Peripheral nervous system (PNS) expression of mRNAs encoding myelin proteins and Fc γ RIII during experimental allergic neuritis

Journal of Neuroimmunology, 41 (1992) 43-50 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00

43

JNI 02240

Peripheral nervous system (PNS) expression of mRNAs encoding myelin proteins and FcyRIII during experimental allergic neuritis Giancarlo Conti

a Christian Vedeler b, Peter Bannerman c, A b d o l m o h a m m a d Rostami c and David Pleasure c

a Institute of Neurology, University of Milan, Italy, b Department of Neurology, Haukeland Hospital, Bergen, Norway, and c Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA (Received 3 February 1992) (Revision received 16 April 1992) (Accepted 17 April 1992)

Key words: Experimental allergic neuritis; P2 basic protein; Immunoglobulin-binding protein; Myelin protein; Demyelination

Summary Experimental allergic neuritis (EAN) was induced in Lewis rats by injection of 'SP26', a peptide homologous to amino acids 53-78 of bovine myelin P2 protein, in complete Freund's adjuvant. The rats developed signs of EAN which began on day 14, were maximal on day 18, and had subsided by day 30. RNA content of cauda equina and sciatic nerves increased more than 2-fold at the height of EAN. Expression of myelin Po and PI mRNAs did not fall during EAN, nor rise during recovery. F c y R mRNA, which encodes FcyRIII, an immunoglobulin-binding protein mediating activation of natural killer cells and macrophages by immune complexes, was transiently, but markedly induced in scattered endoneural cells, presumably macrophages, in cauda equina and sciatic nerves during the period of increasing weakness.

Introduction Experimental allergic neuritis (EAN) can be induced in susceptible animals by injection of myelin P2 basic protein, or neuritogenic peptides derived from P2, emulsified in complete Freund's adjuvant (CFA), or by passive transfer of lymphocytes from syngeneic animals sensitized to P2. The animals develop symmetrical weakness, and demonstrate nerve conduction block or slowing, endoneurial inflammatory infiltrates, segmental demyelination, and scattered axonal degeneration (Brostoff et al., 1980; Szymanska et al., 1981; Rostami et al., 1984, 1990, 1991; Izumo et al., 1985; Heininger et al., 1986; Shin et al., 1989; Olee et al., 1990; Lassmann et al., 1991). These clinical, electrical, and pathological features resemble those of human idiopathic demyelinative polyradiculoneuritis (Asbury et al., 1969; Arnason, 1984; Brosnan et al., 1988).

Correspondence to: D. Pleasure, Neurology Research, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

In the present investigation, young adult Lewis rats were injected with the neuritogenic P2 peptide, 'SP26', emulsified in CFA. This produced a reproducible polyneuropathy which permitted us to address two questions about the pathophysiology of EAN. First, does suppression of Schwann cell myelin-synthetic capacity contribute to segmental demyelination? This was examined by assays of the nerves and roots of the SP26-EAN rats for mRNAs encoding the major structural proteins of PNS myelin. Second, how do the cellular and humoral arms of the immune system interact within endoneurium during the evolution of EAN? We approached this by measuring nerve root and sciatic nerve levels of F c y R mRNA (Zeger et al., 1990), and by localizing expression of this mRNA by in situ hybridization. F c y R mRNA encodes FcyRIII, a protein expressed by NK cells and macrophages which binds aggregated immunoglobulin and activates phagocytosis, antibody-dependent cell-mediated cytotoxicity, cytokine production, and other cell-mediated immune processes (Simmons and Seed, 1988; Anderson, 1989; Kiner, 1989).

44 Materials and Methods

SP26 synthetic peptide SP26 was prepared by solid phase synthesis (Rostami and Gregorian, 1991). The sequence of this peptide is: NH 2-Thr-Glu-Ser-Pro-Phe-Lys-Asn-Thr-Glu-Ile-SerPhe-Lys-Leu-Gly-Gln-Glu-Phe-Glu-Glu-Thr-Thr-AlaAsp-Asn-Arg-COOH. Test groups Lewis rats, 6-7 weeks post-natal and weighing 110130 g, were obtained from Charles River. Hind footpad subcutaneous injection was with 0.1 mg/ml of the SP26 peptide in 0.25 ml of phosphate-buffered saline plus 0.25 ml of CFA containing 5 mg/ml of Mycobacteriurn tuberculosis. CFA controls received an injection of CFA without SP26 by the same route. Groups of five SP26EAN rats and of two CFA control rats were killed on days 9, 14, 18, 23, 30, and 60 after injection (Table 1). Non-injected 6- and 15-week post-natal Lewis rats were also examined. Clinical evaluation Each rat was examined daily by two observers. Clinical signs of EAN were scored as: 0, normal; 1, mild (more than 5% weight loss, flaccid tail); 2, moderate (previous signs plus ataxia and inability to spread the toes); or 3, severe (paraplegia-tetraplegia) (Rostami et al., 1990). RNA extraction and quantification Sciatic nerves (from sciatic notch to popliteal fossa) and caudae equinae were harvested immediately after the animals were killed, frozen in liquid nitrogen, and stored at -80°C. Total RNA was extracted by a guanidinium-phenol-chloroform method (Chomczynski and Sacchi, 1986) from groups of four pooled sciatic nerves or two pooled caudae equinae of SP26-EAN, CFA control, and normal rats at the time-points indicated in the figures. Total RNA in the various preparations was assayed by ultraviolet spectroscopy at 260 and 280 nm (Sambrook et al., 1989). Northern blotting RNA was resolved by electrophoresis on 1% agarose formaldehyde gels (Sambrook et al., 1989). The sample lanes were loaded with RNA equivalent to 25% of that obtained from a single cauda equina (Fig. 2) or to 100% of that from one sciatic nerve (Fig. 3). Since the yield of RNA per cauda equina or nerve varied during the course of illness (Fig. 1), the absolute amounts of RNA loaded per lane also varied, as can be seen in the ethidium bromide-stained gels (upper panels in Figs. 2 and 3). After electrophoresis, RNA was transferred to nylon filters and immobilized by exposure to UV light (Khandjian, 1986). Prehybridization of the filter-bound

RNA was for 4 h at 42°C in a buffer containing 50% formamide (v/v), 5 x SSPE (1 X SSPE = 0.15 M NaC1/10 mM sodium phosphate/1 mM EDTA, pH 7.4), 5 x Denhardt's solution (1 x Denhardt's = 1% (w/v) Ficoll/1% (w/v) polyvinylpyrrolidone/1% (w/v) bovine serum albumin (BSA)), and 0.25 mg/ml denatured salmon sperm DNA. Hybridization with [3ap]_ random primer-labelled cDNA probes (Feinberg and Vogelstein, 1983) was for 18 h in the same buffer. These probes encoded rat FcTR (Zeger et al., 1990), rat myelin Po protein (Lemke and Axel, 1985), or human myelin basic protein, equivalent to P1 PNS basic protein (Kamholz et al., 1986). Blots were washed at 60°C in several changes of 2 x SSC (1 x SSC = 150 mM NaC1/75 mM sodium citrate) in 0.1% (w/v) sodium dodecylsulfate (NaDodSO4), with a final wash of 0.2 x SSC, 0.1% NaDodSO 4, then exposed to X-ray film at -70°C. Autoradiogram band intensities were estimated using a Zeineh Soft Laser Scanning Densitometer (Biomed Instruments, Fullerton, CA). At least two autoradiograms with differing exposure times were prepared from each blot and scanned in order to confirm that the film response was linear. After studies with the first cDNA probe were completed, the nylon filter was 'stripped' by incubation at 70°C for 20 min in hybridization buffer, then rehybridized with a second of the above cDNA probes. This process of stripping and rehybridization was repeated up to five times without perceptible degradation of the RNA immobilized on the blot. In each instance, the last cDNA probe employed encoded the 'housekeeping gene', cyclophilin (Danielson et al., 1988), in order to provide an estimate of the amount and quality of mRNA loaded per lane.

In situ hybridization Sciatic nerves and spleen were frozen in isopentane precooled with liquid nitrogen. 6-10/zm sections from these tissues were mounted on gelatin-coated slides that had previously been heated to inactivate traces of ribonuclease. In situ hybridization was performed as described by Jordan et al. (1989) with minor modifications. Briefly, the sections were fixed in 3% formaldehyde for 6 rain, incubated in 0.1 M triethanolamine/ 0.25% (v/v) acetic anhydride for 5 rain, in 0.1 M glycine/0.1 M Tris. HCl for 30 rain, dehydrated through a series of graded alcohols, and air-dried. The [35S]-labelled sense and antisense Po or F e y R riboprobes (Lemke and Axel, 1985; Sambrook et al., 1989; Zeger et al., 1990) were dissolved in a buffer containing 2 x SSC, 50% (v/v) formamide, 1 mg/ml yeast tRNA, 1 mg/ml single-stranded salmon sperm DNA, 10 mM Dq"T, and 2 mg/ml BSA. 1-2 x 10 6 dpm of sense or antisense probe were applied to each slide. The slides were covered with Parafilm and incubated for 3.5 h in an humid chamber at 52°C. After hy-

45

bridization, the coverslips were treated with 2 × SSC/50% (v/v) formamide at 52°C for 30 min, 100 # g / m l RNAase A for 30 min at 37°C, 2 × SSC/50% (v/v) formamide for 5 min, 0.1 ×SSC/0.1% (v/v) NaDodSO4 for 15 min, and 2 × SSC/10 mM DTI" overnight. The sections were dehydrated in ethanol, air-dried, and then dipped into Kodak NTB2 emulsion. Autoradiographic exposures were for 7 or 14 days. The slides were developed in Kodak D-19, rinsed in 2% (v/v) acetic acid, fixed in Kodak fixer, and counterstained with toluidine blue. Specificity was established by comparing autoradiograms obtained with antisense and sense riboprobes.

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200

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150

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8

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Results

Clinical features of SP26-EAN Weight loss, mild ataxia, and tail weakness first appeared in the SP26-EAN rats on day 14 post-injection. In most rats, motor deficits peaked on day 18. The animals began to gain weight and strength by day 23. Almost all had returned to normal neurological function by day 30, all by day 60. CFA controls showed no neurological abnormalities (Table 1). Yields of total RNA and of cyclophilin mRNA from cauda equina and sciatic nerves of SP26-EAN and control rats The yield of total RNA, estimated spectrophotometricaily, was more than two-fold greater from cauda equina (Fig. 1A) and sciatic nerve (Fig. 1C) of rats at the height of clinical EAN than from normal or CFA control rats. Levels of mRNA encoding the housekeeping gene product, cyclophilin, were also increased in both cauda equina (Figs. 1B, 2) and sciatic nerves (Figs. 1D, 3). Spectrophotometric scanning of these autoradiograms indicated that SP26-EAN cauda equina cyclophin mRNA content peaked at 4 times that in CFA control rats on day 14, and reached 3.5 times the

TABLE 1 Clinical features of SP26-EAN Days post-

N u m b e r of

Clinical score

immunization

animals examined

Median

Maximum

9 14 18 23 30 60

30 25 20 15 10 5

0 1 2 1 0 0

0 3 3 2 1 0

Clinical scoring was as described in the Materials and Methods. Twelve C F A controls were also examined; all had clinical scores of 0 at all time points.

Fig. 1. Effects of SP26-EAN on total R N A and cyclophilin m R N A contents of cauda equina and sciatic nerve. A, C. Spectrophotometric estimates of the yields of total R N A per cauda equina (A) or sciatic nerve (B) as a function of days post-injection of SP26 in CFA. The triangle in (A) indicates the yield obtained from caudae equinae of rats injected 14 days prior to sacrifice with C F A without SP26. The square in (C) indicates the yield obtained from sciatic nerves of 15-week post-natal non-injected rats. All data points are the m e a n s of the results of two independent experiments. B, D. Densitometric estimates of cyclophilin m R N A in Northern blots prepared from caudae equinae (B) or sciatic nerves (D). Results are expressed as the ratio of the SP26-EAN to C F A control cauda equina cyclophilin m R N A (B) or the ratio of SP26-EAN to normal control sciatic nerve cyclophilin m R N A (D). Each point represents a single ratio determination, but similar results were obtained in another series of animals (data not shown).

level in sciatic nerves of non-injected rats on day 18. Cyclophilin mRNA contents of both cauda equina and sciatic nerves fell to near normal levels during recovery (Figs. 1B-D, 2, 3).

Myelin Po and P1 mRNAs in SP26-EAN and control rats Northern blot analysis showed that content of Po mRNA per cauda equina was approximately 50% higher on days 14 and 18 (mean of two independent experiments) than at earlier or later time points or in controls. Results of one of these experiments are illustrated in Fig. 2. Cauda equina P1 mRNA was not assayed, because spinal cord contains high levels of an mRNA species indistinguishable from PNS P1 mRNA, and it was difficult to avoid inclusion of small fragments of spinal cord in the cauda equina samples. Amounts of Po and P1 mRNA per sciatic nerve remained unchanged in the SP26-EAN rats during the first 14 days post-injection, but then, during recovery, fell to 70% (mean of two independent experiments) of the level in age-matched control sciatic nerves. Results of one of these experiments are illustrated in Fig. 3. In situ hybridization with a [35S]-labelled Po antisense riboprobe was employed to examine the distribution of Po mRNA in sciatic nerves of SP26-EAN and normal rats. In situ hybridization with a [35S]-labelled

46 Po sense r i b o p r o b e was used to confirm specificity. E x a m i n a t i o n of l o n g i t u d i n a l sections of sciatic nerves hybridized with the a n t i s e n s e r i b o p r o b e showed that the f r e q u e n c y of cells with the e l o n g a t e d oval nuclei characteristic of S c h w a n n cells a n d with intensely r a d i o l a b e l l e d p e r i n u c l e a r regions in nerves from SP26E A N rats r e s e m b l e d that in a g e - m a t c h e d control rats (Fig. 4). Extensive hypercellularity is also illustrated in the S P 2 6 - E A N n e r v e section. P a n e l C of Fig. 4 is r e p r e s e n t a t i v e of m a n y sections of sciatic nerve from S P 2 6 - E A N rats with n e u r o l o g i c a l deficits that were e x a m i n e d by in situ hybridization; n o e n d o n e u r i a microscopic fields were f o u n d that were devoid of S c h w a n n ceils expressing Po m R N A .

FcyR mRNA in SP26-EAN and control caudae equinae and sciatic nerves

N

9

14

18

30

60

- 28S

- 18S

,,

Cyclophilin

F C -yR

-- lkb

- 1.4 kb

N o r t h e r n blot analysis showed that F c y R m R N A expression was t r a n s i e n t l y b u t m a r k e d l y i n d u c e d in the CFA 9

14 18 2 3 3 0

60

-28S

-18S

Cyclophilin

-lkb

FC1,R

-1.4kb

Po

-1.9kb

Fig. 2. Effects of SP26-EAN on FcyR and Po mRNA contents of cauda equina. The top panel shows the appearance of an agarose formaldehyde gel after electrophoresis and ethidium bromide staining. Each lane was loaded with total RNA equivalent to that from 25% of a cauda equina (1/8th of the yield from two pooled caudae equinae). The 28S and 18S ribosomal bands are indicated. The next three panels show autoradiograms of the blot following hybridization with [32p]-labelled cyclophilin, FcyR, and Po cDNA probes. Days post-injection of SP26 in CFA are indicated at the top; the CFA sample was from rats injected 14 days prior to sacrifice with CFA without SP26. The autoradiograms, exposed for two different times, were analysed with a laser scanning densitometer to estimate relative levels of the mRNAs.

Po

-1.9kb

P1

- 2.1kb

Fig. 3. Effects of SP26-EAN on FcyR, Po, and P1 mRNA contents of sciatic nerve. The top panel shows the appearance of an agarose formaldehyde gel after electrophoresis and ethidium bromide staining. Each lane was loaded with total RNA equivalent to that from one sciatic nerve (1/4 of the yield from four pooled sciatic nerves). The 28S and 18S ribosomal bands are indicated. The next four panels show autoradiograms of the blot following hybridization with [32p]-labelled cyclophilin, FcyR, Po, and P1 cDNA probes. Days post-injection with SP26 in CFA are indicated at the top. The 'N' (normal control) sample was from non-injected 15-week post-natal rats. The autoradiograms were exposed and scanned as described in Fig. 2. PNS of S P 2 6 - E A N rats. T h e a m o u n t of F c y R m R N A per c a u d a e q u i n a p e a k e d on day 14 at 16 times that in controls ( m e a n of two i n d e p e n d e n t experiments), a n d the a m o u n t of F c y R m R N A per sciatic nerve p e a k e d o n day 18 at six times that in controls ( m e a n of two i n d e p e n d e n t experiments). Examples of these results are shown in Figs. 2 a n d 3. I n situ hybridizations with an [35S]-iabeiled F c y R a n t i s e n s e r i b o p r o b e showed that F c y R m R N A was intensely expressed by scattered ceils with large, r o u n d nuclei within e n d o n e u r i u m (pres u m a b l y m a c r o p h a g e s ) of sciatic nerves from day 14 S P 2 6 - E A N rats (Fig. 5); such cells were rare in the e n d o n e u r i u m of n o r m a l rat sciatic nerves.

Discussion As previously r e p o r t e d ( R o s t a m i et al., 1990; Rostami a n d G r e g o r i a n , 1991), injection of Lewis rats with

47

A

B

C

Fig. 4. Demonstration of Po mRNA by in situ hybridization in longitudinal sections of SP26-EAN and normal sciatic nerves. A. Normal sciatic nerve hybridized with a [35S]-labelled antisense Po riboprobe; there is dense perinuclear labelling of a proportion of cells with elongated, oval nuclei, likely to be Schwann cells ( x 180). B. Normal sciatic nerve hybridized with a [35S]-labelled sense Po riboprobe; in this specificity control, very little labelling is noted ( × 180). C. This sciatic nerve was obtained 14 days post-injection of SP26 and CFA. The section was hybridized with a [3ss]-Iabelled antisense Po riboprobe. A small field of spleen from the same animal, frozen adjacent to the sciatic nerve, is indicated by the asterisk at the top of the panel. Considerable infiltration of endoneurium by cells with small, round nuclei is apparent. The frequency of intensely labelled Schwann cells is similar to that in the normal control (A). No radiolabelling is visible over the small area of spleen shown in the figure ( x 180).

SP26 in CFA produced a moderately severe polyneuropathy. The reproducibility and reversibility of this syndrome facilitated longitudinal analysis of the expression in nerves and nerve roots of mRNAs encoding myelin and immunoglobulin-binding proteins during the development of and recovery from EAN. The yield of R N A from the PNS of the SP26-EAN rats rose first in cauda equina, and slightly later in sciatic nerves. This increase in total RNA content likely reflected both the infiltration by blood-borne inflammatory cells and the hypertrophy and hyperplasia of glia and other ceils within the nervous tissue itself (Strigard et al., 1987; Griffin et al., 1990). A rise in total RNA content of similar proportions, and with a similar time-course, takes place in the CNS in experimental allergic encephalomyelitis (Aquino et al., 1990).

This prominent rise in PNS total R N A content during clinically active SP26-EAN necessitated a choice between two procedures for preparation of the Northern blots to be used for m R N A analyses; we could load each lane with either a variable amount of R N A corresponding to a constant number of nerves or nerve roots, or with a constant amount of RNA corresponding to a variable number of nerves or nerve roots. The former approach was selected, so that the intensity of the hybridization signal obtained by probing a Northern blot would be directly proportional to the amount of the m R N A of interest per nerve or root. This facilitated comparisons between the myelin protein synthetic capacities of nerves or roots at various stages of the evolution of SP26-EAN. Results demonstrated that amounts of the mRNAs encoding myelin structural proteins did not fall during development of EAN. Indeed, the total content of Po m R N A per cauda equina rose slightly at the point of peak clinical deficit. Furthermore, amounts of PNS Po and P1 mRNAs did not rise during recovery, suggesting that a generalized increase in Schwann cell synthesis of these myelin components is not necessary to support remyelination. The failure to detect a decrease in mRNAs encoding myelin structural proteins in cauda equina or sciatic nerve during the development of sp26-EAN might have been due to focality of the demyelination. This possibility was evaluated by in situ hybridization of sciatic nerve sections with Po riboprobes. Numbers of Schwann cells expressing Po m R N A per unit area were

Fig. 5. Demonstration of F c y R mRNA by in situ hybridization in longitudinal sections of SP26-EAN and normal sciatic nerves. This sciatic nerve was obtained 14 days post-rejection of SP26 and CFA, and the longitudinal section illustrated was hybridized with a [35S]labelled antisense F c y R riboprobe, and counterstained with toluidine blue. Exposure time was 7 days at 4°C. Three intensely radiolabelled cells are shown. Similar radioautograms with shorter exposure times (not shown) demonstrated the silver grains to be associated with cells with large round nuclei, and not with cells with the long oval nuclei characteristic of longitudinally sectioned Schwann cells. Radiolabelling of sections hybridized with a [3sSl-labeUed sense F c y R riboprobe was faint and not localized to individual cells (not shown) ( × 400).

48

similar in the EAN and control nerves, and large endoneurial foci devoid of Po mRNA positive Schwann cells were not detected in the EAN nerves. Thus, we were unable to demonstrate a diminution in Schwann cell expression of genes encoding myelin structural proteins by either Northern blotting or in situ hybridization. This suggests that segmental demyelination in SP26-EAN is not attributable to a generalized depression of Schwann cell myelin protein synthetic capacity. However, a detailed in situ hybridization study of teased nerve fibers (Griffiths et al., 1989) will be necessary to rule out the possibility that inhibition of myelin gene expression in occasional, scattered Schwann cells contributes to segmental demyelination in SP26-EAN. The sensitization protocol used to induce EAN in this study elicits axonal degeneration as well as segmental demyelination (Rostami et al., 1990). It is probable that the persistent, late diminution in amounts of Po and P1 mRNAs in sciatic nerve, but not in cauda equina, was attributable to such axonal degeneration (Gupta et al., 1988; Trapp et al., 1988). However, the normal steady-state levels of Po and P1 mRNAs in both cauda equina and sciatic nerves of the SP26-EAN rats during the time of greatest clinical deficit suggests that most axons remained intact in these rats during this period (Trapp et al., 1988; Gupta et al., 1988). FcyRIII, a 50-70-kDa plasma membrane IgG-binding glycoprotein expressed by NK cells and macrophages, participates in the initiation of cytotoxic responses by NK cells and phagocytosis by macrophages (Simmons and Seed, 1988; Anderson, 1989; Kiner, 1989; Zeger et al., 1990). In the present study, Northern blot analysis demonstrated a sharp, transient increase in PNS content of F c y R mRNA, which encodes FcyRIII (Zeger et al., 1990), in the SP26-EAN rats. This was first visible in cauda equina and shortly thereafter in sciatic nerves, and did not occur in rats injected with CFA alone. Though previous immunohistological studies demonstrated that Schwann cells can express FcyRIII, we have found that the steady-state level of F c y R mRNA in cultured, Schwann ceils is less than 1% of that in rat peritoneal macrophages and that, by in situ hybridization, accumulation of FcyR mRNA in the distal stumps of transected nerves is restricted to cells with the phenotypic properties of macrophages (Vedeler et al., 1992). In situ hybridization studies of the distribution of FcyR mRNA in SP26-EAN suggest that the increase in F c y R mRNA in the PNS of the SP26-EAN rats, as in nerves undergoing Wallerian degeneration, reflects the accumulation of FcyR-positive macrophages and other effector cells of the immune system in these tissues, rather than induction of FcTR mRNA expression by the Schwann ceils themselves. The marked induction of F c y R mRNA just as clini-

cal signs of SP26-EAN appear suggests that FcyRIIImediated immune responses play a role in the pathophysiology of EAN. It is possible, furthermore, that the therapeutic effects of immunoglobulin infusion or plasma exchange in patients with EAN and other immune-mediated demyelinative polyneuropathies (Guillain-Barr6 Study Group, 1985; Dyck et al., 1986; Harvey et al., 1988, 1989; Vedanarayanan et al., 1991) are due to inhibition by these treatments of the interaction between FcyRIII and immune complexes (Vedeler et al., 1988; Jungi et al., 1990; Vriesendorp et al., 1991) within endoneurium. In summary, the present study illuminates the pathophysiology of immune-mediated EAN in two respects. First, there is no generalized diminution in the capacity of Schwann cells in EAN nerves to express genes encoding the principal structural proteins of myelin during the period of development of deficits in EAN rats, nor is there evidence that myelin proteinsynthetic capacity is enhanced during recovery from EAN. Second, expression of the gene encoding the immunoglobulin-binding protein, FcyRIII, is transiently but markedly induced in the nerves and roots during the period of maximal clinical deficit. The pathophysiological significance of this induction, which is likely to correlate with increased immune complex binding capacity by endoneurial macrophages and NK cells, remains to be determined.

Acknowledgements This research was supported by NIH Grants NS25044 and NS08075, by a Sandie Altman Research Fellowship to G.C., and by the Associazione Amici Centro Dino Ferrari.

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