Journal of Neuroimmunology, 40 (1992), 1- 18
1
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02220
Interactions between CD4 + T-cells and rat Schwann cells in vitro 1. Antigen presentation by Lewis rat Schwann cells to Pz-specific CD4 + T-cell lines Kristin G. Argall a, Patricia J. Armati a, John D. Pollard b, Eilean Watson b and Jim Bonner b School of Biological Sciences and h Department of Medicine, The University of Sydney, Sydney', Australia (Received 8 January 1992) (Revised, received 23 March 1992) (Accepted 24 March 1992)
Key words: Schwann cell; Inflammatory demyelinating neuropathy; CD4+ T-cell line; Major histocompatibility complex molecules; Antigen presentation
Summary Interactions between CD4+ P2-specific T-cell lines and Schwann cells were examined in vitro by scanning electron microscopy (SEM) and T-cell proliferation studies. CD4+ T-cell lines clustered around and attached to Schwann cells which expressed Major histocompatibility complex (MHC) class II molecules. Only those Pz-specific T-cell lines capable of inducing experimental allergic neuritis (EAN) when injected into adult Lewis rats clustered around the Schwann ceils. T-cell lines responsive to P2 but not able to induce EAN did not cluster around Schwann cells. The addition of exogenous P2 protein inhibited in a dose-dependent way clustering and proliferation of the P2-specific T-cell lines. Cytoplasmic P2 was detected in Schwann cells by immunofluorescent labelling and the results of proliferation assays in this study suggest that endogenous P2 protein was processed by the Schwann cells and presented to T-cell lines in association with MHC class II molecules. The clustering and proliferation of class II-restricted CD4+ Pz-specific T-cell lines in the presence of Schwann cells provides evidence for a role for Schwann cells as facultative antigen presenting cells, processing and presenting 'self' endogenous antigen to CD4+ T-cell lines capable of inducing EAN.
Introduction Considerable evidence exists indicating a major pathogenic role for T-cell lines in inflamma-
Correspondence to: K. Argall, School of Biological Sciences, Zoology Building A08, The University of Sydney, Sydney N.S.W. 2006, Australia.
tory demyelinating neuropathies (IDN) of the peripheral nervous system (PNS) such as GuillainBarr6 syndrome (GBS) and chronic idiopathic demyelinating polyneuropathy (CIDP), although knowledge about the mechanism of pathogenesis remains elusive. It is known that both T-cells and macrophages participate in the disruption of normal Schwann cell function and destruction of the myelin sheath.
The pathological hallmark of IDN is the occurfence, throughout the nerve roots and peripheral nerve, of scattered foci containing inflammatory cells and demyelinated nerve fibres. In acute and chronic IDN in man, both C D 4 + and C D S + T-cells are present within these foci although macrophages are the dominant cells (Cornblath et al., 1987; Pollard et al., 1986, 1987). In IDN, myelin removal is effected by macrophages (Prineas, 1971), but whether this results from immune targeting of myelin components (e.g., by antibody) or is secondary to Schwann cell damage is unknown. Similar findings have been reported in the animal model of IDN, experimental allergic neuritis (EAN) (Olsson et al., 1983). EAN may be induced in rats by injection with components of the PNS such as myelin or the myelin proteins P2 or P0 (Kadlubowski and Hughes, 1979; Milner et al., 1987) and studies using Px-specific C D 4 + T-cell lines have shown it to be a T-cell-mediated disorder (Linington et al.. 1984; Rostami et al., 1984). In this passive transfer model, macrophage-monocytes also appear to be the major effector cells (Kolb et al., 1987; Hartung and Toyka, 1990). Additional evidence for T-cell mediation comes from experiments in which administration of anti-interleukin-2 receptor antibodies prevented the development of the clinical signs of EAN in rats with adoptively transferred EAN (Hartung et al., 1989). Also, when thymectomy and irradiation are used to render rats T-cell deficient there is a decreased susceptibility to the induction of EAN by active immunization (Brosnan et al., 1987). T-cell-mediated pathogenesis of IDN could involve C D 8 + or C D 4 + T-cells or both, depending on the expression of Major histocompatibility complex (MHC) class I or class 1I molecules, respectively, by cells in the PNS. In biopsies from patients with GBS and CIDP, M H C class 1 and II molecules have been found on the surface of Schwann cells (Pollard et al., 1986, 1987; Mancardi et al., 1988; Mitchell et al., 1991). In addition, rat and human Schwann cells in vitro express M H C class 1 and II molecules in the presence of y-interferon (Armati et al., 1988,1990) or activated T-lymphocytes (Kingston et al., 1989; Tsai et al., 1991). As Schwann cells have been
shown to express both class I and 11 MHC molecules in vitro and in inflamed ncrve in man (Pollard et al., 1986, 1987; Mancardi ct al., 1988), an interaction between C D 4 + a n d / o r C D S + T-cells leading to demyelination is a distinct possibility. As P2-specific C D 4 + T-cell lines have been shown to be responsible for the adoptive transfer of EAN in Lewis rats in vivo, it was pertinent to examine the interaction between such C D 4 + Tcell lines and cells from PNS tissue in vitro.
Materials and methods
Source of rat tissue Newborn to 5-day-old inbred Lewis J.C. rats were bred in the University of Sydney's Bosch Animal House. Skin grafts were routinely carried out in the colony to confirm the effectiveness of the inbreeding program. Dorsal root ganglia (DRG) cultures D R G were removed aseptically and placed in H a n k s ' calcium- and magnesium-free saline (HCMF). Tissue was transferred into a mixture of 0.025% trypsin and 0.05% collagenase in HCMF, chopped finely for 5 rain, then incubated at 36.5°C for 25-55 min in a 5% CO 2 humidified incubator (standard culture conditions). After incubation, the mixture was washed by centrifugation for 4 rain at 600 × g and the supernatant discarded. Medium containing 10% foetal calf serum (FCS) (CSL, Australia) was added to the tube and the pellet of cells was resuspended. This step was repeated and the cells were resuspended in Eagle's minimum essential medium containing 10% FCS, 50 U / m l penicillin G and 50 m g / m l streptomycin sulphate and 1% L-glutamine (EMEM), and plated out on 13-mm collagen-coated (Bornstein, 1958) glass coverslips (Lomb) in 24-well tissue culture grade plates (Linbro). Cultures were incubated at standard culture conditions. Isolated Schwann cell cultures Sciatic and brachial plexes were removed aseptically and placed in H C M F saline. The nerves were cleaned and teased to remove the
epineurium and processed as for the DRG. After the final wash, the cells were resuspended in EMEM and seeded in a 96-well tissue culture grade plate (Greiner) at densities appropriate for the experiments taking place. Cultures were incubated at standard culture conditions.
Identification of Schwann cells Schwann cells were identified by their characteristic spindle shape and immunofluorescent labelling by the monoclonal antibody, 217-c (a kind gift of Professor Kay Fields, Dept. Health & Human Services, Rockville MD). Light microscope examination after 72 h in vitro (h.i.v.) showed that cultures contained less than 5% fibroblasts.
Preparation of P2-specific T-cell lines 12-14-week-old Lewis J.C. rats were injected in each hind footpad with 0.05 ml (total of 0.10 ml) of an emulsion containing bovine P2 protein (2.5 mg/ml) purified from bovine PNS myelin (Shin et al., 1989) in complete Freund's adjuvant (1.0 mg of Mycobacterium strain H37Ra and 2.5 mg of Mycobacterium tuberculosis subsp, bot~is per ml of normal saline). 9-11 days post-injection the animals were killed and the draining popliteal lymph nodes removed. A lymph node cell (LNC) suspension was then incubated in medium containing P2 protein (20 mg/ml) after the method of Linington et al. (1984) and incubated for 3 days. Lymphoblasts were isolated by centrifugation on a Ficoll density gradient and cultured for a further 5-7 days in growth medium containing T-cell growth factor (prepared from the supernatant of concanavalin A-stimulated Lewis J.C. rat spleen cells). Cells were then restimulated with 20/~g/ml P2. The proliferative response was measured weekly by tritiated thymidine uptake and assessed by standard liquid scintillation beta counter procedures. Cells responding specifically to P2 were collected at 2-6 cycles of stimulation. A typical stimulation cycle consisted of 7 days of stimulation with antigen (in RPMI medium containing P:, antigen-presenting cells, normal rat serum, sodium pyruvate, 2-mercaptoethanol, penicillin/streptomycin and L-glutamine), followed by 3 days of resting phase. T-cells were harvested for use in experiments in the latter part
of the stimulation phase, and were not considered to be 'resting' T-cells. The capacity of these cell lines to induce EAN was tested by intravenous or intraperitoneal injection of 5 × 106 to 20 × 10 6 cells into adult Lewis rats. Pz-specific, but non-EAN-producing T-cell lines, were used as controls. Rats were examined daily post-injection for clinical signs of EAN and were also assessed by electrophysiological studies including motor conduction velocity, amplitude of compound muscle action potential and spinal evoked responses.
Preparation of macrophages Adult Lewis rats were killed and washed with 70% ethanol. 20 ml of sterile Dulbecco's phosphate-buffered saline (DPBS) was injected aseptically into the peritoneal cavity of the rat followed by gentle palpation of the abdominal region. The DPBS solution containing peritoneal macrophages was then withdrawn carefully and washed by centrifugation at 400 × g for 4 min. The supernatant was discarded and the pellet resuspended in DPBS and the cells washed as described previously. This procedure was repeated, followed by resuspension of the pellet for counting. The cells were suspended in an appropriate amount of EMEM.
Indirect immunofluorescent staining of cell cultures Cultures were microwave-fixed for 8 s on 'high' after the method of Argall and Armati (1990) and double-labelled by incubating with antibodies for 30 min at room temperature in dim light in the following order: (i) Mouse anti-rat MHC class I or II molecules (MCA 51 or MCA 46, respectively) (Serotec, 1 : 100 dilution) or rabbit polyclonal antibody against bovine P2 protein (1:100 dilution kindly provided by Professor Richard Hughes, Guy's Hospital, London). (ii) B i o t i n y l a t e d goat a n t i - m o u s e IgG (Amersham, 1 : 25 dilution) or anti-rabbit IgG (Amersham, 1 : 50 dilution). (iii) Texas red-Streptavidin (Amersham, 1 : 100 dilution). (iv) 217c (Ran-l) (1:500 dilution; kindly provided by Professor K. Fields). (v) Fluorescein-conjugated sheep anti-mouse IgG (Amersham, 1 : 25 dilution).
Control cultures were prepared by the omission of primary antibody or inappropriate mouse IgG. Rabbit lgG was used as a control for rabbit P~ polyclonal antibody. When labelling for cytoplasmic Pc, ceils were permeabilized by immersion in 0.1 ~4 Triton-X- 100 in DPBS, immediately fixed by the addition of an equal amount of 1% paraformaldehyde in DPBS and incubated for 20 rain at 4°C. The diluent used for Texas red streptavidin was DPBS with 1%, bovine serum albumin (BSA). For all other antibodies the diluent was E M E M + 10% heatinactivated FCS. After each incubation, the cultures were rinsed three times in Hepes-buffered saline (HBS). Following the final rinse, cultures were drained and mounted in Dabco anti-fade glycerol (Johnson et al., 1982) and sealed with nail polish to prevent dehydration. Neurons were identified by their characteristic morphology and fibroblasts by their negative 217c staining.
Preparation of cell cultures for observation with the scanning electron microscope D R G cultures were removed with their glass coverslips, placed in wells containing HBS, and prepared following the method of Argall and Armati (1990). Briefly, ceils were microwave fixed in DPBS for 8 s on 'high', followed by post-fixation in osmium for 1 h, dehydration in graded alcohols and air-drying in the clearing agent, histolene. Once the specimens were completely dry they were sputter-coated with gold to a thickness of 20 nm and viewed with a Jeol 35-C scanning electron microscope at 15 kV.
Tritiated thymidine proliferation assay Schwann cell cultures were established in 96well plates at a concentration of 2 x 104 cells per well in EMEM. After 24 h in vitro, Schwann cell cultures were incubated with various treatments for a further 16-18 h. 0.5 p.Ci of [SH]thymidine was added to each well 16 h prior to harvesting onto filter paper and assessment was by standard liquid scintillation beta counter procedures.
Experimental design For SEM and proliferation studies, each treatment was replicated at least four times and each experiment repeated a minimum of four times.
Experimental treatments with EAN-inducing P•-specific T-cell lines plus Schwann cells included: (a) T-cells plus antigen plus macrophages: (b) T-cells plus antigen: (c) T-cells plus macrophagcs; (d) T-cells alone. Negative controls included: (a) cultured Schwann cells with no added T-cells; (b) P~specific T-cells not capable of ilfducing EAN; and (c) wells with Schwann cells plus macrophagcs but no T-cells. The two types of cell lines used in this study were Pz-specific C D 4 + T-cells that (1) caused EAN and (2) did not cause EAN.
Treatment details SEM studies on DRG cultures. P2-specific T-cell lines, both EAN- and non-EAN-inducing, were added at a concentration of 5 × l0 s per well of a 24-well tissue culture plate. Macrophages were added at a concentration of 2 x l0 s per well. Antigen was added at a concentration of 20 /~g per ml of medium, a concentration previously determined in our laboratory for maximum T-cell response.
Proliferation studies (SC cultures). 4 / 105 Tcells were added to each well, at a ratio of 20 T-cells to one Schwann cell after the method of Kingston et al. (1989). P2 antigen was added at 0, 2. 4 or 8 p,g per 200 #1 well. Schwann cells or T-cells alone were cultured to determine the level of non-specific proliferation. MHC blocking experiments To test for M H C class II-restricted cytotoxicity by P2-specific T-cell lines, OX6 mouse antibody to rat M H C class II molecules (Serotec, MCA 46) was added to the relevant treatment wells at a dilution of 1:200. OX18 mouse anti-rat M H C class 1 antibody (MCA 51, Serotec) was used in the same manner as a control. Before use, the antibodies were dialysed aseptically at a 1:10 dilution through a 1000 MW dialysis membrane for 24 h to remove sodium azide. Culture treatments were incubated for 12, 24 or 48 h, during which time clumping and proliferation became visually evident. Photographs were taken with an Olympus inverted light microscope
Fig. 1. Light micrographs of rat Schwann cells (S) (48 h in vitro (h.i.v.)) incubated with control medium for 24 h.i.v., double-labelled for M H C class I and 217c. a. Phase contrast, h. Immunofluorescence showing Schwann cell staining (FITC). c. Immunofluorescence showing low-level M H C class I expression by Schwann cells (Texas red). Bar = 13 ~ m .
:a T
J
b
z
g'
T Fig. 2. Light micrographs of rat Schw~mn cells (S) (48; h.i.v.) incubated with Pe specific T-cell lines IT) for 24 h.i.v., double-labelled for MHC class I and 217c. a. Phase contrasl, b. lmmunofluorescence showing Schwann cell slaining (FIT('). c. lmmunofluorescence showing upregulation of Mtt(" class I expression by Schwann cells (Texas red). Bar = 13/xm.
$
b
C Fig. 3. Light micrographs of rat Schwann cells (S) (48 h.i.v.) incubated with control medium for 24 h.i.v., double-labelled for M H C class II and 217c. a. Phase contrast, b. Immunofluorescence showing Schwann cell staining (FITC). c. Immunofluorescence showing no M H C class II expression by Schwann cells (Texas red). Bar = 13 p,m.
Fig, 4. Light micrographs of rat Schwann cells (S) (48 h.i.v.) incubated wilh Pz-specific T-cell lines (T) tk~r 24 h.i.v,, double-labelled fl)r MI I C class 11 and 217c. a. Phase contrast, b. lmmunofluorcscence showing Schwann cell staining (FIT(~). c. lmmunofluorescence showing induction of MHC class 11 expression by Schwann cells (Texas red). Note the patchiness of MH(" class 11 expression compared with the moru uniform distribution of MIt (; cla~s I molecules, l']zu 1.3/~ m.
and camera attachment using Kodak 100 ASA colour reversal film.
Results
Acti~,ated CD4 + P2-specific T-cell lines upregulate MHC expression by Schwann cells Actil,'ated P2-specific T-cell lines upregulate expression of MHC class I molecules by Schwann cells In control Schwann cell cultures (SC), all Schwann cells were positive for M H C class I molecule expression. The same was also true for D R G cultures, where fibroblasts as well as Schwann cells expressed low levels of M H C class I (Fig. 1). After 24 h of incubation with P2-specific T-cell lines, the intensity of M H C class I expression by Schwann cells in SC and D R G cultures and fibroblasts ( D R G cultures) increased markedly (Fig. 2). Neurons remained negative in both control and experimental treatments. Upregulation of M H C class I expression did not appear to depend on direct contact with the T-cells, as many Schwann cells and fibroblasts showed increased expression without T-cells in the vicinity. In addition, the upregulation of M H C class I expression did not depend on the ability of a T-cell line to induce EAN.
Actit,ated P2-specific T-cell lines induce the expression of MHC class H molecules by Schwann cells In contrast to M H C class I expression, there was no detectable M H C class II expression by Schwann cells, fibroblasts or neurons in control SC or D R G cultures (Fig. 3). After 24 h incubation with Pz-specific T-cell lines, M H C class II molecules were evident on Schwann cells in SC and D R G cultures, with neurons and fibroblasts remaining negative (Fig. 4). As was found for M H C class I, the induction of M H C class II expression did not depend on the ability of a T-cell line to induce EAN. Induction of M H C class II expression did not appear to depend on direct contact with the T-cells, as some Schwann cells expressed these molecules without T-cells in the vicinity.
~-specific T-cell lines cluster around Schwann cells expressing MHC class H molecules By 12 h in vitro, T-cells were observed by light microscopy to be clustered around most Schwann cells in SC cultures expressing M H C class II molecules (Fig. 4). Occasional fibroblasts appeared unaffected. In D R G cultures with neurons, Schwann cells and fibroblasts, only Schwann cells were seen in contact with, or surrounded by T-cells. SEM showed clustering of T-cells around Schwann ceils in D R G cultures which was followed by attachment of T-cells to the Schwann cell with extension of their processes over the Schwann cell m e m b r a n e (Fig. 5a). Again, T-cells were only observed clustering around Schwann cells, with fibroblasts and neurons remaining unaffected (Fig. 5b, c).
Only EAN-inducing Pz-specific T-cell lines cluster around Schwann cells In our study, the ability of the T-cells to cluster around Schwann cells in SC and D R G cultures as assessed by observation with the SEM, varied (i) between different P2-specific T-cell lines, and (ii) at different antigen-stimulation cycles of individual T-cell lines. In experiments (n = 6) using D R G cultures (Table 1), activated P2-specific T-cell lines clustered around Schwann cells within 12 h after their addition. A small number of Schwann cells and all neurons and fibroblasts remained free of clustering T-cells. In a further four experiments on D R G cultures, no clustering or attachment to Schwann cells or other cells by the P2-specific T-cells was observed (Fig. 6a). When samples of each P2-specific T-cell line were injected into adult Lewis rats, only some lines induced EAN Only cells from those T-cell lines which clustered around Schwann cells induced EAN (Table 1). T-cell lines that did not cluster did not induce EAN. T-cell effects also varied with the antigenstimulation cycle. With an increasing number of cycles, the ability of T-cell lines to induce EAN appeared to decrease, as did the ability to cluster
Ill TABLE 1 RELATIONSHIP BETWEEN CLUSTERING BY I'~SPECIFIC CD4+ T-CELL LINES AND INDUCTION ()F EAN Pc-Specific T-cell line
Antigenstimulation
clustering
EAN induction
yes yes yes yes yes yes no no
yes yes yes yes yes yes
4
no
no
4
no
no
cyclc EW2 EW2 EW2 EW2 EW2 EW5 EW4 EW6 EW6 EW3
2 3 3 4 5 3 2 2
110
no
The presence q[' macrophages does not ~(l,,'ft'ct clusterin,e hy Pjspecti[)'c T-cell lines A s m a c r o p h a g e s h a v e b e e n p r o p o s e d as t h e m a j o r e f f e c t o r cells in lEAN a n d G B S , t h e y w e r e a d d e d to c u l t u r e s a l o n e o r w i t h E A N - i n d u c i n g P2-specific T - c e l l lincs. T - c e l l s o n l y w e r e s e e n a t t a c h e d to t h e S c h w a n n cells. M a c r o p h a g c s , i d e n t i f i e d by t h e i r c h a r a c t e r i s t i c m o r p h o l o g y a n d t h e i r l a r g e r size o v e r t h e T - c e l l s , w e r e n o t o b s e r v e d in a n y s i g n i f i c a n t n u m b e r a t t h e S E M level, h a v i n g b e e n w a s h e d o f f w i t h e x c e s s T - c e l l s during SEM preparation. Those DRG cultures w i t h m a c r o p h a g e s a l o n e s h o w e d s o m e cells r a n d o m l y s c a t t e r e d ()vet t h e c u l t u r e (Fig. 6b).
EAN-inducing P,-specific T-cell lines prol([~'rate in the presence of Schwann cells a r o u n d S c h w a n n cells. A loss in a n t i g e n r e s p o n s e w a s u s u a l l y o b s e r v e d a f t e r 6-7 a n t i g e n s t i m u l a t i o n cycles ( d a t a n o t s h o w n ) .
T o t e s t w h e t h e r S c h w a n n cells c o u l d r e a c t i v a t e and induce the proliferation of Pc-specific T-cell
Fig. 5. Scanning electron micrographs of CD4+ Pc-specific T-cells (T) capable of inducing EAN attached to Schwann cells (S) m Lewis rat DRG cultures, a. T-cells (3 days in vitro (d.i.v.)) attached to Schwann cells (9 d.i.v.), b a r - 10 #m. b. T-cells (3 d.i.v.) clustering around a Schwann cell (9 d.i.v.) with a nearby fibroblast (F) untouched. Bar - 10 p_m. c. High power magnification of (B) noting alignment of T-cells (T) along Schwann cell process (S). Bar I /*m.
11
Fig. 5 (continued).
lines, Schwann ceils were incubated with P2specific T-cell lines with and without P2 antigen. Figure 7a shows the result of a representative experiment where Schwann cells did induce pro-
liferation of EAN-inducing P2-specific T-cells. Proliferation was statistically signifcant when compared with control treatments using a Student-Newman-Keuls analysis of variance ( P
1
© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00 JNI 02220
Interactions between CD4 + T-cells and rat Schwann cells in vitro 1. Antigen presentation by Lewis rat Schwann cells to Pz-specific CD4 + T-cell lines Kristin G. Argall a, Patricia J. Armati a, John D. Pollard b, Eilean Watson b and Jim Bonner b School of Biological Sciences and h Department of Medicine, The University of Sydney, Sydney', Australia (Received 8 January 1992) (Revised, received 23 March 1992) (Accepted 24 March 1992)
Key words: Schwann cell; Inflammatory demyelinating neuropathy; CD4+ T-cell line; Major histocompatibility complex molecules; Antigen presentation
Summary Interactions between CD4+ P2-specific T-cell lines and Schwann cells were examined in vitro by scanning electron microscopy (SEM) and T-cell proliferation studies. CD4+ T-cell lines clustered around and attached to Schwann cells which expressed Major histocompatibility complex (MHC) class II molecules. Only those Pz-specific T-cell lines capable of inducing experimental allergic neuritis (EAN) when injected into adult Lewis rats clustered around the Schwann ceils. T-cell lines responsive to P2 but not able to induce EAN did not cluster around Schwann cells. The addition of exogenous P2 protein inhibited in a dose-dependent way clustering and proliferation of the P2-specific T-cell lines. Cytoplasmic P2 was detected in Schwann cells by immunofluorescent labelling and the results of proliferation assays in this study suggest that endogenous P2 protein was processed by the Schwann cells and presented to T-cell lines in association with MHC class II molecules. The clustering and proliferation of class II-restricted CD4+ Pz-specific T-cell lines in the presence of Schwann cells provides evidence for a role for Schwann cells as facultative antigen presenting cells, processing and presenting 'self' endogenous antigen to CD4+ T-cell lines capable of inducing EAN.
Introduction Considerable evidence exists indicating a major pathogenic role for T-cell lines in inflamma-
Correspondence to: K. Argall, School of Biological Sciences, Zoology Building A08, The University of Sydney, Sydney N.S.W. 2006, Australia.
tory demyelinating neuropathies (IDN) of the peripheral nervous system (PNS) such as GuillainBarr6 syndrome (GBS) and chronic idiopathic demyelinating polyneuropathy (CIDP), although knowledge about the mechanism of pathogenesis remains elusive. It is known that both T-cells and macrophages participate in the disruption of normal Schwann cell function and destruction of the myelin sheath.
The pathological hallmark of IDN is the occurfence, throughout the nerve roots and peripheral nerve, of scattered foci containing inflammatory cells and demyelinated nerve fibres. In acute and chronic IDN in man, both C D 4 + and C D S + T-cells are present within these foci although macrophages are the dominant cells (Cornblath et al., 1987; Pollard et al., 1986, 1987). In IDN, myelin removal is effected by macrophages (Prineas, 1971), but whether this results from immune targeting of myelin components (e.g., by antibody) or is secondary to Schwann cell damage is unknown. Similar findings have been reported in the animal model of IDN, experimental allergic neuritis (EAN) (Olsson et al., 1983). EAN may be induced in rats by injection with components of the PNS such as myelin or the myelin proteins P2 or P0 (Kadlubowski and Hughes, 1979; Milner et al., 1987) and studies using Px-specific C D 4 + T-cell lines have shown it to be a T-cell-mediated disorder (Linington et al.. 1984; Rostami et al., 1984). In this passive transfer model, macrophage-monocytes also appear to be the major effector cells (Kolb et al., 1987; Hartung and Toyka, 1990). Additional evidence for T-cell mediation comes from experiments in which administration of anti-interleukin-2 receptor antibodies prevented the development of the clinical signs of EAN in rats with adoptively transferred EAN (Hartung et al., 1989). Also, when thymectomy and irradiation are used to render rats T-cell deficient there is a decreased susceptibility to the induction of EAN by active immunization (Brosnan et al., 1987). T-cell-mediated pathogenesis of IDN could involve C D 8 + or C D 4 + T-cells or both, depending on the expression of Major histocompatibility complex (MHC) class I or class 1I molecules, respectively, by cells in the PNS. In biopsies from patients with GBS and CIDP, M H C class 1 and II molecules have been found on the surface of Schwann cells (Pollard et al., 1986, 1987; Mancardi et al., 1988; Mitchell et al., 1991). In addition, rat and human Schwann cells in vitro express M H C class 1 and II molecules in the presence of y-interferon (Armati et al., 1988,1990) or activated T-lymphocytes (Kingston et al., 1989; Tsai et al., 1991). As Schwann cells have been
shown to express both class I and 11 MHC molecules in vitro and in inflamed ncrve in man (Pollard et al., 1986, 1987; Mancardi ct al., 1988), an interaction between C D 4 + a n d / o r C D S + T-cells leading to demyelination is a distinct possibility. As P2-specific C D 4 + T-cell lines have been shown to be responsible for the adoptive transfer of EAN in Lewis rats in vivo, it was pertinent to examine the interaction between such C D 4 + Tcell lines and cells from PNS tissue in vitro.
Materials and methods
Source of rat tissue Newborn to 5-day-old inbred Lewis J.C. rats were bred in the University of Sydney's Bosch Animal House. Skin grafts were routinely carried out in the colony to confirm the effectiveness of the inbreeding program. Dorsal root ganglia (DRG) cultures D R G were removed aseptically and placed in H a n k s ' calcium- and magnesium-free saline (HCMF). Tissue was transferred into a mixture of 0.025% trypsin and 0.05% collagenase in HCMF, chopped finely for 5 rain, then incubated at 36.5°C for 25-55 min in a 5% CO 2 humidified incubator (standard culture conditions). After incubation, the mixture was washed by centrifugation for 4 rain at 600 × g and the supernatant discarded. Medium containing 10% foetal calf serum (FCS) (CSL, Australia) was added to the tube and the pellet of cells was resuspended. This step was repeated and the cells were resuspended in Eagle's minimum essential medium containing 10% FCS, 50 U / m l penicillin G and 50 m g / m l streptomycin sulphate and 1% L-glutamine (EMEM), and plated out on 13-mm collagen-coated (Bornstein, 1958) glass coverslips (Lomb) in 24-well tissue culture grade plates (Linbro). Cultures were incubated at standard culture conditions. Isolated Schwann cell cultures Sciatic and brachial plexes were removed aseptically and placed in H C M F saline. The nerves were cleaned and teased to remove the
epineurium and processed as for the DRG. After the final wash, the cells were resuspended in EMEM and seeded in a 96-well tissue culture grade plate (Greiner) at densities appropriate for the experiments taking place. Cultures were incubated at standard culture conditions.
Identification of Schwann cells Schwann cells were identified by their characteristic spindle shape and immunofluorescent labelling by the monoclonal antibody, 217-c (a kind gift of Professor Kay Fields, Dept. Health & Human Services, Rockville MD). Light microscope examination after 72 h in vitro (h.i.v.) showed that cultures contained less than 5% fibroblasts.
Preparation of P2-specific T-cell lines 12-14-week-old Lewis J.C. rats were injected in each hind footpad with 0.05 ml (total of 0.10 ml) of an emulsion containing bovine P2 protein (2.5 mg/ml) purified from bovine PNS myelin (Shin et al., 1989) in complete Freund's adjuvant (1.0 mg of Mycobacterium strain H37Ra and 2.5 mg of Mycobacterium tuberculosis subsp, bot~is per ml of normal saline). 9-11 days post-injection the animals were killed and the draining popliteal lymph nodes removed. A lymph node cell (LNC) suspension was then incubated in medium containing P2 protein (20 mg/ml) after the method of Linington et al. (1984) and incubated for 3 days. Lymphoblasts were isolated by centrifugation on a Ficoll density gradient and cultured for a further 5-7 days in growth medium containing T-cell growth factor (prepared from the supernatant of concanavalin A-stimulated Lewis J.C. rat spleen cells). Cells were then restimulated with 20/~g/ml P2. The proliferative response was measured weekly by tritiated thymidine uptake and assessed by standard liquid scintillation beta counter procedures. Cells responding specifically to P2 were collected at 2-6 cycles of stimulation. A typical stimulation cycle consisted of 7 days of stimulation with antigen (in RPMI medium containing P:, antigen-presenting cells, normal rat serum, sodium pyruvate, 2-mercaptoethanol, penicillin/streptomycin and L-glutamine), followed by 3 days of resting phase. T-cells were harvested for use in experiments in the latter part
of the stimulation phase, and were not considered to be 'resting' T-cells. The capacity of these cell lines to induce EAN was tested by intravenous or intraperitoneal injection of 5 × 106 to 20 × 10 6 cells into adult Lewis rats. Pz-specific, but non-EAN-producing T-cell lines, were used as controls. Rats were examined daily post-injection for clinical signs of EAN and were also assessed by electrophysiological studies including motor conduction velocity, amplitude of compound muscle action potential and spinal evoked responses.
Preparation of macrophages Adult Lewis rats were killed and washed with 70% ethanol. 20 ml of sterile Dulbecco's phosphate-buffered saline (DPBS) was injected aseptically into the peritoneal cavity of the rat followed by gentle palpation of the abdominal region. The DPBS solution containing peritoneal macrophages was then withdrawn carefully and washed by centrifugation at 400 × g for 4 min. The supernatant was discarded and the pellet resuspended in DPBS and the cells washed as described previously. This procedure was repeated, followed by resuspension of the pellet for counting. The cells were suspended in an appropriate amount of EMEM.
Indirect immunofluorescent staining of cell cultures Cultures were microwave-fixed for 8 s on 'high' after the method of Argall and Armati (1990) and double-labelled by incubating with antibodies for 30 min at room temperature in dim light in the following order: (i) Mouse anti-rat MHC class I or II molecules (MCA 51 or MCA 46, respectively) (Serotec, 1 : 100 dilution) or rabbit polyclonal antibody against bovine P2 protein (1:100 dilution kindly provided by Professor Richard Hughes, Guy's Hospital, London). (ii) B i o t i n y l a t e d goat a n t i - m o u s e IgG (Amersham, 1 : 25 dilution) or anti-rabbit IgG (Amersham, 1 : 50 dilution). (iii) Texas red-Streptavidin (Amersham, 1 : 100 dilution). (iv) 217c (Ran-l) (1:500 dilution; kindly provided by Professor K. Fields). (v) Fluorescein-conjugated sheep anti-mouse IgG (Amersham, 1 : 25 dilution).
Control cultures were prepared by the omission of primary antibody or inappropriate mouse IgG. Rabbit lgG was used as a control for rabbit P~ polyclonal antibody. When labelling for cytoplasmic Pc, ceils were permeabilized by immersion in 0.1 ~4 Triton-X- 100 in DPBS, immediately fixed by the addition of an equal amount of 1% paraformaldehyde in DPBS and incubated for 20 rain at 4°C. The diluent used for Texas red streptavidin was DPBS with 1%, bovine serum albumin (BSA). For all other antibodies the diluent was E M E M + 10% heatinactivated FCS. After each incubation, the cultures were rinsed three times in Hepes-buffered saline (HBS). Following the final rinse, cultures were drained and mounted in Dabco anti-fade glycerol (Johnson et al., 1982) and sealed with nail polish to prevent dehydration. Neurons were identified by their characteristic morphology and fibroblasts by their negative 217c staining.
Preparation of cell cultures for observation with the scanning electron microscope D R G cultures were removed with their glass coverslips, placed in wells containing HBS, and prepared following the method of Argall and Armati (1990). Briefly, ceils were microwave fixed in DPBS for 8 s on 'high', followed by post-fixation in osmium for 1 h, dehydration in graded alcohols and air-drying in the clearing agent, histolene. Once the specimens were completely dry they were sputter-coated with gold to a thickness of 20 nm and viewed with a Jeol 35-C scanning electron microscope at 15 kV.
Tritiated thymidine proliferation assay Schwann cell cultures were established in 96well plates at a concentration of 2 x 104 cells per well in EMEM. After 24 h in vitro, Schwann cell cultures were incubated with various treatments for a further 16-18 h. 0.5 p.Ci of [SH]thymidine was added to each well 16 h prior to harvesting onto filter paper and assessment was by standard liquid scintillation beta counter procedures.
Experimental design For SEM and proliferation studies, each treatment was replicated at least four times and each experiment repeated a minimum of four times.
Experimental treatments with EAN-inducing P•-specific T-cell lines plus Schwann cells included: (a) T-cells plus antigen plus macrophages: (b) T-cells plus antigen: (c) T-cells plus macrophagcs; (d) T-cells alone. Negative controls included: (a) cultured Schwann cells with no added T-cells; (b) P~specific T-cells not capable of ilfducing EAN; and (c) wells with Schwann cells plus macrophagcs but no T-cells. The two types of cell lines used in this study were Pz-specific C D 4 + T-cells that (1) caused EAN and (2) did not cause EAN.
Treatment details SEM studies on DRG cultures. P2-specific T-cell lines, both EAN- and non-EAN-inducing, were added at a concentration of 5 × l0 s per well of a 24-well tissue culture plate. Macrophages were added at a concentration of 2 x l0 s per well. Antigen was added at a concentration of 20 /~g per ml of medium, a concentration previously determined in our laboratory for maximum T-cell response.
Proliferation studies (SC cultures). 4 / 105 Tcells were added to each well, at a ratio of 20 T-cells to one Schwann cell after the method of Kingston et al. (1989). P2 antigen was added at 0, 2. 4 or 8 p,g per 200 #1 well. Schwann cells or T-cells alone were cultured to determine the level of non-specific proliferation. MHC blocking experiments To test for M H C class II-restricted cytotoxicity by P2-specific T-cell lines, OX6 mouse antibody to rat M H C class II molecules (Serotec, MCA 46) was added to the relevant treatment wells at a dilution of 1:200. OX18 mouse anti-rat M H C class 1 antibody (MCA 51, Serotec) was used in the same manner as a control. Before use, the antibodies were dialysed aseptically at a 1:10 dilution through a 1000 MW dialysis membrane for 24 h to remove sodium azide. Culture treatments were incubated for 12, 24 or 48 h, during which time clumping and proliferation became visually evident. Photographs were taken with an Olympus inverted light microscope
Fig. 1. Light micrographs of rat Schwann cells (S) (48 h in vitro (h.i.v.)) incubated with control medium for 24 h.i.v., double-labelled for M H C class I and 217c. a. Phase contrast, h. Immunofluorescence showing Schwann cell staining (FITC). c. Immunofluorescence showing low-level M H C class I expression by Schwann cells (Texas red). Bar = 13 ~ m .
:a T
J
b
z
g'
T Fig. 2. Light micrographs of rat Schw~mn cells (S) (48; h.i.v.) incubated with Pe specific T-cell lines IT) for 24 h.i.v., double-labelled for MHC class I and 217c. a. Phase contrasl, b. lmmunofluorescence showing Schwann cell slaining (FIT('). c. lmmunofluorescence showing upregulation of Mtt(" class I expression by Schwann cells (Texas red). Bar = 13/xm.
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b
C Fig. 3. Light micrographs of rat Schwann cells (S) (48 h.i.v.) incubated with control medium for 24 h.i.v., double-labelled for M H C class II and 217c. a. Phase contrast, b. Immunofluorescence showing Schwann cell staining (FITC). c. Immunofluorescence showing no M H C class II expression by Schwann cells (Texas red). Bar = 13 p,m.
Fig, 4. Light micrographs of rat Schwann cells (S) (48 h.i.v.) incubated wilh Pz-specific T-cell lines (T) tk~r 24 h.i.v,, double-labelled fl)r MI I C class 11 and 217c. a. Phase contrast, b. lmmunofluorcscence showing Schwann cell staining (FIT(~). c. lmmunofluorescence showing induction of MHC class 11 expression by Schwann cells (Texas red). Note the patchiness of MH(" class 11 expression compared with the moru uniform distribution of MIt (; cla~s I molecules, l']zu 1.3/~ m.
and camera attachment using Kodak 100 ASA colour reversal film.
Results
Acti~,ated CD4 + P2-specific T-cell lines upregulate MHC expression by Schwann cells Actil,'ated P2-specific T-cell lines upregulate expression of MHC class I molecules by Schwann cells In control Schwann cell cultures (SC), all Schwann cells were positive for M H C class I molecule expression. The same was also true for D R G cultures, where fibroblasts as well as Schwann cells expressed low levels of M H C class I (Fig. 1). After 24 h of incubation with P2-specific T-cell lines, the intensity of M H C class I expression by Schwann cells in SC and D R G cultures and fibroblasts ( D R G cultures) increased markedly (Fig. 2). Neurons remained negative in both control and experimental treatments. Upregulation of M H C class I expression did not appear to depend on direct contact with the T-cells, as many Schwann cells and fibroblasts showed increased expression without T-cells in the vicinity. In addition, the upregulation of M H C class I expression did not depend on the ability of a T-cell line to induce EAN.
Actit,ated P2-specific T-cell lines induce the expression of MHC class H molecules by Schwann cells In contrast to M H C class I expression, there was no detectable M H C class II expression by Schwann cells, fibroblasts or neurons in control SC or D R G cultures (Fig. 3). After 24 h incubation with Pz-specific T-cell lines, M H C class II molecules were evident on Schwann cells in SC and D R G cultures, with neurons and fibroblasts remaining negative (Fig. 4). As was found for M H C class I, the induction of M H C class II expression did not depend on the ability of a T-cell line to induce EAN. Induction of M H C class II expression did not appear to depend on direct contact with the T-cells, as some Schwann cells expressed these molecules without T-cells in the vicinity.
~-specific T-cell lines cluster around Schwann cells expressing MHC class H molecules By 12 h in vitro, T-cells were observed by light microscopy to be clustered around most Schwann cells in SC cultures expressing M H C class II molecules (Fig. 4). Occasional fibroblasts appeared unaffected. In D R G cultures with neurons, Schwann cells and fibroblasts, only Schwann cells were seen in contact with, or surrounded by T-cells. SEM showed clustering of T-cells around Schwann ceils in D R G cultures which was followed by attachment of T-cells to the Schwann cell with extension of their processes over the Schwann cell m e m b r a n e (Fig. 5a). Again, T-cells were only observed clustering around Schwann cells, with fibroblasts and neurons remaining unaffected (Fig. 5b, c).
Only EAN-inducing Pz-specific T-cell lines cluster around Schwann cells In our study, the ability of the T-cells to cluster around Schwann cells in SC and D R G cultures as assessed by observation with the SEM, varied (i) between different P2-specific T-cell lines, and (ii) at different antigen-stimulation cycles of individual T-cell lines. In experiments (n = 6) using D R G cultures (Table 1), activated P2-specific T-cell lines clustered around Schwann cells within 12 h after their addition. A small number of Schwann cells and all neurons and fibroblasts remained free of clustering T-cells. In a further four experiments on D R G cultures, no clustering or attachment to Schwann cells or other cells by the P2-specific T-cells was observed (Fig. 6a). When samples of each P2-specific T-cell line were injected into adult Lewis rats, only some lines induced EAN Only cells from those T-cell lines which clustered around Schwann cells induced EAN (Table 1). T-cell lines that did not cluster did not induce EAN. T-cell effects also varied with the antigenstimulation cycle. With an increasing number of cycles, the ability of T-cell lines to induce EAN appeared to decrease, as did the ability to cluster
Ill TABLE 1 RELATIONSHIP BETWEEN CLUSTERING BY I'~SPECIFIC CD4+ T-CELL LINES AND INDUCTION ()F EAN Pc-Specific T-cell line
Antigenstimulation
clustering
EAN induction
yes yes yes yes yes yes no no
yes yes yes yes yes yes
4
no
no
4
no
no
cyclc EW2 EW2 EW2 EW2 EW2 EW5 EW4 EW6 EW6 EW3
2 3 3 4 5 3 2 2
110
no
The presence q[' macrophages does not ~(l,,'ft'ct clusterin,e hy Pjspecti[)'c T-cell lines A s m a c r o p h a g e s h a v e b e e n p r o p o s e d as t h e m a j o r e f f e c t o r cells in lEAN a n d G B S , t h e y w e r e a d d e d to c u l t u r e s a l o n e o r w i t h E A N - i n d u c i n g P2-specific T - c e l l lincs. T - c e l l s o n l y w e r e s e e n a t t a c h e d to t h e S c h w a n n cells. M a c r o p h a g c s , i d e n t i f i e d by t h e i r c h a r a c t e r i s t i c m o r p h o l o g y a n d t h e i r l a r g e r size o v e r t h e T - c e l l s , w e r e n o t o b s e r v e d in a n y s i g n i f i c a n t n u m b e r a t t h e S E M level, h a v i n g b e e n w a s h e d o f f w i t h e x c e s s T - c e l l s during SEM preparation. Those DRG cultures w i t h m a c r o p h a g e s a l o n e s h o w e d s o m e cells r a n d o m l y s c a t t e r e d ()vet t h e c u l t u r e (Fig. 6b).
EAN-inducing P,-specific T-cell lines prol([~'rate in the presence of Schwann cells a r o u n d S c h w a n n cells. A loss in a n t i g e n r e s p o n s e w a s u s u a l l y o b s e r v e d a f t e r 6-7 a n t i g e n s t i m u l a t i o n cycles ( d a t a n o t s h o w n ) .
T o t e s t w h e t h e r S c h w a n n cells c o u l d r e a c t i v a t e and induce the proliferation of Pc-specific T-cell
Fig. 5. Scanning electron micrographs of CD4+ Pc-specific T-cells (T) capable of inducing EAN attached to Schwann cells (S) m Lewis rat DRG cultures, a. T-cells (3 days in vitro (d.i.v.)) attached to Schwann cells (9 d.i.v.), b a r - 10 #m. b. T-cells (3 d.i.v.) clustering around a Schwann cell (9 d.i.v.) with a nearby fibroblast (F) untouched. Bar - 10 p_m. c. High power magnification of (B) noting alignment of T-cells (T) along Schwann cell process (S). Bar I /*m.
11
Fig. 5 (continued).
lines, Schwann ceils were incubated with P2specific T-cell lines with and without P2 antigen. Figure 7a shows the result of a representative experiment where Schwann cells did induce pro-
liferation of EAN-inducing P2-specific T-cells. Proliferation was statistically signifcant when compared with control treatments using a Student-Newman-Keuls analysis of variance ( P
