Isolation and purification of a Thy-1-like glycoprotein from sheep brain and its distribution in ovine tissues

Research in Veterinary Science /987, 41, 358-364

Isolation and purification of a Thy-I-like glycoprotein from sheep brain and its distribution in ovine tissues R. F. SHELDRAKE*, B. D. CROCKER, A. J. HUSBAND, J.A.P. ROSTAS, Faculty of Medicine, University of Newcastle, New South Wales 2308, Australia

Standard methods for the purification of Thy-l were applied to sheep brain to purify a sheep brain membrane glycoprotein (SBMG). On 12 per cent sodium dodecylsulphate polyacrylamide gels this glycoprotein was shown to be a doublet with apparent molecular weights 24,000 and 25,000. Bya number of physicochemical criteria SBMG was shown to have properties similar to those previously reported for Thy-l isolated from other species. Immunological investigations, however, revealed that SBMG did not react with rabbit anti-rat Thy-l and rabbit anti-sBMG did not recognise rat or chicken brain. By fluorescent antibody techniques the tissue distribution of SBMG appeared to be similar to that of Thy-l in other species. A small population of peripheral blood and intestinal lymph lymphocytes were stained with antiSBMG. These results suggest that sheep have an immunologically distinct Thy-l homologue. WHILE the sheep is an excellent experimental model for the study of cell recirculation, little work has been conducted on movements of non-B cells because of the inadequacy in techniques for their identification. In the mouse the Thy-I antigen has been widely used to distinguish between thymus-derived lymphocytes and other lymphohaemopoietic cells. The Thy-I alloantigens (Thy-I. I and Thy-I.2) were first described in mouse thymic and neuronal tissue (Reif and Allen 1964). More recently, homologues to mouse Thy-l have been detected in other species, namely the rat (Douglas 1972, Williams et al 1977), man and dog (Dalchau and Fabre 1979), chicken (Rostas et al 1983), cattle, carp and hamster (Shalev and Zuckerman 1984), frog (Mansour and Cooper 1984a), and in an invertebrate (Mansour and Cooper 1984b). To date a homologous molecule to Thy-I has not been reported in sheep. Preliminary experiments conducted in this laboratory showed that sheep lymphocytes were negative when stained with rabbit anti-rat Thy-I. However,

• Present address: New South Wales Department of Agriculture. Central Veterinary Laboratory, Glenfield, NSW 2167, Australia

between species so far studied there is a lack of uniformity in the expression of Thy-Ion lymphocytes and other tissues with the only uniformly Thy-Ipositive tissue being brain (Campbell et al 1981, Williams and Gagnon 1982). In addition, homologous Thy-I molecules from at least one species have been shown not to cross react immunologically (Rostas et al 1983). Thus the possibility exists that sheep lymphocytes express a form of the Thy-I glycoprotein which, like that of chickens, is not cross reactive with rat Thy-I. Alternatively sheep, like humans, may not express Thy-Ion lymphocytes. To distinguish between these two alternatives, standard biochemical methods were applied for the purification of Thy-I from sheep brain and a polyvalent heterologous antiserum raised to the Thy-I-like glycoprotein to test its cross species reactivity and tissue distribution. The purified glycoprotein was partly characterised using biochemical procedures to determine if it is homologous to the well characterised rat Thy-l glycoprotein. Materials and methods

Animals Adult sheep were Merino cross Border Leicester approximately one or two years old. Tissues were also obtained from a 12-day-old lamb and from fetuses at approximately day 60 and day 80 of gestation. Rat brain was obtained from an inbred adult piebald Viral Glaxo (PVG) rat.

Chemicals Sodium deoxycholate (DOC), bovine serum albumin, ovalbumin grade III, Tris-hydrochloric acid buffer (Tris-HCl) and l-methyl-alpha-D-glucopyranoside were purchased from Sigma Chemical Company, St Louis. Lentillectin-Sepharose 4B and Sephacryl S-200 (superfine) were purchased from Pharmacia Fine Chemicals, Uppsala. Concanavalin A was from Boehringer-Mannheim, Munich, and sodium dodecyl sulphate (SDS) was from DOH 358

ThY-I-like glycoprotein from sheep Chemicals, Poole. 125 1 was obtained as [1251] sodium iodide from New England Nuclear, Sydney, New South Wales.

Purification of sheep brain membrane glycoprotein (SBMG)

The method used for the preparation of the membrane glycoprotein from ovine brain was an adaptation of previously published methods (Barclay et al 1975, McClain et al 1978, Rostas et al 1983). Briefly, adult ovine brain devoid of cerebellum and excess white matter (30 g) was homogenised with a motor driven teflon pestle in seven volumes of 0·32 M sucrose, pH 7· O. After removal of nuclei and cellular debris by centrifugation at 1000 g for 10 minutes the membrane pellet obtained following centrifugation at 1 x 105 g for 60 minutes was delipidated with 200 ml of acetone at - 20°C in a Sorvall Omnimixer, was extracted with 2 per cent w/v DOC in O' 01 M Tris HCl pH 8· 0, (2' 0 per cent DOC buffer) and recentrifuged to remove insoluble material. The DOC concentration in the solubilised extract was reduced from 2 per cent (w/v) to O' 5 per cent (w/v) by the addition ofO' 26 per cent (w/v) DOC in 0'01 M Tris HCl pH 8 (0'26 per cent DOC buffer) yielding a final total volume of about 600 ml.

359

Autoradiography of concanavalin A-binding glycoprotein Con A-binding glycoproteins present in samples run on sDs-polyacrylamide gel electrophoresis were detected by the lectin overlay technique (Rostas et al 1977) using 125I-Con A labelled by the chloramine-T method (Bolton 1977).

Western transfer and immunoblots Electrophoretic transfer to nitrocellulose paper was performed by the method of Towbin et al (1979). Rabbit anti-sheep brain membrane glycoprotein (SBMG) (lgG fraction) and horseradish peroxidase conjugated goat anti-rabbit IgG (GARIgG) (Cappel) were used at 1120 and 11100 dilutions, respectively, and colour was visualised using 4-chloro-l-naphthol.

Circular dichroism spectra Circular dichroism spectra of the SBMG in O' 5 per cent DOC buffer were obtained on a Jasco 500 c spectropolarimeter (Jasco, Japan) using a 1 mm quartz cell (Markson Scientific, USA). Samples were scanned from 275 to 195 nm at 20 nm min - 1 and 35°C. The spectrum shown in Fig 2 is the average of 16 scans.

Chromatography

Antisera

The detergent extract was added to 50 ml of lentil lectin Sepharose 4B equilibrated with O' 5 per cent DOC in 0'01 M Tris HCl, pH 8'0 (0'5 per cent DOC buffer) and rotated overnight at 4°C. The beads were allowed to settle at unit gravity for one hour, the supernatant discarded and the beads washed three times with the original volume of O' 5 per cent DOC buffer. The bound glycoproteins were eluted with 0'2 M 1-methyl-alpha-D-glucopyranoside in 1·0 per cent DOC, 0·01 M Tris HCl, pH 8·0 (four times, 15 minutes each), the final glycoprotein fraction was concentrated to a volume of 5 ml and the solute changed to O' 5.per cent DOC buffer using an Amicon ultrafiltration cell with a PMIO membrane (Amicon Corporation, Lexington). The concentrate was applied to a Sephacryl S200 column as described by Rostas et al (1983).

Antiserum to the purified SBMG was prepared in a rabbit using the immunisation procedure previously described (Rostas et al 1983). Blood was collected from the marginal ear vein and the IgG fraction prepared by elution of an ammonium sulphateprecipitated globulin preparation on DEAE-Sephacel (Pharmacia). Rabbit anti-rat Thy-I was obtained as a gift from Dr R. J. Morris, National Institute for Medical Research, Mill Hill, London.

SDS

polyacrylamide gel electrophoresis

Polyacrylamide slab gels (12 per cent) were run as previously described (Rostas et al 1983) and stained with Coomassie brilliant blue or silver. An 8 to 17 per cent gradient gel was used to enhance protein separation for the Wesfern transfer. All gels were run with molecular weight standards (Pharmacia).

Immunofluorescent histology Tissues were collected from brain, thymus, spleen. adrenal gland, supramammary lymph node, mammary gland, jejunum, Peyer's patches and retina from adult non-lactating ewes. Some of these tissues were also collected from fetuses at days 60 and 80 of gestation, and a lamb 12 days post partum. Cryostat sections (10 JAm) were fixed with ice cold 70 per cent ethanol and incubated with rabbit anti-SBMG (which had previously been exhaustively absorbed with washed sheep liver homogenate) and subsequently with fluorescein isothiocyanate (FITc)-conjugated GARIgG (diluted 1120 in phosphate buffered saline). For some sections the initial reagent was rabbit anti-

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R. F. Sheldrake, B. D. Crocker, A. J. Husband, J. A. P. Rostas

rat Thy-I. Sections of rat and chicken brain were studied using the same protocol. On all occasions a negative control using normal rabbit serum as the first reagent in the above protocol was included. Pre-immune serum from the rabbit used to produce the polyvalent anti-SBMG antiserum gave identical reactivity to normal rabbit serum. As a further control, rabbit anti-SBMG and rabbit anti-rat Thy-l were absorbed with excess amounts of membrane suspensions prepared from a number of tissues by incubating for one hour at room temperature. The suspension was then centrifuged at 9000 g for three minutes in a Beckman microfuge and the supernatant used as the initial reagent following the method outlined above.

Flow cytometric analysis A Spectrum III automated flow cytometer (Ortho Diagnostics Systems, Raritan, New Jersey) was used in the analysis of positively fluorescing sheep cell suspensions. The photomultiplier tube settings for the forward light scatter, right angle scatter and red and green fluorescence were 110, 300, 178 and 695, respectively, while the sample rate used was 0·26 ml min- 1 and the sheath rate 12 ml min -I. Single cell suspensions of adult and fetal peripheral blood, adult intestinal lymph and adult and fetal spleen and thymus were prepared. The reagents used were either normal rabbit serum or rabbit anti-SBMG as the first label followed by FITc-conjugated GARIgG as the second. The results were expressed as the difference in percentage fluorescing cells between suspensions stained initially with rabbit anti-SBMG and those stained with normal rabbit serum. Between 2000 and 5000 cells were counted from each sample.

Results Electrophoresis of the SBMG fraction on 12 per cent SOS polyacrylamide gels revealed a protein doublet of apparent molecular weight 24,000 and 25,000 which constituted the overwhelming majority of the protein in the purified SBMG fraction (Fig 1). The major contaminants were lentil lectin (which leached from the affinity column to a variable extent and is not observed in the sample shown in Fig 1) and an unknown protein of appoximately 65 kDa. When the gel was incubated with 12sI-Con A (Fig 1), the majority of the lectin bound to the SBMG doublet. The remainder bound to a glycoprotein doublet of approximately 40 kDa which corresponds to a very minor protein component of the SBMG. The polyvalent rabbit antiserum gave a single precipitin line by double immunodiffusion against the SBMG fraction (not shown) and its specificity was further characterised by immunoblots. The preimmunisation rabbit

serum showed no precipitin arcs. Western blots of gels were incubated with the polyvalent antiserum against the SBMG fraction and at least 80 per cent of the antibodies bound to the SBMG protein doublet (Fig 1). The remainder bound to lentil lectin, lower molecular weight minor degradation products of SBMG and the 40 kDa doublet whose identity and distribution is currently under investigation. As judged by immunofluorescence histology the anti-SBMG serum did not cross react with Thy-l molecules from rat or chicken brain (Table 1). This was confirmed by absorption experiments in which excess quantities of rat brain and chicken brain homogenates could not absorb out any significant proportion of the anti-SBMG activity in a rabbit anti-SBMG serum whereas it was completely removed by absorption with sheep brain homogenate (Table 1). The circular dichroism spectrum of the SBMG frac-

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251< .,""';'' , 241(,.,.,-

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(a)

(b)

lcl

FIG 1: Biochemical analysis of SBMG fraction. (al Protein content determined by electrophoresis on 12 per cent polyacrylamide gels stained with Coomassie blue; (bl glycoprotein content determined by autoradiography of 125I-Con A bound to SGMB separated on 12 per cent polyacrylamide gels; (c) reactivity of rabbit anti-SBMG serum determined by immunoblotting of SBMG transferred to nitrocellulose from an 8 to 17 per cent gradient polyacrylamide gel

Thy-I-like glycoprotein from sheep

361

TABLE 1: Fluorescent staining of selected tissues with rabbit anti-sheep brain membrane glycoprotein (SBMG) and rabbit anti-rat Thy-1 Fluorescence in the following tissues stained with: Rabbit anti-rat Thy-1 Adult chicken brain Adult rat brain Adult sheep liver Adult sheep brain Lamb brain (12 days post partum) Fetal sheep brain (60 days gestation) Fetal sheep brain (80 days gestation) Adult sheep thymus Lamb thymus (12 days post partum) Fetal sheep thymus (80 days gestation) Adult sheep spleen Adult sheep adrenal gland (medulla) Adult sheep mammary gland (non-lactating) Adult sheep intestinal lamina propria Adult sheep supramammary lymph node Adult sheep Peyer's patch Adult sheep retina

Rabbit antiSBMG

+++ Not +++ ++ + +

Fluorescence in sheep brain stained with anti-SBMG absorbed with the following tissues:

+++ +++ +++

+

NO NO NO NO

++ ++ + +++ ++ +++ + +

+

NO NO NO NO NO

• All sections were additionally stained with FllC-conjugated GARlgG and fluorescence assessed by incident light microscopy. t NO Not done No fluorescence was detected in any of the above tissues if NRS was used instead of anti-Thy-1 or anti-SBMG

tion (Fig 2) was almost identical to the spectra obtained under the same conditions for rat and human Thy-I and the Thy-I-like glycoprotein from chickens which appears to be a homologue but is immunologically not cross reactive (Rostas et al 1983). There was a single negative peak at about 212 nm, which probably corresponds to the peak of the beta sheet spectrum which has been shifted from its normal position at 217 nm because of the presence of detergent (Campbell et al 1979, Rostas et al 1983). There was no evidence of negative peaks at 208 nm or 222 nm (peaks characteristic of alpha helix) consistent with Thy-I in other species (Campbell et al 1979, Rostas et al 1983). The steep rise in the spectrum below 210 nm is not due to alpha helix because it is also found with' rat Thy-I, which has been shown by analysis of its primary structure to contain no significant alpha helix (Williams and Gagnon 1982), but may be due to the optical activity of the bound carbohydrates, since solutions of the appropriate monosaccharides show similar spectra in this region (unpublished data). Overall, the spectrum is consistent with a predominantly beta sheet structure with no detectable alpha helix. Immunofluorescence histology showed strong reactivity between rabbit anti-SBMG and sheep cerebral cortex. The fluorescence pattern (not shown) consisted of a mass of small dots and ringlets very similar to that observed when rat brain was stained

with rabbit anti-rat Thy-I and also similar to that observed in the chicken (Rostas and Jeffrey 1977).

210

230 250 Wavelength (nm)

270

FIG 2: Circular dichroism (Cd) spectrum for SBMG after subtracting the background effects of the 0·5 per cent DOC Tris buffer. To calculate the molar concentration of the SBMG, molecular weight was assumed to be 17,000

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R. F. She/drake, B. D. Crocker, A. J. Husband, J. A. P. Rostas

This was thought to be due to reaction with neuronal processes. The fluorescence in retina was confined to the optic fibre and inner plexiform layers (not shown), as is the case in rat (Perry et al 1984) and chicken (Jeffrey et ai, unpublished observations), however, the intensity of the fluorescence was low. As sheep were found to have a substantially lower density of retinal ganglion cells than rat or chicken, this lower intensity of fluorescence is consistent with the suggestion that the Thy-I in retina is expressed predominantly only on retinal ganglion cells (Perry et al 1984 Barnstable and Drager 1984). In the fetal and neonatal thymus staining with rabbit anti-SBMG was restricted to the medulla and trabeculae (Fig 3a) while staining in the cortex was equivalent to background staining observed in sections stained with normal rabbit serum or anti-rat Thy-I instead of anti-sBMG. In the adult thymus this differential pattern was not obvious and was more noticeable in foci-like regions with fluorescence occurring in the intercellular space. A similar observation was made for adult spleen with fluorescence being detected in regions analogous to splenic corpuscles. Both these tissues showed considerable autofluorescence. Fluorescence in Peyer's patches was homogeneous and restricted to intercellular spaces and appeared to be associated with cell surface membranes. In the adrenal gland (Fig 3b) fluorescence was bright and restricted to the medulla, while in the jejunal lamina propria (Fig 3c) connective tissue and some cells were seen to be positively stained.

la)

Ib)

Similarly, in the non-lactating mammary gland fluorescence was generally associated with connective tissue. The tissue distribution of the SBMG antigen was confirmed by absorption experiments in which samples of anti-sBMG were absorbed with excess quantities of various tissue homogenates and subsequently tested on adult sheep cerebral cortex by immunofluorescence histology. The staining was compared with sections incubated with unabsorbed antiserum. All the tissues that showed no reactivity by immunofluorescence (sheep liver, rat brain, chicken brain) were unable to absorb out any significant proportion of the anti-sBMG activity. Conversely all tissues, even those whose reactivity by immunofluorescence was weak, were able to absorb totally the anti-SBMG activity (Table I). The differences in proportion of fluorescing lymphocytes in preparations stained with rabbit antiSBMG compared with normal rabbit serum for various tissues and fluids are presented in Table 2.

Discussion To determine whether sheep possess a glycoprotein antigen which is analogous or identical to Thy-I in other mammals, a similar purification procedure to that used for preparation of Thy-I glycoprotein from rat brain was used (Barclay et al 1975). The authors' biochemical and physical studies of

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Thy-i-like glycoprotein from sheep the SBMG fraction show that the protein doublet, which constitutes the majority of the fraction, is a Thy-I-like glycoprotein. Its abundance in brain and apparent molecular weight correspond well with other Thy-I glycoproteins (Barclay et a1I975, Letarte-Muirhead et a1I975, Kuchel et a1I978, Cotmore et aII98I). Its secondary structure is predominantly beta sheet with no detectable alpha helix. Its quantitative binding to lentil lectin affinity columns indicates that all the molecules have an N-linked carbohydiate chain with a fucose linked to the internal N-acetylglucosamine (Kornfeld et al 1981). All the molecules also bind Con A and therefore must have a high mannose or biantennary N-linked carbohydrate chain (Baenziger and Fiete 1979). Preliminary analysis of the N-linked carbohydrate chains by stepwise digestion with endoglycosidase F (Elder and Alexander 1982) showed that the molecules had at least two such chains and that, after the removal of the first chain, the glycoprotein migrated as a single band (data not shown). Thus the doublet formation appears to be due to carbohydrate heterogenei ty. The polyvalent antiserum against SBMG was used to determine the tissue distribution of this glycoprotein in ovine tissues. The vast majority of the antibodies were directed against the Thy-I-like SBMG but a minor antigen of approximately 40 kDa was also detected. In using immunofluorescence as a semiquantitative assay of antigen content, low antigen concentrations may be missed and the relative contribution of the minor antigen is not known. In this regard it is important to note that all fluorescence positive tissues were able to absorb out all immunoreactivity in the antiSBMG suggesting that none of the positive tissues consisted of structures containing only the contaminating antigen. Also, every tissue which was fluorescence negative also failed to absorb significant amounts of immunoreactivity. Furthermore, experience with the

363

chicken brain Thy-I-like glycoprotein suggests that this contaminating antigen may have a limited effect on assays of quantitative distribution. The polyvalent antiserum to the chicken molecule recognised an analogous 40 kDa contamInant but gave very similar results in quantitative assays to those obtained with a monoclonal antibody to the chicken Thy-I-like glycoprotein which did not recognise the contaminant (P. L. Jeffrey, D. 1. Greig, C. M. Sinclair and J. A. P. Rostas, unpublished). Since the distribution of Thy-I between various tissues varies greatly even within mammals (Campbell et al 1981) the identity of the individual tissues which were immunoreactive did not aid in the identification of SBMG as a Thy-I-like glycoprotein. However, the distribution of SBMG within each tissue was similar to that described for Thy-I. SBMG was found associated with connective tissue within the jejunal lamina propria, non-lactating mammary gland and thymus. These findings concur with those of Ritter and Morris (1980) who showed that, in the rat, Thy-I was associated with connective tissue, mainly basement membrane associated, surrounding some blood vessels in lymphoid and non-lymphoid organs. In the spleen and lymph node the Thy-I was primarily associated with blood vessels associated with recirculation of lymphocytes, and these authors suggested that Thy-I may be involved in migration of small lymphocytes into peripheral lymphoid organs. They recorded a similar observation regarding blood vessels and reticulin fibres in the gut lamina propria, and dermis, both major sites of lymphocyte activity. Figs 3a and c show in the present study the relationship between SBMG and trabaculae within the fetal thymus, and connective tissue within the gut lamina propria. The findings of this study suggest that sheep do possess a homologue of the Thy-I glycoprotein isolated from other species and that an antiserum

TABLE 2: Distribution of sheep brain membrane glycoprotein (SBMG) molecules on lymphocyte populations from various sheep tissues and fluids

Proportion of lymphocyte population fluorescing 1%)'

Cell population Adult peripheral bloodt Adult intestinal lymph Adult supramammary lymph node Adult spleen Adult thymus Fetal peripheral blood Fetal thymus Fetal spleen

Rabbit anti·SBMG; FITC-GARlgG 11)

Normal rabbit serum; FITC·GARlgG 12)

Difference between III and 12)

15·0 ± 1·0 18·8± 1·6 50·0 39·0 66·4 5·1 3·2 17·4

3·0 ± 0·7 2·2± 0·1 7·5 22·0 58·7 3·0 0·7 19·7

12·0 ± 0·9 17·6± 1·6 42·5 17·0 7·7 2·1 2·5

o

• Detected using flow cytometry t Values shown are means ± standard errors of measurements on four samples of adult peripheral blood and two samples of adult intestinal lymph and are single measurements for all other samples

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R. F. Sheldrake, B. D. Crocker, A. J. Husband, J. A. P. Rostas

raised against the SBMG preparation containing this molecule is able to identify a subpopulation of sheep lymphocytes. While detailed studies have not yet been undertaken to examine the functional characteristics of the population of cells recognised by this antiserum, the results in Table 2 indicate differences in their distribution between peripheral blood and efferent intestinal lymph. Since the SBMG fraction contained a minor glycoprotein of 40 kDa which was also recognised by this antiserum, it is possible that some of the cells expressed this glycoprotein in addition to, or instead of, the Thy-I-like molecule. Nevertheless, this molecule may prove useful as a marker for a functional subset of sheep lymphocytes. A monoclonal antibody to the molecule has been produced for this purpose and it will be interesting to compare its pattern of reactivity with that of the monoclonal antibodies directed against ovine cell surface antigens described by MacKay et al (1985) and Maddox et al (1985). Acknowledgements This work was supported by grants from the Australian Meat Research Committee, the Australian Dairy Research Committee (ADRC) and the National Health and Medical Research Council of Australia. R. F. S. acknowledges financial support from the ADRC and the NSW Public Service Board. We are grateful to Dr G. Mendz, department of biochemistry, University of Sydney, for providing the circular dichroism spectra and for valuable discussions. We are. also grateful to Mrs A. Tyler for excellent technical assistance. References BAENZIGER, J. U. & FIETE, D. (1979) Journal of Biological Chemistry 254. 2400-2407 BARCLAY. A. N., LETARTE-MUIRHEAD, M. & WILLIAMS, A. F. (1975) Biochemical Journal 151. 699-706 BARNSTABLE. C. J. & DRAGER, U. C. (1984) Neuroscience II. 847-855

BOLTON, A. E. (1977) Radioiodination Techniques: Review 18. The Radiochemical Centre, Amersham CAMPBELL, D. G., GAGNON, J., REID, K. B. M. & WILLIAMS, A. F. (1981) Biochemical Journal 195, 5-30 CAMPBELL, D. G., WILLIAMS, A. F., BAYLEY. P. M. & REID, K. B. M. (1979) Nature 282.341-342 COTMORE. S. F., CROWHURST. S. A. & WATERFIELD, M. D. (1981) European Journal of Immunology II. 597-603 DALCHAU, R. & FABRE. J. w. (1979) Journal of Experimental Medicine 149. 576-591 DOUGLAS, T. C. (1972) Journal of Experimental Medicine 136. 1054-1062 ELDER. J. H. & ALEXANDER, S. (1982) Proceedings of the National Academy of Sciences, USA 79, 4540-4544 KORNFELD, K., REITMAN, M. L. & KORNFELD, R. (1981) Journal of Biological Chemistry 256. 6633-6640 KUCHEL. P. w., CAMPBELL, D. G., BARCLAY, A. N. & WILLIAMS, A. F. (1978) Biochemical Journal 169. 411-417 LETARTE·MUIRHEAD. M.• BARCLAY, A. N. & WILLIAMS, A. F. (1975) Biochemical Journal 151. 685-697 McCLAIN, L. D., TOMANA. M. & ACTON. R. T. (1978) Brain Research 159. 161-171 MacKAY, C. R.• MADDOX, J. F., GOGOLIN-EWENS, K. J. & BRANDON. M. R. (1985) Immunology 55.729-737 MADDOX. J. F.• MacKAY, C. R. & BRANDON, M. R. (1985) Immunology 55. 739-748 MANSOUR, M. H. & COOPER, E. L. (I 984a) Journal of Immunology 132. 2515-2523 MANSOUR, M. H. & COOPER, E. L. (1984b) European Journal of Immunology 14.1031-1039 PERRY, P. H.• MORRIS, R. J. & RAISMAN. G. (1984) Journal Jf NeurocYlOlogy 13. 809-824 REIF. A. E. & ALLEN, J. M. V. (1964) Journal of Experimental Medicine 1201.413-433 RITTER. M. A. & MORRIS, R. J. (1980) Immunology 39.85-91 ROSTAS, J. A. P. & JEFFREY. P. L. (1977) Neurochemistry Research 2, 59-85 ROSTAS, J. A. P .• KELLY, P. T. & COTMAN. C. w. (1977) Analytical Biochemistry 80,366-372 ROSTAS. J. A. P .• SHEVENAN. T. A.• SINCLAIR, C. M. & JEFFREY, P. L. (1983) Biochemical Journal 213. 143-152 SHALEV, A. & ZUCKERMAN. F. (1984) Immunology 50. 667-670 TOWBIN. H., STAEHELlN, T. & GORDON. J. (1979) Proceedings of the National Academy of Sciences, USA 76. 4350-4354 WILLIAMS. A. F., BARCLAY, A. N., LETARTE-MUIRHEAD, M. & MORRIS. R. J. (1977) Cold Spring Harbor Symposium on Quantitative Biology 41.51-61 WILLIAMS, A. F. & GAGNON, J. (1982) Science 216.696-703

Accepted June 23, 1986