and iodohistidine 3, and iodotyrosine 4, are known to be eluted.
BEP, a peak in the low molecular weight region and a peak of radioactivity where iodotyrosine would be eluted (Fig. 7). Without incubation with serum this last peak of iodotyrosine was absent, but with increasing periods of incubation this peak increased proportionally and the peak of 125I BEP decreased proportionally. Incubation of t25I BEP with normal rat, rabbit, bovine, foetal calf and human serum and cerebrospinal fluid (CSF) gave a similar result. Paper chromatography of the products of digestion by human serum showed monoiodotyrosine, monoiodohistidine and iodinated peptides, although with CSF the pattern of radioactive spots on a chromatogram of the digest was different. We concluded that normal serum and CSF have enzymes capable of hydrolysing the encephalitogenic polypeptides. DISCUSSION Kibler and Barnes/12~ working with a mixture of proteins and polypeptides extracted from rabbit spinal cord and labelled with l~tI, found that the encephalitogenic activity of their preparation was not destroyed by iodination. Caspary ~18~ labelled an encephalitogenic protein from human nervous tissue and found that it reacted immunologically with serum from guinea pigs immunized with the same but unlabelled encephalitogen. Caspary did not test the encephalitogenic activity of his labelled protein. In both of these experiments 131I was used as the label and the preparations were of very low specific radioactivity. We used lZSI rather than xatI to label BEP because x25I is better for localizing an antigen in tissues by autoradiography. Although our preparations of iodinated polypeptide retained their encephalitogenic activity, it remains to be determined whether complete iodination of
280
Communication to the Editors
tyrosine and histidine would destroy the activity. Hence we anticipate that encephalitogenic polypeptides labelled with 125I will become important experimental tools for further studies on the pathogenesis of EAE. BEP is readily iodinated, yet part of the iodine appears to dissociate easily from lzsI BEP and is then found in the solution as free iodine. It is known that in proteins, in addition to tyrosine, histidine can be iodinated uS~ and histidine is present in BEP in a relatively high concentration. When histidine is iodinated the iodine is initially attached to an N in the imidazole ring and only slowly forms a bond with C. In contrast to the C-iodination, the N-iodination is unstable.tt4,15~ Dissociation of an N - - I bond in iodohistidine could account for the appearance of a small amount of free iodide when iodinated BEP is stored. It is significant that BEP was hydrolysed by proteinases or peptidases in normal serum from various animals and human CSF. Moreover our finding that 125I BEP disappeared rapidly from the circulation after intravenous injection (unpublished observations) could indicate that proteolysis of BEP also occurs in vivo. BEP injected into guinea pigs without FCA does not induce EAE. uT~T h e Lewis rat, which is very sensitive to EAE, will develop EAE with only one injection of whole nervous tissue without FCA, t18~ but even in this strain 'purified' encephalitogens without FCA were not active; ~19~ conceivably some factor in whole nervous tissues could have an adjuvant effect (see, for example Levine and Wenk and KiesC19~). Our inference is that one of perhaps a number of effects of F C A in the induction of EAE is to protect encephalitogens from enzymatic digestion by proteinases. If so, proteolysis of the supposedly sequestered autoantigens of myelin may be a normal means of preventing them from reaching the immunological system.
Acknowledgements--We wish to acknowledge the invaluable assistance of Mr J. Pye and
Dr G. Ada in the iodination procedures and Professor V. M. Trikojus for iodinated amino acids. Glaxo Group Limited, England, generously provided the radioactive iodine. We are most grateful to Dr I. R. Mackay for reviewing the manuscript. This work was conducted during tenure of a Fellowship from the Institut de Microbiologie et d'Hygi6ne de l'Universit6 de Montr6al (G.L.) and supported in part by Grant No. 421 from the National Multiple Sclerosis Society, New York (P.R.C.).
The Clinical Research Unit of the Walter and Eliza Hall Institute of Medical Research and the Royal Melbourne Hospital, Parkville, Victoria, Australia and the Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia
G. LAMOUREUX* P. R. CARNEGIE T. A. MCPHERSON
REFERENCES x CAaNSGmP. R., BENCINAB. and LAMOURSUXG., submitted for publication. LAMOUREUXG., CARNEGmP. R., McPHERSON T. A. and JOI~STON D., submitted for publication. 8 CAlU~GmP. R. and LUMSD~ C. E., Immunoloffy 12, 133 (1967). 4 CARfare P. R., LAMOLU~UXG. and BENCINAB., Nature, Lond., in press. 5 HUNTERW. M. and GmmNWOODF. C., Nature, Lond. 194, 495 (1962). ¢ Present address: Institut de Microbiologie et d'Hygi6ne de l'Universit6 de Montr6al, Case Postale 100, Laval-des-Rapides, P. Qu6, Canada.
Experimental Allergic Encephalomyelitis
281
8 CAmc~om P. R., Nature, Lond. 206, 1128 (1965). E^SLEV C. W., Biochim. Biophys. Acta 107, 386 (1965). 80UCHa~aLONY O., Acta path. microbiol. Scand. 26, 507 (1949). 9 OVARY Z., Prog..4l/ergy 5, 459 (1958). 10 COWLT-II. and WOLF J., Biochemistry 5, 860 (1960). 11 SMITH I., Chromatographic and Electrophoretic Techniques. Heinemann (1960). 12 KmLER R. F. and BARNESA. E.,J. exp. Med. 116, 807 (1962). 18 CAsP~da'¢E. A . , J . Neurol. Neurosurg. Psychiat. 29, 103 (1966). 14 HUGHESW. L., Ann. N. Y..4cad. Sci. 70, 3 (1957). 15 BntrNINGS K. J.,J. Am. chem. Soc. 69, 205 (1947). 16 ~ m L - C O N n A T , H. F., Archs Biochem. 27, 109 (1950). iv LAMotrnmrX G., Cam~cIE P. R., McPn~aSON T. A. and MAcKaY I. R., submitted fol publication. is LEVlNE S. and WssK E. J., Ann. N. Y..4cad. Sci. 122, 209 (1965). 19 L~CINE S., Wm;K E. J. and Kms M. W., Neurology, Minnea. 15, 560 (1965).
TO THE
EDITORS
Experimental allergic encephalomyelitis--Properties of an iodinated encephalitogenic polypeptide* (Received 3 March 1967) Abstract~A bovine encephalitogenic polypeptide fraction (BEP) from ox spinal cord was iodinated with 195I and 1~I by the technique of Hunter and Greenwood.~ Iodinated BEP retained its electrophoretic and chromatographic properties and encephalitogenic activity. Hydrolysis of 1~I BEP showed that tyrosine and histidine were labelled. Delayed hypersensitivity reactions with BEP used as antigen performed on guinea pigs immunized with BEP trace labelled with 1~5I were positive. However the incidence of delayed hypersensitivity reactions on animals immunized with heavily iodinated la6I BEP was low, in spite of their normal encephalitogenic response. Foetal calf serum, the serum of adult animals and man and human cerebrospinal fluid appeared to contain a proteinase or peptidase capable of digesting BEP. Proteolysis of supposedly sequestered autoantigens of myelin may be a normal means of preventing them from reaching the immunological system. INTRODUCTION EXPERIMENTAL allergic encephalomyelitis (EAE) can be produced by injecting a bovine encephalitogenic polypeptide fraction (BEP) with Freund's complete adjuvant (FCA) into guinea pigs. BEP is isolated from an acid extract of an acetone dried powder of bovine spinal cord. There are three polypeptides in BEP all of relatively low molecular weight (3500). These polypeptides are thought to be derived from a basic protein of myelin by the action of an acid proteinase during the extraction with acid. tl~ The encephalomyelitis produced by immunization with BEP is preceded by cutaneous delayed hypersensitivity to BEP but no humoral antibody to BEP has been detected by conventional methods, t2~ We labelled BEP with radioactive iodine to aid the study of its immunological properties. This paper describes the iodination of BEP and the effects of iodination on the encephalitogenic and immunological activity of BEP. In addition we studied the effect of normal serum and cerebrospinal fluid on iodinated BEP.
MATERIALS AND METHODS Carnegie and Lumsden 'a~ isolated a group of polypeptides from bovine spinal cord and subsequently a more highly purified fraction was isolated tl~ as follows. A mixture of basic proteins and polypeptides was obtained by extracting an acetone dried powder of bovine spinal cord with HCI. This extract was neutralized and applied to carboxymethyl-Sephadex G25. The retained fraction was eluted with HCI and the proteins and polypeptides precipitated with 90 per cent acetone. This 'basic fraction' was passed repeatedly through a column of Sephadex G50, using formic acid : acetic acid : water (1 : 4 : 45) as solvent, to isolate the protein and polypeptide fractions. Bovine encephalitogenic polypeptide (BEP) was obtained as an apparently homogeneous fraction with an elution volume equivalent * Publication No. 1131 from The Walter and Eliza Hall Institute of Medical Research. 273
274
C o m m u n i c a t i o n to the Editors
to a polypeptide with a molecular weight of 3500. Amino acid analysis showed a high content of lysine, arginine, histidine, serine and glycine. On electrophoresis in polyacrylamide gel it was possible to observe three zones with only minor differences in mobility, the mobility with reference to cytochrome C being 1.85-2.05. ta~ The tyrosine content of BEP is 0.21/~-mole per mg and the histidine content 0.44 v-mole per mg. tl~ Iodination of BEP. Labelling with 1~5I was performed by the technique of Hunter and Greenwood. tS~ In a typical experiment 20/~g of BEP in 20/~1 of 1 M phosphate buffered saline (pH 7.3) was mixed with 2 mc (30/~1) of carrier-free radioactive sodium iodide (Radiochemical Centre, Amersham, England). Five microlitres of chloramine T, 10 mg/ml in water were added and allowed to react for 3 min. To stop the iodination 5/~1 of sodium metabisulfite, 24 mg/ml in water, were added. The la5I BEP was separated from inorganic iodide by chromatography of the mixture on a small column (6 cm × 0.6 cm) of Sephadex G25 fine (Pharmacia, Uppsala, Sweden). The column was washed several times with phosphate buffered saline (pH 7.3) and treated with 50/~g of BEP to minimize adsorption of 1~5I BEP onto Sephadex and glassware. The column was washed with 3 to 6 volumes of 0.1 N HC1 prior to fractionating the iodination mixture. The 125I BEP was eluted with 0.1 N HC1 and collected in tubes numbered 4 to 7 (fraction size 0.2 ml) and immediately buffered to pH 7.3 with a 1 M solution of sodium phosphate buffered saline (Fig. 1). The radioactivity in each fraction was determined using a sodium iodide scintillation crystal (Type No. 883A, Ekco Electronics Ltd., England). Other samples of low radioactivity were counted in the well of a sodium iodide scintillation crystal counter with an efficiency of about 60 per cent (Type No. 664A, Ekco Electronics Ltd., England). Electrophoresis of BEP on polyacrylamide gel was as described. ~4} Peptide maps were made from tryptic digests of BEP mixed with a trace amount of t25I BEP as described by Carnegie, Bencina and Lamoureux. tl~ The digests were examined by electrophoresis and chromatography. A typical experiment is described in Fig. 6. The digests were examined also by a peptide mapping technique whereby the sample is separated first on a micro-column of Sephadex G25 in phenol-acetic acid-water (1 : 1 : 1, w/v/v) followed by paper chromatography with n-butanol : pyridine : acetic acid : water (30 : 30 : 6 : 24 by volume) as solvent. 161 The papers were examined using the reagents described by Easley. c7~ Chromatograms were placed in contact with Kodak X-ray film for 3 to 24 hr to obtain autoradiographs. The encephalitogenic activity of BEP and the iodinated preparations of BEP were tested by immunizing guinea pigs as described by Lamoureux et al. ~2~ in doses shown in Table 1. Guinea pigs which develop EAE become incontinent and show loss of righting reflexes, hind limb paresis or hind limb paralysis; however guinea pigs which did not develop hind limb paresis or paralysis were not scored as having 'clinical' EAE. The brain and upper spinal cord of all animals were examined microscopically for our histological criteria of EAE which were perivascular aggregates of mononuclear cells in the brain substance in several separate fields. Cutaneous sensitivity to encephalitogen was tested 9 to 10 days after immunization, using 10 to 25 #g of BEP. The cutaneous injection site was examined at 3 hr
Experimental Allergic Encephalomyelitis
275
for an immediate reaction and at 24 and 48 hr for delayed reactions, as described by Lamoureux et aL <2~ Humoral antibody. Pooled sera were tested by immunodiffusion in agar geF s~ and passive cutaneous anaphylaxis (PCA)(9~ using BEP as antigen. For immunodiffusion we used small petri dishes containing 4 ml of melted Noble agar (Difco, Detroit, Michigan, U.S.A.) at a concentration of 0.3, 0.5 and 0.75 per cent and the gels were buffered at pH 4.8 with 0.2 M sodium acetate, at pH 6.0, 6.5 and 7.3 with 0.2 M sodium phosphate and at pH 8.6 with 0.05 M veronal buffer. EXPERIMENTAL RESULTS
Iodination of BEP. Iodination of BEP with 125I occurred readily. The total recovery of radioactivity was at least 85 per cent after chromatography on Sephadex G25 with 0.1 N HC1 as eluent (Fig. 1). In every case all the radioactivity which was recovered was attached to BEP and no peak of free 125I was detected. The specific activity of 125I BEP in eight iodination experiments ranged from 10 to 180 ~c per tzg of BEP. Because of the low molecular weight of BEP, preparations even of high specific activity had only a small proportion of molecules labelled.
o 1500
--
¢0
"
1
\%. I
10
~5
2O
Frocli0n number
FIG. 1. Chromatography of BEP on Sephadex G25 after iodination with x~5I (for conditions, see text). Fraction size 0"2 rnl; column size 6 cm ×0"6 era; eluent 0.1 N HCI. Fractions between the cross lines were pooled to give 125I BEP.
BEP was more heavily labelled with a mixture of 127I and a trace amount of 125I. Two preparations were obtained, one with a ratio of 0.3 atoms of iodine per mole of tyrosine in BEP (1271 BEPa) and the other with a ratio of 1.68 atoms of iodine per mole of tyrosine (187I BEPb). In the latter experiment a distinct peak of free iodide was obtained when the mixture was fractionated after iodination (Fig. 2). Chemical properties of iodinated BEP. Chromatography of 1251 BEP on Sephadex G50 three weeks after iodination showed that 75-80 per cent of the radioactivity
Communication to the Editors
276
was still bound to BEP and the peak of radioactivity coincided with the peak of the non-iodinated BEP (Fig. 3); a second peak in the salt region contained radioactivity. Electrophoresis on polyacrylamide gel of lzsI BEP three weeks after labelling showed that the mobility of 125I BEP relative to cytochrome C was unchanged, the I000 --
g
\
,-o~/
I
2O
Fraction
number
FIO. 2. Separation of iodinated BEP (600 ~g) from iodide. In this experiment a ratio of three atoms of iodide 18¢I per mole of tyrosine in BEP was used in the iodination. A trace of lasI was added to the iodide used for labelling. T h e fLrst peak is 125I BEP and the second peak is excess iodide. For conditions of chromatography, see Fig. 1.
--
3000C
LI~
O-O6O
Ila5 B E P
E --~040
2000C
N >,
1 0 IO00C
--
-
).O~n
(~
Fraction number
FIG. 3. Chromatography of a mixture of BEP (2-4 vg) and 1~51 BEP (0-08 ~g, 70 ~ / ~ g ) on Sephadex G50 (10g) in formic acid:acetlc a c i d : w a t e r (1]: 4 : 45). Radioactivity shown by ( ), optical density at 280 m~ (. . . . ).
l
FIG. 4. Electrophoresis on polyacrylamide gel of BEP (2), BEP mixed with 125I BEP (3) and mixture of basic protein and polypeptides (4). Cytochrome C (1) was used as reference. T h e gel contained 15 per cent polyacrylamide in formic acid : acetic acid : water (1 : 4 : 45) and 4 M urea p H 2"5.
FIG. 5. Autoradiography of the polyacrylamide gel shown in Fig. 4. Conditions and samples as in Fig. 4. (Facing p. 276)
Experimental Allergic Encephalomyelitis
277
Mc-value being 1.90 to 2.05 (Fig. 4); an autoradiograph of this particular gel showed only one radioactive spot corresponding to BEP (Fig. 5). t2¢I BEPa and 12¢I BEPb also had unchanged Mc-values. t~sI BEP was digested for 5 hr at 37°C with pronase at pH 7.5 and the digest examined by paper chromatography in butanol : acetic acid : water (5 : 1 : 5). '1°~ Iodine was found to be present as monoiodo-tyrosine, monoiodo-histidine and diiodo-tyrosine. When 125I BEP was left for 3 months at pH 2 the spot corresponding to monoiodo-histidine was greatly diminished and a spot of free iodide was evident. We concluded that iodination with a ratio of up to 1.68 atoms of iodine per mole of tyrosine in BEP did not significantly alter the chromatographic or electrophoretic properties of BEP and that most of the iodine remained bound to the BEP molecules for at least 3 weeks. Peptide mapping oftryptic digest of125I BEP. Peptide mapping was performed on tryptic digest of BEP together with a trace of 12~I BEP as a preliminary step to a study of the encephalitogenic determinant. The position of the peptides which stained with ninhydrin and spots which contained radioactivity are shown in Fig. 6. The spots of radioactivity did not coincide with any ninhydrin positive spots; the radioactive spots had a slightly higher Rf than the spots which contained tyrosine. These radioactive spots possibly correspond to iodotyrosine spots; if so the presence of the iodine in the peptide molecules would have caused an increase in the Rf-value in a manner similar to the change caused in the Rf of free tyrosine when it is iodinated. ~m Peptide mapping by the alternative procedure wherein the
© -r a
o O
a, T t3
5
X
5
I0
cm
ELECTROPHORE51S
FIG. 6. Tryptic digest of BEP (400 ~,g) mixed with trace of t~sI BEP (3 ~ , specific activity 35/~c/~g). Electrophoresis in pyridine acetate p H 6"5, 40 min at 50 V/cm. Chromatography in n-butanol : pyridine : acetic acid : water (30 : 20 : 6 : 24). Ninhydrin positive spots are shown by O. Spots which stained positive for tyrosine are shown by ~ and those which were radio° active by • (strong) and ~ (weak).
278
Communication
to the Editors
peptides were first separated according to molecular size and then by chromatography t6~ showed that the main spot of radioactivity migrated in Sephadex G25 as a peptide composed of five amino acids. We concluded that in the tryptic digest x85I was attached to relatively small peptides which were neutral or only slightly cationic at pH 6.5 and no radioactivity was associated with the strongly basic peptides. Immunological activity of iodinated BEP. The preparations studied were BEP, 125I BEP with a specific activity of 35/~c per ~g of BEP and the more heavily labelled preparations 127I BEPa and 1~7I BEPb which are described above. All preparations produced EAE in guinea pigs (Table 1). With the iodinated preparations it is possible that the encephalitogenic activity could be accounted for by small amounts of unlabelled BEP, since 50 ng of BEP are able to produce EAE. c2~ None of the preparations induced precipitating antibody to BEP as measured by immunodiffusion. Pooled sera from guinea pigs immunized with 127I BEPa gave positive PCA reactions with BEP, although sera from guinea pigs immunized with unlabelled BEP did not. Guinea pigs immunized with BEP, x25I BEP and 1~'7I BEPa developed typical cutaneous delayed hypersensitivity to BEP (Table 1). However delayed hypersensitivity reactions in animals immunized with heavily iodinated BEP (x27I BEPb) were less decisive than those obtained with guinea pigs immunized with other preparations, since only two of seven animals gave positive reactions (Table 1), yet five had lesions of EAE. We can at present offer no explanation for these apparent differences. TABLE 1. (DHS)
EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS (EAE), DELAYED HYPERSENSITIVITY AND CIRCULATING ANTIBODY IN GUINEA PIGS AFTER IMMUNIZATION W I T H BEP AND IODINATED BEP IN FREUND'S COMPLETE ADJUVANT
Fractions injected (dose)
EAE
Incidence of Time of neurological onset disorder (days) BEP (50 ~g) 9/11 12-18 12sI BEP 35 p.c//zg 6/10 14--19 (2-5 t,g) lZ7I BEPa (40-50 t'g) 5/6 12-18 1~7I BEPb (40-50 tLg) 4/7 14-18
DHS to BEP Incidence of histological lesions 10/11 5/10 6/6 5/7
Incidence of positive reactions 8/11
Antibody* to BEP IDt
PCA++
-
-
6/10
-
:k
4/5
-
+
2/7t
--
:k
* Pooled sera. t ID --immunodiffusion; ++Weak reactions. PCA--passive cutaneous anaphylaxis.
Degradation of 1~5I BEP by normal serum and cerebrospinalfluid. Chromatography on Sephadex G50 was used to study the effect of normal serum on 1251 BEP. Fifty to 1000 ng of l~sI BEP were incubated for 30 rain at room temperature with 0.2 ml of adult guinea pigs serum; chromatography of the mixture on Sephadex G50 revealed a small peak of radioactivity in the protein region, a peak of 125I
Experimental Allergic Encephalomyelitis
279
6oc 2
w
3
4
30'
I
--
o
I Time,
I
2
0'
hr
FIG. 7. Chromatography of a mixture of 1~5I BEP mixed with normal guinea pig serum and applied immediately to the column (0') and after 30 min incubation (30'). Column was prepared from 10 g Sephadex G50 with 0.1 M Tris buffer pH 7"5. Flow rate 60 ml per hour, radioactivity monitored in a Nuclear Chicago flow counter. Peaks occurred where protein 1, 300(0-4000 molecular weight polypeptides 2, iodide and iodohistidine 3, and iodotyrosine 4, are known to be eluted.
BEP, a peak in the low molecular weight region and a peak of radioactivity where iodotyrosine would be eluted (Fig. 7). Without incubation with serum this last peak of iodotyrosine was absent, but with increasing periods of incubation this peak increased proportionally and the peak of 125I BEP decreased proportionally. Incubation of t25I BEP with normal rat, rabbit, bovine, foetal calf and human serum and cerebrospinal fluid (CSF) gave a similar result. Paper chromatography of the products of digestion by human serum showed monoiodotyrosine, monoiodohistidine and iodinated peptides, although with CSF the pattern of radioactive spots on a chromatogram of the digest was different. We concluded that normal serum and CSF have enzymes capable of hydrolysing the encephalitogenic polypeptides. DISCUSSION Kibler and Barnes/12~ working with a mixture of proteins and polypeptides extracted from rabbit spinal cord and labelled with l~tI, found that the encephalitogenic activity of their preparation was not destroyed by iodination. Caspary ~18~ labelled an encephalitogenic protein from human nervous tissue and found that it reacted immunologically with serum from guinea pigs immunized with the same but unlabelled encephalitogen. Caspary did not test the encephalitogenic activity of his labelled protein. In both of these experiments 131I was used as the label and the preparations were of very low specific radioactivity. We used lZSI rather than xatI to label BEP because x25I is better for localizing an antigen in tissues by autoradiography. Although our preparations of iodinated polypeptide retained their encephalitogenic activity, it remains to be determined whether complete iodination of
280
Communication to the Editors
tyrosine and histidine would destroy the activity. Hence we anticipate that encephalitogenic polypeptides labelled with 125I will become important experimental tools for further studies on the pathogenesis of EAE. BEP is readily iodinated, yet part of the iodine appears to dissociate easily from lzsI BEP and is then found in the solution as free iodine. It is known that in proteins, in addition to tyrosine, histidine can be iodinated uS~ and histidine is present in BEP in a relatively high concentration. When histidine is iodinated the iodine is initially attached to an N in the imidazole ring and only slowly forms a bond with C. In contrast to the C-iodination, the N-iodination is unstable.tt4,15~ Dissociation of an N - - I bond in iodohistidine could account for the appearance of a small amount of free iodide when iodinated BEP is stored. It is significant that BEP was hydrolysed by proteinases or peptidases in normal serum from various animals and human CSF. Moreover our finding that 125I BEP disappeared rapidly from the circulation after intravenous injection (unpublished observations) could indicate that proteolysis of BEP also occurs in vivo. BEP injected into guinea pigs without FCA does not induce EAE. uT~T h e Lewis rat, which is very sensitive to EAE, will develop EAE with only one injection of whole nervous tissue without FCA, t18~ but even in this strain 'purified' encephalitogens without FCA were not active; ~19~ conceivably some factor in whole nervous tissues could have an adjuvant effect (see, for example Levine and Wenk and KiesC19~). Our inference is that one of perhaps a number of effects of F C A in the induction of EAE is to protect encephalitogens from enzymatic digestion by proteinases. If so, proteolysis of the supposedly sequestered autoantigens of myelin may be a normal means of preventing them from reaching the immunological system.
Acknowledgements--We wish to acknowledge the invaluable assistance of Mr J. Pye and
Dr G. Ada in the iodination procedures and Professor V. M. Trikojus for iodinated amino acids. Glaxo Group Limited, England, generously provided the radioactive iodine. We are most grateful to Dr I. R. Mackay for reviewing the manuscript. This work was conducted during tenure of a Fellowship from the Institut de Microbiologie et d'Hygi6ne de l'Universit6 de Montr6al (G.L.) and supported in part by Grant No. 421 from the National Multiple Sclerosis Society, New York (P.R.C.).
The Clinical Research Unit of the Walter and Eliza Hall Institute of Medical Research and the Royal Melbourne Hospital, Parkville, Victoria, Australia and the Russell Grimwade School of Biochemistry, University of Melbourne, Parkville, Victoria, Australia
G. LAMOUREUX* P. R. CARNEGIE T. A. MCPHERSON
REFERENCES x CAaNSGmP. R., BENCINAB. and LAMOURSUXG., submitted for publication. LAMOUREUXG., CARNEGmP. R., McPHERSON T. A. and JOI~STON D., submitted for publication. 8 CAlU~GmP. R. and LUMSD~ C. E., Immunoloffy 12, 133 (1967). 4 CARfare P. R., LAMOLU~UXG. and BENCINAB., Nature, Lond., in press. 5 HUNTERW. M. and GmmNWOODF. C., Nature, Lond. 194, 495 (1962). ¢ Present address: Institut de Microbiologie et d'Hygi6ne de l'Universit6 de Montr6al, Case Postale 100, Laval-des-Rapides, P. Qu6, Canada.
Experimental Allergic Encephalomyelitis
281
8 CAmc~om P. R., Nature, Lond. 206, 1128 (1965). E^SLEV C. W., Biochim. Biophys. Acta 107, 386 (1965). 80UCHa~aLONY O., Acta path. microbiol. Scand. 26, 507 (1949). 9 OVARY Z., Prog..4l/ergy 5, 459 (1958). 10 COWLT-II. and WOLF J., Biochemistry 5, 860 (1960). 11 SMITH I., Chromatographic and Electrophoretic Techniques. Heinemann (1960). 12 KmLER R. F. and BARNESA. E.,J. exp. Med. 116, 807 (1962). 18 CAsP~da'¢E. A . , J . Neurol. Neurosurg. Psychiat. 29, 103 (1966). 14 HUGHESW. L., Ann. N. Y..4cad. Sci. 70, 3 (1957). 15 BntrNINGS K. J.,J. Am. chem. Soc. 69, 205 (1947). 16 ~ m L - C O N n A T , H. F., Archs Biochem. 27, 109 (1950). iv LAMotrnmrX G., Cam~cIE P. R., McPn~aSON T. A. and MAcKaY I. R., submitted fol publication. is LEVlNE S. and WssK E. J., Ann. N. Y..4cad. Sci. 122, 209 (1965). 19 L~CINE S., Wm;K E. J. and Kms M. W., Neurology, Minnea. 15, 560 (1965).