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The normal neuromuscular synapse is formed by the intimate association of nerve endings, postsynaptic end-plate foldings in the muscle fiber, and nonmyelinating Schwann cells (SC) sealing the synaptic ramifications. Because SC have been recognized recently to have an immunogenic potential inducible to present protein autoantigens to autoimmune Tlymphocytes, and considering their close proximity to the acetylcholine receptor (AChR)-bearing postsynaptic membranes, presentation of soluble and membrane vesicle-bound AChR to appropriate Tcells was investigated. Shortterm monolayer cultures of SC isolated from neonatal rat sciatic nerves, as well as cells of an immortalized SC line ofsimilar origin, werefully able to present the relevant molecular epitopes to major histocompatibility complex (MHC) compatible AChR-specific T line lymphocytes immunogenically. Presentation ofAChR was restricted by RTI.B (I-A) MHC class IIproducts. Both types of cultured rat SC were inducible to expression of MHC class I and IIproducts, and they were able to phagocytose AChR-enriched membrane vesicles preferentially. In contrast, phagocytosis of latex particles by SC was negligible. These data qualify perisynaptic SC as potential presenter cells of autoimmunogenic AChR in myasthenia gravis. Thus, SC may play a critical and as-yet unpredicted regulatory role in the cellular pathogenesis of myasthenia gravis. (A m l Pathol 1990, 13 6:111-122)
Most current studies of the immunologic properties of the acetylcholine receptor (AChR) in experimental and clinical myasthenia gravis (MG) have focused on determination of the major immunogenic region, 1-3 or, more recently, on the T cell response to dominant autoimmunogenic epitopes411 on AChR molecules. Much less attention has been paid to the mechanisms of autoantigen presentation by the cell elements forming the local microenvironment around AChR-bearing membranes. Recent studies have analyzed the thymic microenvironments, which are most likely the site of the initial stages of the myasthenogenic autoimmune pathogenesis,12-14 but, to our knowledge, no comparable work has been focused on the cellular elements surrounding the neuromuscular end-plate. Apart from the end-plate and nerve endings, nonmyelinating Schwann cells (SC, 'telocytes") are the cells most closely associated with the neuromuscular synapse,15 and thus they are most closely located to the potential source of the myasthenogenic autoantigen, AChR. The immunogenic processing and presentation of AChR has been studied using normal neonatal rat SC and the immortalized SC line 1. 17, which displays most of the functional characteristics of primary SC cultures.16'17 This study demonstrates that AChR-bound vesicles are engulfed by SCs within three hours. In interferon-gamma (IFN--y)-induced SC, soluble and membrane-bound AChR are processed and their autoimmunogenic epitopes are presented to appropriate AChR-specific T lymphocytes. Thus, SC are qualified as potential local modulatory elements in the cellular pathogenesis of MG.
Supported by the Hermann and Lilly Schilling Foundation. SP was the recipient of a Junior Investigator Award from the National Neurofibromatosis Foundation, New York, NY. Accepted for publication August 23, 1989. Address reprint requests to Prof. Dr. Hartmut Wekerle, Max-Planck Institute for Psychiatry um Klopferspitz 18, D-8033 Martinsried, FRG.
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Culture Media
For lymphocyte cultures, we used as the complete medium (CM), Dulbecco's modified Eagle's medium (GIBCO, Grand Island, NY), supplemented with 5 X 10-5 M 2-mercaptoethanol (Serva Chemie, Heidelberg, FRG), 100 U/ml penicillin, 100 jig/ml streptomycin, 2 nM L-glutamine, and 1% vol/vol nonessential amino acids (1OOX), all from GIBCO. For SC line culture, the N2 medium was used.18 It is a 1:1 mixture of a Dulbecco's modified Eagle's medium and F12 medium supplemented with insulin (5 ,g/ml), transferrin (100 ,ug/ml), progesterone (20 nM), putrescine (100 nM), and sodium selenite (30 nM).
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cording to the principles of Ben-Nun, Wekerle, and Cohen,23 from the lymph nodes of a Lewis rat immunized with AChR in Freund's complete adjuvant (FCA). The primed lymphocytes were incubated for three days in CM plus 1 % normal syngeneic rat serum in the presence of Torpedo AChR (2 ,g/ml). After three days in culture, the activated blast cells were separated from the irrelevant resting cells by gradient centrifugation. Cells at the gradient interface were collected, washed three times, and cultured in CM with 10% T cell growth factor (TCGF, supernatant of ConA-activated mouse spleen cultures), and 15% heat-inactivated horse serum. The cells were restimulated with antigen and irradiated thymus cells after ten days. Restimulation and expansion cycles were repeated every 10 to 14 days.
rate (Dil, Molecular Probes, Inc, Eugene, OR), according to the method of Pitas et al.24 Briefly, 50 Al of freshly dissolved Dil (2 mg/ml in absolute ethanol) was added to 200 gg protein of membrane vesicles in total volume of 4 ml serum-free CM to a final Dil concentration of 25 Ag/ml. The sterile mixture was incubated at room temperature for 20 minutes and agitated gently. The Dil-labeled vesicles were washed three times with cold serum-free CM (2000 rpm centrifugation for ten minutes). The pellets were resuspended in 1 ml CM and sonicated in a waterbath ultrasonicator.
Phagocytosis Assays
Both monodisperse carboxylated green fluorescent microspheres (diameter, 1.72 ,u; Polysciences, Inc., Warrington, PA) and freshly washed, red fluorescent, Dil-labeled, AChR-rich membrane vesicles were used as probes. The SCs and PEC cultures used for uptake assays were grown in chamber slides and pretreated with INF-y for 24 hours. After three rinsings with prewarmed serum-free medium, 20 ,u of a 1:1 mixture of vesicles (3 ,ug protein content/ml) and spheres (0.01%) was added to the chambers. The cultures were incubated at 37 C, in 10% C02 for three hours. After another triple rinsing round with prewarmed PBS, the slides were fixed with 4% formaldehyde at 4 C for 30 minutes. Phagocytosis was monitored under a Zeiss universal fluorescent microscope with epifluorescent illumination using either a FITC (450 to 490 nm) or a rhodamine (510 to 560 nm) filter package. Photomicrographs were taken using Kodak Tri-X panchromatic film (ASA 400).
Results
tadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlo-
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/m1t )
0
0.03
0.1
0.3
ent both soluble and membrane - bound ACbR to ACbR specific T cells. Lympbocyte proliferation induced by soluble AChR (o or AChR-V (* ---*) was determined as described in Materials and Methods. Each dot represents the mean cpm value of triplicate cultures.
---o)
1.17 behaves in a way similar to short-term SC lines isolated from neonatal rat nerves.26'27
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Figure 2. Expression ofMHC class I and class II molecules on the SC line 1. 17. MHC antigetn expression was assessed before (left side) and after (right side) IFN-y (50 U/ml) treatment (37 Cfor 36 hours) by indirect staining with monoclonal antibodies OX 18 (class I), 0 X 3, or 0 X 6 (class II, monomorphic or polymorphic, respectively). Fluorescence intensity was measured with a cytofluorograph system.
Fluoes I
gers similar SC/T cell interactions (Figure 3A3, B3). These processes can be enhanced and accelerated by IFN--y
pretreatment of SCs. Proliferation assays show that activation of the T-lines cell in the presence of SC and AChR reflects classical immunogenic presentation and recognition of the relevant AChR epitopes (Figure 4). These responses critically depend on the dose of soluble antigen (Table 1). Furthermore, as expected, they can be blocked by MAbs binding to products of the RT1.B subregion. In contrast, antibodies directed to MHC class I determinants or to class 11 RT1-D, corresponding to l-E molecules, are ineffective (Table 2, 3). Line 1.17 SCs not only are able to present soluble AChR to T helper line cells immunogenically, but also, much like "professional" thymic APC, and certain hybridoma cells,25 they readily present membrane-incorporated AChR (Figure 4). In fact, soluble and vesicle-bound AChR are presented with equal efficiency by immune associated antigen (la)-induced SCs (Table 1).
Discussion
SCs, the glial elements of peripheral nerves, have interesting immunologic potential. Rat and human SC can be
116
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Figure 3. AChR-mediated Schwann-cell/T-cell interaction. Both, fresh SC cultures (Nr 107; A) and 1.17 SC(B) were cocultured with AChR-specific Lewis rat T helper line cells in the absence (Al, B1), presence (A2, B2) ofAChR protein (3 ,sg/ml), or of Con A (5Mugl ml, A3, B3). After 36 hours, both antigen and lectin initiate Iymphoblast transformation. In the absence ofantigen, T cells remained in their resting state. Bar: 50g m
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10
Control AChR
AChR-V
ConA
Figure 4. AChR presenting capacity offresh SC cultures. T-cell activation was determined in the presence of SC cultures (Nr. 109) and soluble AChR (2 JAg/ml) or membrane-bound AChR (AChR- Vprotein concentration 2 Agl/ml). SC without antigen, or with ConA (5 Aig/ml) were negative or positive controls. Each bar represents the mean cpm and standard deviation of triplicate cultures.
readily induced to synthesize and express la determinants on their membranes.26-28 After la induction, SC are able to present not only soluble foreign antigen to specific syngeneic T lymphocytes, but also endogenous peripheral nerve myelin proteins.-6 These observations were the basis for the conclusion that SC play a central role, not
only in the induction of myelin-specific peripheral autoimmune neuropathies, but also by acting as targets for cytotoxic autoimmune T lymphocytes. SC are located not only in myelinated parts of peripheral nerves, however. Apart from SC sheathing unmyelinated nerve fibers, SC seal the terminal branchings of motor nerve endings overlaying muscular end-plates.15 The perijunctional SC seem to be specialized, failing to produce myelin, but expressing high levels of ion channels.29 At least some of these specialized functions may be induced by contact with skeletal muscle cell; SC cocultured in vitro with myotubes are induced to express elevated levels of choline acetyl transferase.30 We were intrigued by the proximity between AChR-containing membrane parts of the neuromuscular synapse and SC, and we suspected that AChR presentation by SC could have a role in local activation of T lymphocytes involved in MG. As discussed previously,14 there is evidence suggesting that AChR-specific T cells, originally expanded within the thymic medulla, are required in MG as helpers in the production of pathogenic autoantibodies. To possibly present AChR to autoimmune T cells, SC must fulfil several functional criteria. Most T lymphocytes, especially those acting as helpers in antibody production, recognize specific antigenic epitopes only in the molecular context of MHC class 11 la antigens. SCs, like any APC, are therefore required to synthesize and express on their membrane la antigens. This has been established before both in vitro26-28 and in viVo.31,32 la expression is necessary, but by no means sufficient, for presentation of all protein antigens, as shown by transfection experiments, for example.33 Large protein molecules or particles must be engulfed by an APC and degraded into peptide fragments, and some of these fragments are then reexpressed in physical association with membrane la anti-
gens.-'
Our finding that SC are able to ingest, process, and present AChR in particle form is significant. Ultrastructural
Table 1. Rat Schwann Cells Present Soluble and Membrane-BoundAChR to MHC Compatible TLine Cells Antigen presenting cells (CPM SD) Thymus APC SC/-IFN-y SC/+IFN-,y AChR AChR-V AChR AChR-V AChR AChR-V
Dose (ag/ml) 3 1 0.3 0 ConA (5 Ag/ml)
66494 4457 35142 812 61438 4560 25674 866 36355 644 21140 3335 1372 133 116943 4313
19748 1303
47913 3171 35674 2066 36616 1333 21467 3065 20892 142 10320 378 1729 75 28995 2504
Irradiated thymus cells, or 1.17 SCs with or without IFN--y treatment, were cultured with AChR-specific T line cells in the presence of log 3 diluted AChR, AChR-bound membrane vesicles (AChR-V), or ConA (5 Jg/ml) for 72 hours and were harvested with 3H-thymidine for the final 16 hours. CPM values are means obtained from triplicate cultures.
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Antigen
None AChR (3,ug/ml) ConA (5 ,g/ml) AChR (3 Ag/ml) AChR (3 gg/ml) AChR (3,ug/ml) AChR (3 /Ag/ml) AChR (3,g/ml) AChR (3 qg/ml) AChR (3 ,ug/ml) AChR (3,ug/ml)
mAB (dilution)
None None None OX18 (RT1.A) 1:100 OX18 (RT1.A) 1:500 OX6 (RT1.B) 1:100 OX6 (RT1.B) 1:500 OX17 (RT1.D) 1:100 OX17 (RT1.D) 1:500 W3/13 (pan.T) 1 :100 W3/13 (pan.T) 1:500
After preactivation with INF-y (50 U/ml; 24 hours) the SC 1.17 cells were used as APCs. The results are expressed as mean cpm of triplicate cultures. Standard deviation in all cases are in the range of 15%.
investigations of the myasthenic end-plate in acute MG have shown that the synaptic cleft is filled with abundant fuzzy debris, which seems to be shed from the end-plate as the result of autoantibody and complement binding.35 36At the normal neuromuscular synapse, SC, nerve endings, and postsynaptic end-plate are ensheathed by a complex system of basal membranes,37 which may separate distinct spaces within this microenvironment. In myasthenic end-plate areas, especially with strong destructive changes and binding of complement components, at least major parts of the basement membranes seem to break down, apparently allowing direct contact between the vesicular debris of the subsynaptic space and the synaptic SC.-" Thus, it is conceivable that a perisynaptic APC is faced with particle-bound rather than soluble AChR. Consequently, only APCs able to process membrane vesicles are good candidates for locally stimulating AChR-specific T cell responses.
The present studies used two in vitro systems to test AChR/membrane presentation by SC. First, short-term SC cultures derived from neonatal sciatic nerves were used. To definitely exclude effects contributed by any non-SC cellular contaminants, our experiments were paralled using an immortal SC line derived from a neonatal rat. These cells are known to exhibit practically all functional options of normal cultured SC, including ensheathing of axons, and myelination,16 and as shown here, these cells were readily inducible to la expression by IFN-,y. Moreover, it should be noted that the SC line 1.17, although of Sprague-Dawley rat origin, was la compatible with T lines from Lewis rats. "Normal" and immortal SC were therefore indistinguishable in their immunologic potential. Although SC are generally considered poor phagocytes, several reports have demonstrated that SC are able to phagocytose certain particles selectively. For example, myelin particles-6 38 but not axolemmal frag-
Table 3. Presentation of Soluble andMembrane-BoundAChR by Fresh SCs Is RT1.B (I-A) Restricted Combination T cell
L-R L-R L-R L-R L-R L-R L-R L-R L-R L-R L-R L-R
Antigen
None AChR (2 jg/ml) AChR-V (2 ,g/ml) ConA (5 ,ug/ml) AChR (2 Ag/ml) AChR (2 jg/ml) AChR (2 jg/ml) AChR (2,ug/ml) AChR-V (2 jg/ml) AChR-V (2,gg/ml) AChR-V (2 Ag/ml) AChR-V (2 jg/ml)
OX18 (RT1.AO OX3 (RT1.B) OX1 7 (RT1.D) W3/13 (Pan.T) OX18 (RT1.A) OX3 (RT11.B) OX17 (RT1.D) W3/13 (Pan.T)
2510 31528 22233 105185 27055 (14.1) 1069 (96.5) 33253 (0) 33450 (0) 21634 (2.7) 6810 (69.4) 24307 (0) 22730 (0)
APCs are fresh prepared SC cultures (Nr. 115) that have been preactivated with recombinant mean cpm of triplicate cultures. Standard deviations in all cases are in the range of 15%. * The monoclonai antibodies are used at 1:300 dilution.
t The percentage of inhibition is calculated according to the following formula: (control CPM - experimental CPM/control CPM) X 100).
LU
.01
fi'
t ;)
,;\.
t.
C)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~410~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~N
Figure 5. Selective uptake of AChR enriched membrane vesicles by 1. 17 SC line. After IENyj treatment (50 U/12, PECs and SC line 1. 17 were cultured with FITC-microsphere (green fluorescence) and Dil-labeledAChRV (redfluorescence). After three hours, PECs (A) had phagocytosed not only FI TC-microspbhere (B) but also AChR- V(C). Using phase contrast (D) and two-color exposure (E), three of five 1. 17SCs, had incorporatedAChR-V Bar 10gAm.
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Figure 6. Selective uptake of AChR-enriched membrane vesicles by a fresh SC culture. Two partly overlapping fresh SCs (culture Nr. 111; A), show uptake of Dillabeled AChR-V, as identified by red fluorescence (B). The Dil-labeled vesicles (red) are accumulated in the perinuclear area. Bar 10Am.
autoimmunogenically to specific T lymphocytes.'4 It is also clear, however, that, especially in long standing MG, the disease can proceed after removal of the thymus. In these cases, local presentation of AChR in the vicinity of neuromuscular junctions has special importance. SC may have an as-yet unrecognized active role in these processes.
References
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Acknowledgments
The authors thank Mrs. B. Goebel for processing the manuscript, and Dr. G. Adolph, Boehringer Institute, Vienna, for donating the recombinant IFN--y.