Chronic inflammatory demyelinating polyneuropathy: Histological and immunopathological studies on biopsied sural nerves

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Journal of the Neurological Sciences 127 (1994) 170-178

Chronic inflammatory demyelinating polyneuropathy: histological and immunopathological studies on biopsied sural nerves Kenji Matsumuro,

Shuji Izumo

*, F u j i o U m e h a r a ,

Mitsuhiro

Osame

Third Department of Internal Medicine, Faculty of Medicine, Kagoshima Uniz'ersity, 8-35-1 Sakuragaoka, Kagoshirna 890, Japan Received 26 October 1993; revised 12 July 1994; accepted 26 July 1994

Abstract

We undertook histological and immunopathological studies on biopsied sural nerves from 9 patients with chronic inflammatory demyelinating polyneuropathy (CIDP). The diagnosis of CIDP was based on the research criteria proposed by the Ad Hoc Subcommittee of the American Academy of Neurology AIDS Task Force. The nerve pathology in these patients comprised macrophage-associated active demyelination and subsequent remyelination of various proportions. The presence of T cells in the endoneurium correlated with activity of demyelination. An analysis of T cell subsets demonstrated that the number of CD8-positive cells predominated over that of CD4-positive ones. Infiltration of B cells, and depositions of immunoglobulin and complement were not seen. These observations suggest that a T cell-mediated process is of pathogenic significance in CIDP. Furthermore, a double immunofluorescence staining revealed that most HLA-DR antigen-positive cells in the nerves in which active demyelination was seen coexpressed a macrophage-specific determinant. Conversely, HLA-DR-positive Schwann cells were found in the nerves in which remyelination was predominant. The expression of HLA-DR antigen on Schwann cells might not play a pathogenic role in the active demyelination in CIDP.

Keywords: Chronic inflammatory demyelinating polyneuropathy; Sural nerve biopsy; T cell-mediated immunity; HLA-DR expression

1. Introduction

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired neuropathy believed to be caused by immune-mediated damage to peripheral nerves (Dyck et al. 1975; Prineas and McLeod 1976; Dalakas and Engel 1981). The clinical and laboratory findings in C I D P exhibit some similarities to those in acute Guillain-Barr6 syndrome (GBS), suggesting that they are related diseases with a common pathogenesis. However, controversy existed as to whether C I D P was a homogeneous clinicopathological entity or merely a clinical syndrome produced by more than one disease process (Barohn et al. 1989). It is also inconclusive as to how cellular or humoral immunity can participate in the sequence of peripheral nerve damage. Another

* Corresponding author. Present address: Molecular Pathology and Genetic Epidemiology, Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshirna 890, Japan. Tel.: + 81-992-75-5940; Fax.: + 81-992-75-5942. 0022-510X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 2 2 - 5 1 0 X ( 9 4 ) 0 0 1 8 0 - 4

concern in C I D P is which cell type expresses the major histocompatibility complex (MHC) class II antigens in the peripheral nervous system (Pollard et al. 1986; Mancardi et al. 1988; Schroeder et al. 1988; Mitchell et al. 1991). Local presentation of M H C class II antigens is thought to play a role in the pathogenesis of demyelination because reactive lymphocytes can recognize antigens only in association with M H C class 1I antigen. Since there are no firm clinical criteria for diagnosis of CIDP, most reviews have included various types of neuropathies within this category. As a consequence, a better approach in the establishment of specific clinical or laboratory features could not be made. Thus, it is imperative to exclude the other neuropathies in order to characterize typical patients of C I D P and elucidate pathomechanisms of peripheral nerve damage. Recently, a new research criteria for the diagnosis of C I D P has been reported (Ad Hoc Subcommittee of the American Academy of Neurology A I D S Task Force 1991). In the present study, we performed histological and immunopathological investigations on biopsied sural nerves from patients that

K. Matsumuro et al. / Journal of the Neurological Sciences 127 (1994) 170-178

fulfilled these criteria. We have immunohistochemically analyzed the infiltrating cells in the peripheral nerves, and correlated them to the pathological findings. The HLA-DR expressing cells in the endoneurium using double immunofluorescence techniques was also performed.

2. Materials and methods

Patients and tissue samples We examined biopsied sural nerves from 9 patients with CIDP. These patients fulfilled all of the diagnostic criteria reported from Ad Hoc subcommittee of American Academy of Neurology Aids Task Force. The criteria included progressive or relapsing motor and sensory dysfunction with hypo- or areflexia, predominant process of demyelination on electrophysiological studies, unequivocal evidence of de- or remyelination on the routine pathologic studies of the biopsied sural nerves, and high protein and < 10/mm 3 of cell count in CSF. The patients included 6 male and 4 female, and the ages of onset of neurological symptoms ranged from 15 to 76 years (Table 1). In 4 patients the course was relapsing while 5 patients had a progressive course. No patient had risk factors for human imrnunodeficiency virus type I (HIV) infection, nor subsequently developed HIV-associated diseases. We excluded patients with other systemic diseases, such as monoclonal gammopathies, Crow-Fukase syndrome, diabetes, collagen vascular disease, and central nervous system demyelinating diseases. The duration of illness until nerve biopsy ranged from 4 months to 7 years. All patients showed apparent responses to corticosteroid treatment. Except for patients 4 and 7, they underwent plasmapheresis, that resulted in temporary clinical improvement. In 2 relapsing patients (patients 6 and 7),

171

nerve biopsies were performed at the time of exacerbation related to corticosteroid withdrawal. No other patients received corticosteroid nor immunosuppressive treatment before biopsy. The sural nerve biopsy was performed after consenting for procedure. A part of the biopsied nerve was fixed in 3% glutaraldehyde diluted in 0.0625 M cacodylate buffer. Another part of the biopsied sural nerve specimen was snap frozen in isopentane cooled in liquid nitrogen. SIX biopsied sural nerves, which were obtained from patients with clinical symptoms of peripheral nerve diseases but had no histological abnormalities, were also studied as controls.

Histological studies Epon-embedded semithin sections were stained with toluidine blue for general histological observation and morphometry. The morphology of teased fibers was classified according to previously reported criteria (Dyck et al. 1993). Ultrathin sections, contrasted with uranyl acetate and lead citrate, were examined under an electron microscope. Immunohistochemical studies Consecutive cryostat sections were fixed in cooled acetone, and then stained to examine the infiltrated mononuclear cells and depositions of immunoglobulin or complement using the modified ABC method (Nanba et al. 1987). The following mouse monoclonal antibodies were used as primary antibodies for cell typing: anti-Leu-4/CD3 (T cells, 1:200; Becton Dickinson), anti-CD4 (helper/inducer T cells, 1 : 20; Dako), anti-CD8 (cytotoxic/suppressor T cells, 1 : 200; Dako), anti-CD22 (B cells, 1 : 20; Dako), and anti-CD68 (macrophages, EBMll, 1:100, or kp-1, 1:100; Dako). The secondary antibody was biotinylated horse anti-mouse IgG (1 : 200; Vector). Alkaline phosphatase-avidin con-

Table 1 Summary of clinical and pathological findings Patient

Clinical

Onset

Densities of MFs ( n u m b e r / m m 2)

Nerve

Variation

Percent

Electron microscopy

No., age/sex

type

to nerve biopsy

Total

Small ( < 55/xm)

Large ( > 55/~m)

edema

in the MF density between fascicles

of TFs with de-/remyelination

Macrophageassociated demyelination

Demyelinated axons

Onion bulbs

1 76/M 2 40/M 3 63/F 4 15/M 5 22/M 6 51/F 7 24/M 8 60/F 9 29/M Controls (n = 6)

prog. prog. relap. prog. relap. relap. relap. prog. prog.

4 mo. 4 mo. 9 mo. 9 mo. 2 yr. 3 yr. 4 yr. 6 yr. 7 yr. mean SD

3499 6393 5601 4643 5786 3879 7776 4161 6471 8353 1476

1009 2995 2468 1935 2421 1162 4455 3157 5300 3720 652

2490 3398 3133 2708 3365 2717 3321 1004 1171 4633 909

0 + 0 + + + + + + + 0

+ 0 + 0 + + 0 0 0

77 24 21 53 19 39 31 46 27

+ + + + + + + 0 0 0 0 0

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

0 0 0 0 + + + + +

prog. = progressive; relap. = relapsing; MF = myelinated fiber; TF = teased fiber; 0, + , + + , + + + : severity of histologic changes.

K~ Matsurnuro et al. /Journal of the Neurological Sciences 127 (1994) 170-178

172

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Fig. 1. Light and electron microscopic findings. A: light microscopy of the sural nerve specimen from patient 1. Note the myelin debris-containing cytoplasm surrounding a nerve fiber (arrowheads). Epon-embedded semithin section stained with toluidine blue. Bar = 20/xm. B: ultrastructural findings for the sural nerve specimen from patient 2. Note the macrophages invading the myelin lamellae and the myelin disruption. M = macrophage; A = axon. Bar = 1 /.~m. C: ultrastructural findings for the sural nerve specimen from patient 2. Note the early remyelinated fiber and surrounding redundant basement m e m b r a n e (arrowheads). Bar = 1 /xm. D: ultrastructural findings for the sural nerve specimen from patient 8. Note the onion bulb formation. Bar = 2 /~m.

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K. Matsumuro et al. /Journal of the Neurological Sciences 127 (1994) 170-178

subsequent remyelination. The findings of active demyelination, such as myelin debris-containing cytoplasm surrounding myelin sheaths or completely demyelinated axons (Fig. 1A), were abundant in patients 1 and 2, and less frequent in patients 3 and 4. Cellular infiltration, which comprised mononuclear cells, was obvious in patients 1 and 2. Perivascular demyelination was rare. As observed on electron microscopy, invading macrophages penetrated the basement membrane around the fibers and displaced the Schwann cell cytoplasm (Fig. 1B). The myelin lamellae surrounded by macrophages were disrupted and actively stripped by cell processes. However, we could not find myelin disruption in the absence of direct contact with macrophages. Axons remained intact or were only slightly altered. In addition to the demyelinated axons, we also noted a few fibers exhibiting early remyelination, that were surrounded by a redundant basement membrane (Fig. 1C). In patients 5 to 9, completely demyelinated axons were still noted, however, evidence of remyelination became increasingly prominent. This was typified by thin myelin sheaths surrounding normal axons and onion bulb formations (Fig. 1D). In these nerves, a few macrophages containing myelin debris or lipid droplets were seen in the endoneurium. Despite careful observation, we never found widening of myelin lamellae. In the nerves from patients 2 and 8, a few fibers showed uncompacted myelin lamellae. The density of myelinated fibers was generally decreased. However, the density of small myelinated fibers appeared to increase in patients 7 and 9, in which many onion bulbs were seen. Differences in the myelinated fiber density between fascicles and nerve edema were observed in 4 and 6 patients, respectively.

jugate (1 : 100; BioMakor) was then applied. The color was developed using naphthol-AS-BI-phosphoric acid as a substrate and hexazotized new fuchsin as a coupler. The stained sections were examined without knowledge of the treatment protocol, and graded in a semi-quantitative way. At a magnitude of × 100, the number of positive cells per microscopic field (0.0864 mm 2) was graded as follows: 0 = none, + = 1-5, + + = 6-10,+++ =11-20,++++ =20<0. Rabbit polyclonal antiserum to human C l q (1 : 1000; Dako) was used for the detection of complement deposition in the nerves. Biotinylated goat polyclonal antibodies to human IgG (H + L, 1 : 200; Vector), IgM (mu chain specific, 1:200; Vector), and IgA (alpha chain specific, 1 : 200; Vector) were used to demonstrate the deposition of immunoglobulins. Immunofluorescence

For the simultaneous demonstration of macrophages and the H L A - D R antigen or CD4 antigen, frozen sections were sequentially incubated with anti-CD68, biotinylated horse anti-mouse IgG as the first step, and then with fluorescein isothiocyanate (FITC)-conjugated anti-HLA-DR (1 : 10, Becton Dickinson), or FITC-conjugated anti-CD4 (1:20, DAKO), and Texas red-conjugated streptavidin (1:10, Becton Dickinson) as the second step. For demonstration of Schwann cells, we used the anti-S-100 antibody (1:200, Dako) a s the primary antibody and biotinylated anti-rabbit IgG (1:200, Vector) as the secondary antibody in the first step. The sections were examined by fluorescent microscopy. Using double exposure photographs, we counted the double positive cells.

Immunohistochemical studies

3. Results

A few anti-macfophage-positive cells, mainly located in the perivascular area within nerve fascicles were found in control nerves. Other monoclonal antibodypositive cells were not detected.

Histological studies

The most obvious finding in sural nerves was the presence of macrophage-associated demyelination and

Table 2 Immunohistochemical analysis of invading cells and double immunofl~zorescencestaining for HI~-DR-positive cells in the endoneurium* Patient Invading cells Double immunofluorescenc¢staining No. CD3 CD4 CD8 B cells Macrophages H L A - D R+ HLA-DR+ macrophage+ cells S100+ cells 1 ++ + ++ 0 ++++ +++ 0 2 + + + 0 +++ +++ 0 3 + 0 + 0 + 0/+ ++ 4 + 0/+ + 0 + + 0 5 O/+ 0 O/+ 0 + O/+ + 6 + 0 + 0 + 0/+ ++ 7 O/+ 0 O/+ 0 + O/+ + 8

0/+

0

0/+

0

9 + 0 + 0 * See under Materials and methods for the scoring system.

+

+

+ +

+

0/+

+

K. Matsumuro et al. /Journal of the Neurological Sciences 127 (1994) 170-178

174

The results of cell typing in nerves of CIDP patients are presented in Table 2. As indicated, a large number of anti-CD68-positive cells, macrophages, could be noted in the endoneurium of the nerves from patients 1 and 2. Some of these cells had extended processes and surrounded the nerve fibers (Fig. 2A). In patients 3 to 9, anti-CD68-positive cells were present, however, their numbers were reduced. CD3-positive cells were identified in the endoneurium of the nerves from patients 1 and 2 (Fig. 2B). Analysis of T cell subsets in serial sections revealed the presence of CD4-positive

cells and CD8-positive cells. The CD8-positive cells were inore numerous than CD4-positive cells (Fig. 2C, D). CD3-positive cells were also present in vessel walls, where CD4-positive cells and CD8-positive cells were noted in approximately the same ratio. In patients 3 to 9, CD3-positive cells and CD8-positive cells were identified in the endoneurium. CD4-positive cells could not be found in these patients. B cells were not detectable in the endoneurial area of the nerves from any patients. Immunoglobulin deposits (IgG, IgM and IgA), were

L

b

~Q ~o

.(3

....

D

Fig. 2. Consecutive frozen sections of the biopsied sural nerve from patient 1. A: immunostaining with monoclonai antibody to macrophages. Many antimacrophage-positive ceils with extended large processes can be identified in the endoneurium. B-D: immunostaining with monoclonal antibodies to CD3 (B), CD4 (C), and CD8 (D). CD3-positive cells were identified in the endoneurium, and CD8-positive cells predominated over CD4-positive cells. Bar = 50/zm.

I~ Matsumuro et al. / Journal of the Neurological Sciences 12 7 (1994) 170-178

not identified on the myelin sheaths of any of the patients. Clq could not be detected on the myelin or elsewhere in the endoneurium,

175

Double immunofluorescence studies In the endoneurium of the nerves from patients 1 and 2, there were abundant HLA-DR-positive cells

Fig. 3. Double immunofluorescence study on the biopsied sural nerve from patient 2. HLA-DR-positive cells (A, FITC, green) coexpressed a macrophage-specific determinant (B, Texas red, red). The double exposure technique demonstrated the two colors of fluorescence were almost completely superimposed (C). Bar = 50 p,m. Fig. 4. Double immunofluorescence study on the biopsied sural nerve from patient 5. HLA-DR-positive cells were visualized with FITC (green) and S100-positive cells with Texas red (red). The double exposure technique demonstrated that some S100-positive Schwann cells, which co-expressed HLA-DR, were detectable (arrowheads). Bar = 20/,tm.

176

K. Matsurnuro et aL /Journal of the Neurological Sciences 127 (1994) 170-178

(Fig. 3A). These cells coexpressed a macrophagespecific determinant (Fig. 3B). The double exposure technique demonstrated that the two different colors of fluorescence from HLA-DR and macrophages were almost completely superimposed (Fig. 3C). We could not find HLA-DR-positive and S-100-positive cells in these nerves. In the other nerves, HLA-DR-positive and CD68-positive cells were also present, however, their numbers were markedly reduced. Conversely, some S-100-positive Schwann cells, which coexpressed HLA-DR, were detected (Fig. 4). We could not detect CD68-positive cells coexpressing CD4.

4. Discussion

There have been a few studies which attempted to define the pathological and immunological characteristics of CIDP as a distinct clinical entity. Our study confirmed that the nerve pathology in patients who satisfied the newly described criteria for CIDP essentially comprised active demyelination and subsequent remyelination of varying proportions. The difference in nerve pathology between each patient was related to the duration of the illness from the onset. In the nerves from the patients with less than 1 year duration from onset, phagocytic macrophages were observed to be invading the Schwann cell cytoplasm. This finding was also reported in GBS (Prineas 1972) and has been considered as one of the pathological features of autoimmune demyelinating neuropathies. Macrophage invasion in the peripheral nerves persisted for months and years. Although active myelin breakdown was still noted, remyelination was predominant in the nerves from patients who had had the illness for more than 2 years. Although analysis of the infiltrating cells in peripheral nerves is important for understanding the pathogenesis of the diseases, the frequency and specificity of cell infiltration in various types of neuropathies remain unknown (Pollard et al. 1986, 1987; Schroeder et al. 1988; Cornblath et al. 1990). Our study represents a detailed immune characterization of the demyelinating lesions in CIDP. Our data indicated that the mononuclear cell infiltrates were composed of macrophages and T lymphocytes of both helper/inducer and cytotoxic/suppressor phenotypes. This pattern of cellular infiltration is observed in experimental autoimmune neuropathy (EAN), in which a T cell-mediated process is suggested as the primary mechanism underlying the demyelinating lesions (Hartung et al. 1990). The crucial role of CD4-positive cells in the initiation of EAN has been firmly established (Linington et al. 1984; Izumo et al. 1985). Regarding the function of CD8-positive cells in EAN, it is unclear whether this lymphocyte subset may exert cytotoxic effects on Schwann cells or play a

role as suppressor cells (Holmdahl et al. 1985; Strig•rd et al. 1988). In our study of CIDP, analysis of T cell subsets demonstrated that CD8-positive cells predominated over CD4-positive cells in the nerves in which active demyelination was seen. Furthermore, CD8positive cells were constantly noted even in the nerves in which CD4-positive cells could not be detected. The role of CD8-positive cells in CIDP remains inconclusive. However, the results of our present study suggests that CD8-positive cells may have a function to sustain the demyelinating activity of this disease. It is known that macrophages can express CD4 molecules on their surfaces, however, our double immunocytochemical staining failed to demonstrate coexpression of CD4 on macrophages. The result suggested that the macrophages expressed only undetectable amount of CD4 molecules on this immunostaining condition. It may also possible that the number of CD4positive T cells was underestimated because of relatively low sensitivity of the anti-CD4 Ab. In this study, we could not find B cells, and neither were there depositions of immunoglobulin and complement in the nerves. The rapid and predictable response of some CIDP patients to plasmapheresis is well known and a possible role of circulating humoral mediators has been suggested in this disease (Dalakas and Engel 1981; Gross and Thomas 1981; Donofrio et al. 1985; Dyck et al. 1986). In an experimental study, long-term injection to marmosets of immunoglobulins derived from CIDP patients resulted in significant slowing of nerve conduction in the recipient animals (Heininger et al. 1984). However, the nerves from these animals showed minimum histological changes. EAN in rabbits induced by immunization with galactocerebroside (GC) was thought to be an animal model of CIDP (Saida et al. 1981). The intraneural passive transfer of hyperimmune serum from these rabbits may lead to an acute conduction block with paranodal demyelination (Saida et al. 1984). However, the pathology of GC-induced EAN was not consistent with that of CIDP in the present study. Lymphocyte infiltration was characteristically absent in the demyelinating lesions of GC-induced EAN (Saida et al. 1981). It remains to be determined whether or not antibodies, complements or other humoral factors are responsible for the demyelination of nerves in CIDP. It was reported that various proportions of Schwann cells were positive for the MHC class II antigen in inflammatory neuropathies (Pollard et al. 1986, 1987; Schroeder et al. 1988; Mancardi et al. 1988; Scarpini et al. 1990; Mitchell et al. 1991). This finding was considered important for the pathogenesis of the human inflammatory demyelinating neuropathy, because Schwann cells might serve as a potential autoantigen presenter in the peripheral nervous system. Using double immunofluorescence techniques in the present

K. Matsumuro et al. /Journal of the Neurological Sciences 127 (1994) 170-178

study, we carefully examined the expression of the HLA-DR antigen on Schwann cells and macrophages in the endoneurium of CIDP patients. Interestingly, in the nerves in which active demyelination was prominent, the HLA-DR antigen was primarily expressed by macrophages but could not be detected on Schwann cells. It was demonstrated that in EAN, endoneurial macrophages, but not Schwann ceils, expressed the MHC class II antigen (Schmidt et al. 1990). Our result is consistent with this experimental study, and contrasts with reports that aberrant HLA-DR expression by Schwann cells may play a pathogenic role in the active demyelination of CIDP (Pollard et al. 1986; Mitchell et al. 1991). Our study also demonstrated that the expression of HLA-DR antigen on Schwann cells was found in the nerves that predominantly had remyelination. Previous studies indicate that the up regulation of MHC class II antigen-expression was not restricted to inflammatory neuropathies of autoimmune etiology (Mancardi et al. 1988; Schroeder et al. 1988; Scarpini et al. 1990; Mitchell et al. 1991). It was suggested that this up regulation was an indicator of severity and activity of the disease (Schroeder et al. 1988). It is possible that the HLA-DR antigen on Schwann cells may reflect not only an immune-mediated phenomenon but also cellular activation in various pathological conditions.

Acknowledgments The authors thank Dr. Raymond Rosales for his helpful discussion. This study was supported by a grant from the National Center of Neurology and Psychiatry of the Ministry of Health and Welfare, Japan.

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