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Sulfasalizine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells
Journal of Neuroimmunology, 34 (1991) 109-120
109
© 1991 ElsevierScience Publishers B.V. All rights reserved 0165-5728/91/$03.50 JNI 02053
Sulfasalazine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells Jorge Correale 1, Tomas Olsson 1, Jakob Bj6rk 2 G 6 r a n Smedeg~.rd and Hans Link 1
2 Bo H6jeberg
1
1Department of Neurology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden, and 2 Department of Inflammation Research, Kabi Pharmacia Therapeutics, Uppsala, Sweden
(Received 1 April 1991) (Revised, received16 May 1991) (Accepted 17 May 1991)
Key words: Interferon-y;Myelinbasic protein; Immunopharmacology
Summary Sulfasalazine (SASP; 5-(p-(2-pyridylsulfamoyl)phenylazo)salicylic acid) has beneficial effects on certain inflammatory diseases and has been proposed for clinical trials in multiple sclerosis (MS). We have explored the effects of SASP on actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. SASP was given orally at three different doses from the day of immunization to day 40 post-immunization (p.i.). All doses led to a clinically more protracted disease, increased numbers of T cells infiltrating into the central nervous system (CNS) and to increased numbers of interferon-y-secreting cells (IFN-y-sc) in the CNS. The effects of SASP treatment on T cell-mediated autoimmunity against CNS myelin and peptides of myelin basic protein (MBP) were measured by IFN-y secretion and proliferation by lymph node mononuclear cells in response to these antigens. In SASP-treated rats, increased numbers of IFN-y-sc appeared in response to myelin antigens, while the proliferative responses were decreased. We suggest that monitoring cell-mediated immunity with the IFN-y-sc method may be relevant for the evaluation of new immunotherapeutic strategies in inflammatory demyelinating diseases. Furthermore, our results demand caution as to clinical trials with SASP in MS.
Introduction It is important to find effective and relatively safe therapies for inflammatory demyelinating
Address for correspondence: Dr. Tomas Olsson, Department of Neurology,KarolinskaInstitute,HuddingeUniversity Hospital, S-141 86 Huddinge,Stockholm,Sweden.
diseases such as multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) can be used to evaluate various therapeutic strategies a n d / o r specific immunomodulating drugs with potential, usefulness in MS. Sulfasalazine (5-p-(2pyridylsulfamoyl)phenylazo)salicylic acid; SASP) represents one immunomodulating drug that is attractive in this respect, mainly due to its application in other chronic inflammatory diseases and
110
low frequency of serious adverse effects. The use of SASP is thus well documented in the treatment of inflammatory bowel diseases (Peppercorn, 1984). In addition, it is known that SASP acts as a disease-modifying agent in rheumatoid arthritis (Pullar et al., 1983; Situnayake et al., 1987; van der Heijde et al., 1990), and recently beneficial clinical effects have also been reported in other diseases with suspected autoimmune pathogenesis such as ankylosing spondylitis and psoriasis (Nissil~i et al., 1988). SASP was recently shown to have beneficial effects both on the clinical course and the histopathological severity of acute EAE in guinea pigs (Prosiegel et al., 1990). On the basis of these results clinical trials with SASP for treatment of MS have been suggested (Prosiegel et al., 1989). We have now used EAE in Lewis rats to further study the effects of SASP on the clinical course, and the infiltration of immunocompetent cells into the central nervous system (CNS). Since T cells recognizing myelin autoantigens hold a key role in the pathogenesis of EAE (Ben-Nun et al., 1981), we also examined the T cell response to myelin components. This is mostly measured by lymphocyte proliferation in response to antigen. However, lymphocytes are also triggered to secrete cytokines such as interferon-7 (IFN-y) after recognition of antigen in context with the major histocompatibility complex (MHC) (Hecht et al., 1983). The number of IFN-y secreting cells (IFN-y-sc) in response to presented antigen is an alternative measure of specific T cell immunity (Kabilan et al., 1990; Olsson et al., 1990; Mustafa et al., 1991). We measured both proliferation and the number of IFN-y-sc among lymph node mononuclear cells in response to myelin antigens in SASP-treated and untreated EAE rats.
Material and methods
Induction of EAE, assessment of clinical signs and sulfasalazine treatment Lewis rats (150-250 g, locally bred, originally obtained from Alab, Stockholm, Sweden) were injected into each hind foot pad with 100 ~1 of inoculum containing 25 mg of guinea pig spinal
cord, 1 mg Mycobacterium tuberculosis (strain H37RA, Difco, Detroit, MI, U.S.A.), 50/~l saline and 50/zl Freund's incomplete adjuvant (Difco). Body weights and clinical signs were assessed every second day. The clinical grading was as follows: 0 = no illness, 1 = flaccid tail, 2 = moderate paraparesis, 3 = severe paraparesis, 4 = moribund state or death. SASP (Pharmacia, Uppsala, Sweden) was given via the drinking water and initiated on day 0. The following groups were studied; I: 25 m g / k g / d a y (9 rats); II: 100 m g / k g / d a y (19 rats); III: 200 m g / k g / d a y (10 rats); IV: immunized controls, no treatment (19 rats). These rats were killed at day 40 post-immunization (p.i.). For studies of T cell responses, three additional groups of eight animals each were treated with SASP 25 m g / k g / d a y or 100 m g / k g / d a y or water only until killing at day 21 p.i.
Immunohistochemistry Segments of the lumbar spinal cord were snap-frozen in liquid nitrogen at sacrifice on day 40 p.i. (numbers as shown in Table 1). Cryostat sections (8 ~m) were exposed to appropriate dilutions of the following mouse monoclonal antibodies (Mabs): Ox19 = anti-rat CD5, pan T cells (Dallman et al., 1984); Ox8 = anti-rat CD8, T cytotoxic/suppressor cells (Brideau et al., 1980); W3/25 = anti-rat CD4, T helper cells and macrophages (Williams et al., 1987); Ox39 = antirat interleukin 2 (IL-2) receptor (Sedgwick et al., 1987); Ox6 = anti-rat MHC class II (McMaster et al., 1979); Ox18 = anti-rat M H C class I (Fukumoto et al., 1982); Ox33 = anti-rat B cells (Woolett et al., 1985). The antibodies were purified from culture supernatants (Holmdahl et al., 1985) of hybridomas originally obtained from Dr. Allan Williams (Oxford, U.K.), except for Ox33, Ox39 and Ox18 that were purchased from Seralab (Crawley-Down, U.K.). The avidin-biotin peroxidase method was used for final staining, applying the ABC Vectastain Elite Kit (Vector Lab., Burlingame, CA, U.S.A.). Peroxidase staining was performed according to Kaplow (1974). Omission of the primary antibody served as a negative control. Specificity of the staining was also controlled on sections of peripheral lymphoid organs. Ox6, Ox8, Ox18, Ox33 and W3/25
111 stained cells were semiquantitatively scored on coded sections using an arbitrary scale from 0 = no stained cells, to + + + = more than 30 stained cells per visual field at X 100. Ten randomly selected areas of x 100 magnification were evaluated for each antibody in each rat. The numbers of cells stained with Ox19 and Ox39 were counted in ten random visual fields at x 200 magnification and the mean per visual field was calculated.
Antigens Myelin (Kadlubowski et al., 1981) from Lewis rats' CNS and guinea pig myelin basic protein (GP-MBP) (Deibler et al., 1972) were isolated. MBP peptides were synthesized (Olsson et al., 1990). Sequences were chosen to encompass previously reported T cell immunodominant regions in Lewis rats and other species as follows: MBP(63-88) (Lewis rat; Vandenbark et al., 1985), MBP(89-101) (SJL/J mouse; Sakai et al., 1988), MBP(107-124) (strain 13 guinea pig; Ben-Nun et al., 1981), MBP(148-165) (rhesus monkey; Karkhanis et al., 1975). Purified protein derivate (PPD) was purchased from Statens Seruminstitut (Copenhagen, Denmark) and phytohemagglutinin (PHA) from Difco (Detroit, MI, U.S.A.).
Isolation of mononuclear cells from lymph nodes and CNS Rats were killed at day 21 p.i. and mononuclear cells (MNC) from popliteal and inguinal lymph nodes were prepared by standard techniques. MNC from brain and spinal cords were isolated by washing their surfaces as described (Zachau et al., 1989; Mustafa et al., 1991). Both lymph node and CNS MNC suspensions were then washed twice and resuspended in complete Dulbecco's modified Eagle's medium (CDME) (Flow Lab., Irvine, U.K.) containing 1% (v/v) minimum essential medium (Flow), 50 I U / m l penicillin, 60 ~ g / m l streptomycin (Gibco, Paisley, U.K.), 2 mM glutamine (Flow), 5 x 10 -5 M 2-mercaptoethanol (Merck, Darmstadt, F.R.G.) and 5% (v/v) heat-inactivated fetal calf serum (Gibco). Lymph node cells were adjusted to 2 x 106 viable cells/ml. From the CNS wash fluid, between 5 x 10 4 and 10 × 10 4 cells were recovered that were resuspended in 100/zl CDME.
Assay for single cells secreting IFN-7 An immunospot assay that enables detection of single cells secreting IFN-7 (Czerkinsky et al., 1988) was adopted to enumerate rat IFN-y-sc (Mustafa et al., 1991). In principle, nitrocellulosebottomed microtiter plates (Millititer-HAM, Millipore Co., Bedford, MA, U.S.A.) were coated with Mab directed against rat IFN-7 (DB1, a generous gift from Dr. Peter van der Meide, TNO Primate Center, Rijswijk, The Netherlands) (Van der Meide et al., 1986). Then, duplicate aliquots (100 /xl) containing 2 x 105 MNC from lymph nodes were added. CNS wash fluid MNC were sufficient for plating into one well per rat. After 24 h of culture, cells were discarded, and secreted and bound IFN-7 visualized by secondary immunochemicals, resulting in stained spots corresponding to single cells that have secreted the cytokine. The numbers of spots were enumerated and standardized to number per 106 mononuclear cells.
T cell-mediated immunity measured by antigen-induced IFN-7 secretion Lymph node cell suspensions (200/zl aliquots), at a cell concentration of 2 x 106/ml in CDME, were cultured in round-bottomed polystyrene 96well microtiter plates (Nunc, Copenhagen, Denmark). Antigens were added to duplicates to obtain final concentrations as follows: PPD 50 /zg/ml, rat CNS myelin 40 /zg/ml, GP-MBP, MBP(107-124), MBP(148-165), MBP(89-101) and MBP(63-88) 10/zg/ml. These antigen concentrations were found optimal in preliminary experiments both for this assay and the proliferation assay described below. Other duplicate wells received no exogenous antigen to serve as background controls. After 48 h culture at 37°C, and 7% CO 2 in humid atmosphere, the cells were washed and transferred to microtiter plates for detection of IFN-y-sc, as described above. In some experiments, SASP (Pharmacia) was added to obtain final concentrations of 25-100 ~ g / m l .
T cell-mediated immunity measured by antigen-induced lymphocyte proliferation Aliquots in triplicate (200 /.d) of lymph node MNC suspensions were applied in round-bottomed 96-well microtiter plates (Nunc) at a cell
112 3"
density of 2 × 106 cells/ml CDME. The different antigens were added as described above. PHA was used as positive control mitogen at a final dilution of 1:1000 (v/v). In some experiments SASP was added to obtain final concentrations of 25 or 100 /zg/ml. After 60 h of incubation the cells were pulsed for 10 h with [3H]methylthymidine (10 ~1, 100/zCi/ml; Amersham, Little Chalfont, U.K.). Cells were harvested and thymidine incorporation was counted in a /3-scintillation counter.
o AA ~
2"
•
CONTROL 25 mg
E
10
20
30
40
i 50 Days PI
Statistical methods Statistical evaluation was performed with the non-parametric Mann-Whitney's U-test. 2'
Results
I
Clinical course of EAE A survey of these data is presented in Fig. 1. The non-treated E A E rats showed an acute monophasic disease. Rats treated with 100 or 200 mg S A S P / k g / d a y showed neurological deficits similar to the untreated controls until day 22 p.i. Thereafter they were, however, more severely affected (Fig. lb and c). Around day 26 p.i. and onwards clinical scores were significantly higher in SASP-treated rats ( p < 0.02) as compared to the controls. The rats given 25 mg S A S P / k g / d a y exhibited a delay in onset of E A E with less severe clinical signs than controls on days 10 and 12 p.i. (Fig. la). However, during the ensuing days this group showed more severe neurological deficits with a maximum at day 18 p.i. After a slight improvement the rats once again deteriorated and remained diseased until sacrifice at day 40. CNS immunopathology At day 40 p.i. CD5 + T cells in perivascular inflammatory infiltrates were conspicuously more numerous in SASP-treated rats than in controls (Table 1; Fig. 2a and b). The same was true for IL-2 receptor expressing cells (Fig. 2c and d). The CD4 antigen is well known to be expressed both on round lymphocyte-like cells and on irregularly shaped macrophage-like cells in organspecific autoimmune diseases (Klareskog et al.,
CONTROL
1'
100rag
i
10
20
30
40
50
Days PI
3
o~ CONTROL
200 rng
] -
O" 10 C
20
30
40
50 Days
PI
Fig. 1. Clinical scores (mean values) during the course of EAE in untreated animals (filled circles) and rats treated with SASP (open circles). Dose of SASP in m g / k g body weight/day as indicated. Statistics comparing controls and treated rats are indicated: • p < 0.05; • • p < 0.02; * p < 0.002; * * p < 0.001.
1983; Olsson et al., 1983). This was the case also presently and we did not attempt to numerically differentiate these two stained cell types. The overall number of CD4-stained ceils did not appear to differ between controls and SASP-treated rats. CD8 may as well appear on non-lymphocytic cells in local inflammation (Olsson et al., 1987; Strig~rd et al., 1987). Presently, CD8 staining
113
appeared both on lymphocyte-like cells and irregularly shaped cells, although in this case the number of CD8 + cells was higher in the SASP-
treated rats than in the controls. Also B cells were more numerous in treated rats than in controis. Both treated and control animals showed
Fig. 2. Immunostained spinal cord cryosections from a SASP-treated (a, c, e) and an untreated EAE rat (b, d, f ) on day 40 p.i. Panels (a) and (b) show infiltrating Oxl9-1abelled T cells, (c) and (d) IL-2 receptor-expressing Ox39-1abelled cells, while (e) and ( f ) show MHC class II-expressing cells detected by the Ox6 antibody. Note more conspicuous infiltration of T cells and IL-2 receptor-expressing cells in the treated rat, while the MHC class II immunoreactivity is approximately similar in the treated and untreated rat.
114
similar large numbers of cells stained for MHC class I and MHC class II (Fig. 2e and f).
4001 300
Spontaneous and antigen-induced IFN-y-secreting cells To enumerate cells that produce IFN-y or are triggered to this production already in vivo, lymph node or CNS wash fluid MNC from SASP-treated and control animals obtained at day 21 p.i. were analyzed without preculture by direct application into nitrocellulose plates. IFN-y-sc were significantly more numerous among CNS wash fluid MNC from SASP-treated animals than among CNS MNC from the untreated EAE rats (Fig. 3). The number of IFN-y-sc in the regional lymph nodes also tended to be higher in SASP-treated animals as compared to controls (Fig. 3). In order to quantitate the number of primed T
TABLE
~
:~:~
• Lymph o~gans [] CNS
11111-
0
CONTROL
25 mg
100 mg
Fig. 3. N u m b e r o f I F N - y - s e c r e t i n g cells a m o n g M N C recovered from lymph nodes (lymph organs) and CNS from unt r e a t e d E A E r a t s ( c o n t r o l ) a n d S A S P - t r e a t e d r a t s (25 a n d 100 mg/kg/day) o n d a y 21 p.i. m e a s u r e d by t h e i m m u n o s p o t assay. C o l u m n s a n d b a r s r e p r e s e n t m e a n v a l u e s a n d S E M , respectively. N o t e s i g n i f i c a n t i n c r e a s e in n u m b e r o f IFN-3,s e c r e t i n g cells a m o n g C N S M N C in t r e a t e d rats, * p < 0.01; • * p < 0.001.
1
ESTIMATION OF IMMUNOHISTOCHEMICALLY DETECTED LYMPHOCYTES SPINAL CORDS OF NON-TREATED EAE CONTROLS AND SULFOSALAZINE
AND MHC ANTIGEN EXPRESSION (SASP)-TREATED EAE RATS
1N
E a c h r o w s h o w s o b s e r v a t i o n f r o m o n e a n i m a l . In t h e m o r e d e t a i l e d q u a n t i t a t i v e a n a l y s i s ( O x 1 9 a n d O x 3 9 ) cells w e r e c o u n t e d at a m a g n i f i c a t i o n o f × 2 0 0 . T h e a r b i t r a r y s c a l i n g w a s as follows: 0 = n o s t a i n e d cells; + = 1 - 4 cells; + + = 5 - 3 0 cells; + + + = m o r e t h a n 30 s t a i n e d cells. R e s u l t s a r e e x p r e s s e d as t h e a v e r a g e n u m b e r o f s t a i n e d cells p e r visual field at a m a g n i f i c a t i o n o f × 100. Monoclonal antibodies and labelled antigens Group
Controls
Animal number
2 5 6 8 9
Mean ± SD 25 m g S A S P / k g / d a y
Mean ± SD
43.3 _+ 27.7 6 . 0 + 1.2 14.4 + 12.2 11.6 ± 17.1 16.1 + 11.6 18.3 _+ 14.5
Ox39 IL-2 rec
5.2 0.0 1.3 3.5 4.2 2.8
± 4.6 ± ± ± ±
2.5 2.8 1.5 1.5
W3/25 CD4
+ + + + +
+ + + + + + +
Ox33 B cells
Ox18 MHC
Ox6 MHC
class 1
class II
0 0 0 0 0
+ + ++ + + + + + +
+ + + ++ + + + + + + + +
+ + + + + + +
+ + + + + +++ + + + + + +
+ + + + ++ + + + +
+ + + + + + + + +++
+ + + + +
+ + + +
10 13 14 17 18
23.0 _+ 27.8 93.7 ± 47.5 24.1±10.3 29.7 ± 18.5 34.5 ± 15.4 41.0 + 29.8 *
0.0 10.0 ± 5.7 6.3±2.0 10.3 ± 5.3 9.6 + 4.7 7.4 ± 4.5 *
+ + + + +++ + + + + + +
0 + + + + +
+ + + + +++ + + + +
+ +
19 20 25 26 28
68.3 ± 22.7 56.5 ± 35.3 30.9 ± 11.1 55.9 ± 43.0 35.5±32.3 49.4 ± 15.7 * *
11.4 ± 8.8 9.5 ± 4.1 2.4 ± 2.2 4.7 ± 4.4 1.6±1.7 5.9 ± 4.3 ~
+ + + + + + + + + + + +++
+ 0 0 + + 0
+ + + + + + + + +++
+ + + +
Mean + SD 100 m g S A S P / k g / d a y
Ox19 CD5
# N o t s i g n i f i c a n t ; * p < 0.005; * * p < 0.01.
+ +
Ox8 CD8
+ + + +
+ + + + + +
115
500400-
180
•
CONTROL
• CONTROL [ ] 25mg [ ] 100mg
E
ml~ 25rng tOOmg
so
,.= 100 -
o Ag 0
r-CNS
GP-MBP
MBP
o
107-124 MBP §3-1~8
$~o
Fig. 4. Numbers of primed T cells that in response to antigen secrete IFN-3,, determined on regional lymph node MNC on day 21 p.i. in untreated EAE rats (control) or SASP-treated rats (25 and 100 mg/kg/day). Immunospots were determined after 48 h preculture in vitro without antigen (Ag 0) or with different antigens added: r-CNS = rat CNS myelin, GP-MBP = guinea pig myelin basic protein, MBP 107-124 and MBP 63-88 = synthetic MBP peptides. Columns and bars represent mean values and SEM, respectively. Note dramatic increase in number of IFN-,/-sc after stimulation with all antigens in treated rats.
r-CNS
G~-M~p MBp 107-124MBp 63-85
PHA
Fig. 5. Cell-mediated immunity measured by [3H]thymidine incorporation in response to myelin antigen of lymph node MNC from untreated EAE rats (control) and SASP-treated rats (25 and 100 mg/kg/day) 21 days p.i. Background in unstimulated cultures (Ag 0) is also indicated. Columns and bars represent mean values and SEM, respectively. PHA = phytohemagglutinin; other abbreviations as in Fig. 4. Note decreased proliferative activity in treated rats both in nonstimulated cultures and in most antigen- or lectin-stimulated cultures.
Spontaneous and antigen-induced lymphocyte proliferation cells among lymphoid cells that produced IFN-~/ in response to different antigens, these cells were precultured for 48 h to allow for antigen uptake; processing, presentation and T cell triggering were then counted. The number of spots obtained in antigen-exposed cultures exceeding zero antigen, background control cultures, gives an estimate of the number of antigen-specific T cells. The SASP-treated animals showed approximately 2to 6-fold higher numbers of IFN-y-sc in response to GP-MBP or the different MBP peptides as compared to the control animals (Fig. 4; Table 2).
As shown in Fig. 5, the proliferation of cells from treated rats was less pronounced as compared to those obtained from control animals. In the group treated with 25 m g / k g / d a y a clear decrease was seen in the spontaneous as well as the syngeneic rat CNS myelin and PHA-induced proliferation, while no difference was seen after stimulation with GP MBP and the different peptides of MBP. A still more conspicuous decrease in lymphocyte proliferation, now encompassing all antigens was observed in the group of animals treated with 100 m g / k g / d a y (Fig. 5).
TABLE 2 NUMBERS OF IMMUNOSPOTS PER 106 LYMPH NODE MONONUCLEAR CELLS IN UNTREATED AND SASPTREATED EAE RATS, AFTER STIMULATION WITH DIFFERENT NERVOUS TISSUE AUTOANTIGENS Ag 0 = no antigen added. GP-MBP: guinea pig myelin basic protein; r-CNS: rat central nervous system myelin; MBP: myelin basic protein; PPD = purified protein derivate. Ag 0 Control (n = 8) 25 mg S A S P / k g / day (n = 7) 100 mg S A S P / k g / day (n = 8)
9 5:5
GP-MBP 60+32
r-CNS
MBP (63-88)
61+46
110+ 53
MBP (89-101)
MBP (107-124)
MBP (148-165)
33+15
22+ 7
23+17
PPD 27+13
34 +- 15
200+77 #
154+_64
248+- 81
199+-50"*
126+47 #
154+42 **
40+15
17 _+4
110_+67
128+-54
331+-110 **
138+-47 *
114_+48
125+55 ##
102+-42
# p < 0.03; ## p < 0.02; *p < 0.01; * * p < 0.002.
116 120 •
Discussion ~
80"
~
.
_
.
_
~
Ag 0 PPD MBP 107-124 MBP63.S8
40"
PHA
z. i 0
i 25
i 50
i 100
Suffasallzlnl
concintratlon
(microgr,/mJ)
~o
180
-
E =
80-
~
~
.o E
:~
~
-20
.
, 20
=
.
, 40
-~-e-~
0
.
MBP107-124 MBP63-88
=
, 60
.
, 80
.
, 100
.
, Sulfaza[azlnl
concentration
(rnlcrogrJml)
Fig. 6. Effects in vitro of sulfasalazine at different concentrations on the proliferative activity of (a) lymph node cells from EAE rats measured by [3H]thymidine incorporation, and (b) number of IFN-y-secreting cells in unstimulated cultures (Ag 0), and cultures stimulated with different antigens. For [3H]thymidine incorporation, results are expressed as percent of the incorporation obtained without drug added. Abbreviations are as in Fig. 4. Note dose-dependent decrease of [3H]thymidine incorporation, while there are constant levels of IFN-y-secreting cells both in unstimulated and antigenstimulated cultures.
Effects of sulfasalazine in vitro on antigen-induced IFN-y secretion and lymphocyte proliferation Unstimulated and antigen-stimulated lymph node MNC from control EAE animals were exposed to different concentrations of SASP and the number of IFN-y-sc was analyzed. The number of IFN-y-sc remained unaffected among both unstimulated and antigen-stimulated cells (Fig. 6b). In contrast, a dose-dependent inhibition of spontaneous and antigen-induced lymphocyte proliferation was observed (Fig. 6a).
We here show that SASP treatment intensifies and prolongs EAE in Lewis rats, both clinically and histologically. The number of T cells and interleukin-2 receptor-expressing cells was increased in the CNS tissue. In parallel, the number of IFN-y-sc among mononuclear cells in CNS wash fluid was more elevated in treated rats than in non-treated counterparts. Furthermore, the number of primed 'memory' T cells from regional lymph nodes that produce IFN-y in response to myelin autoantigens was increased in SASPtreated rats. IFN-y shows a number of activities that are important in inflammation, e.g. (i) activation of macrophages (Adams and Hamilton, 1987; Goldberg et al., 1990), (ii) induction of MHC antigens on many cell types (Skoskiewicz et al., 1985) including MHC class II on astrocytes (Fontana et al., 1984), and microglia (Steininger and Van der Meide, 1988), and (iii) induction of T cell homing (Duijvestijn et al., 1986; Issekutz et al., 1988; Oppenheimer-Marks and Ziff, 1988). In vivo blocking of IFN-y with monoclonal antibodies leads to decreased clinical signs of experimental allergic neuritis (Strig~rd et al., 1989; Hartung et al., 1990). IFN-y is produced by activated T ceils that passively transfer EAE (Ando et al., 1989; Karpus and Swanborg, 1989; Sedgwick et al., 1989). Systemic administration of IFN-y leads to exacerbations of multiple sclerosis (Panitch et al., 1987). We detected approximately 10-fold more cells among peripheral blood mononuclear cells that produced IFN-y in response to myelin antigens in multiple sclerosis patients compared to controls (Olsson et al., 1990). Furthermore, we recently observed that IFN-y-producing T cells appeared in the CNS shortly preceding onset of clinical signs of EAE (Mustafa et al., 1991). On the other hand, although these data are compatible with a deleterious effect of IFN-y on EAE, Voorthuis et al. (1990) suppressed EAE by injecting high doses of the cytokine intrathecally. Also, in vivo blocking of IFN-y during EAE with antibodies was found to enhance disease (Billiau et al., 1988). Furthermore, high endogeneous levels of IFN-y that are produced during graft versus host disease (Klimpel et al., 1990) and trypanoso-
117
miasis (Bakhiet et al., 1990) are associated with immunosuppression. Thus, the net effect of IFN-7 in a certain disease might be difficult to predict, and effects may be dependent on time, site and level of production. We speculate that the increased secretion of IFN-y up to a certain level enhances the local inflammatory response, while even higher levels might depress it. Such a mechanism might also explain the drug effects seen presently that did not follow a simple dose-response curve (Fig. 1). It is anyhow tempting to suggest that during SASP treatment the increased numbers of IFN-y-sc, especially within the CNS, may be causally related to the worsened course of EAE. An in vivo expansion of T cells recognizing myelin antigens was indicated by our observation that SASP-treated EAE rats had increased number of lymph node T cells that in response to such antigens produced IFN-y in vitro. At the same time, the proliferative response to these antigens was decreased. SASP is known to inhibit signals involved in cellular activation (Carlin et al., 1989) and to reduce proliferation in vitro (Ali et al., 1982). Since the number of memory T cells secreting IFN-y was expanded in the SASPtreated rats, this would imply a preferential drug effect in vivo on proliferation of cells other than those with potential capacity to produce IFN-y. In mice, T helper cell subsets are defined, where TH1 cells characteristically produce IFN-y and TH2 cells produce IL-4 and IL-5 (Mosmann et al., 1986). Surface marker analysis suggests that a similar distribution may exist also in the rat (Arthur and Mason, 1986). Recently it has been shown that the TH1 and TH2 subsets use different T cell receptor signal transduction mechanisms for the induction of lymphokine gene expression (Gajewski et al., 1990), which could relate to a preferential effect of SASP on different T cell subsets. It will be of interest to determine if SASP inhibits proliferation or function of TH2 cells that normally secrete IL-4, since this cytokine downregulates IFN-y production by TH1 cells (Chr6tien et al., 1990; Vercelli et al., 1990). Our observed increase in the number of T cells responding with IFN-y secretion in response to
MBP peptides during SASP treatment was not restricted to the peptide previously defined encephalitogenic in Lewis rats (Vandenbark et al., 1985). Another region, MBP(89-99) was recently shown encephalitogenic (Offner et al., 1989) and still another MBP peptide was shown to promote EAE in Lewis rats during measles infection (Liebert et al., 1990). We have shown that cellular responses to all MBP peptides used here did indeed occur, and developed late in the course of EAE (Mustafa et al., 1991). Thus, it is possible that any of the MBP peptide responses increased by SASP may contribute to the deterioration of EAE. The observed augmentation of EAE in Lewis rats shown here contradicts previous results on EAE in guinea pigs, where SASP was shown to reduce severity of disease (Prosiegel et al., 1989). Important differences between the two studies include rate of treatment as well as observation times. We observed the animals for 40 days and found no augmentation of clinical signs until day 22 p.i. Prosiegel et al. (1990) observed their animals for only 21 days and a relapse could well occur later also in this model. One must also point to the difference in species between the two studies. It may even be speculated that the strain of rats used could be of importance. A parallel can be drawn to the effect of SASP in experimental models of arthritis where contrasting effects are obtained depending on species, strain and immunization protocol (Klareskog et al., 1987; Bj6rk et al., 1989; Fenfer et al., 1990). However, the present observations as well as a recent case report on onset of MS during treatment with SASP (Gold et al., 1990) demand caution with regard to further applications of this drug in clinical trials on MS. Preferably, before such trials, a variety of treatment protocols should be tested on different EAE models with a chronic course. Finally, our observations strengthen the hypothetical role of IFN-y as a deleterious effector molecule in EAE, and suggest that effects on T cell cytokine secretion may be relevant in the evaluation of new immunotherapeutic strategies in demyelinating diseases.
118
Acknowledgements We greatly appreciate the excellent technical assistance of Mrs. Anita Gustafsson, and we thank Ms. Yvonne Nilsson for secretarial help in preparing the manuscript. This study was supported by grants of the Swedish Medical Research Council (project 7488), the Swedish Multiple Sclerosis Society (NHR) and Karolinska Institutet.
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119 Issekutz, T.B., Stoltz, J.M. and Van der Meide, P.H. (1988) The recruitment of lymphocytes into the skin by T cell lymphokines. The role of gamma-interferon. Clin. Exp. Immunol. 73, 70-75. Kabilan, L., Andersson, G., Lolli, F. et al. (1990) Detection of intracellular expression and secretion of interferon--g at the single cell level after activation of human T cells with tetanus toxoid in vitro. Eur. J. Immunol. 20, 1085-1089. Kadlubowski, M., Hughes, R.A.C. and Gregson, N.A. (1981) Experimental allergic neuritis in the Lewis rat: characterization of the activity of peripheral myelin and its major basic protein P2. Brain Res. 184, 439-454. Kaplow, L.S. (1974) Substitute for benzidine in myeloperoxidase stains. Am. J. Clin. Pathol. 63, 451. Karkhanis, Y.D., Carlo, D.J., Brostoff, S.W. and Eylar, E.H. (1975) Allergic encephalomyelitis isolation of an encephalitogenic peptide active in the monkey. J. Biol. Chem. 250, 1718-1722. Karpus, W.D. and Swanborg, R.H. (1989) CD4 + suppressor cells differentially affect the production of IFN-y by effector cells of experimental autoimmune encephalomyelitis. J. Immunol. 143, 3492-3497. Klareskog, L., Holmdahl, R., Larsson, E. and Wigzell, H. (1983) Role of T lymphocytes in collagen II induced arthritis in rats. Clin. Exp. Immunol. 51, 117-123. Klareskog, L., Holmdahl, R., Goldschmidt, T. and Bj6rk, J. (1987) Immunoregulation in arthritis. Scand. J. Rheumatol. Suppl. 64, 7-15. Klimpel, G.R., Annable, C.R., Cleveland, M.G., Jerrells, T.R. and Patterson, J.C. (1990) Immunosuppression and lymphoid hypoplasia associated with chronic graft versus host disease is dependent upon IFN-~/ production. J. Immunol. 144, 84-93. Liebert, U.G., Hashim, G.A. and Ter Meulen, V. (1990) Characterization of measles virus induced autoimmune reactions against myelin basic protein in Lewis rats. J. Neuroimmunol. 29, 139-142. McMaster, W.R. and Williams, A.F. (1979) Identification of la glycoproteins in rat thymus and purification from rat spleen. Eur. J. Immunol. 9, 426-433. Mosmann, T.R., Cherwinski, M., Bond, M.W., Giedlin, M.A. and Coffman, R.L. (1986) Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348-2357. Mustafa, M.H., Diener, P., H6jeberg, B. et al. (1991) T cell immunity and interferon-3, secretion during experimental allergic encephalomyelitis in Lewis rats. J. Neuroimmunol. 31, 165-177. Nissil~i, M., Lehtinen, K., Leirisalo-Repo, M. et al. (1988) Sulfasalazine in the treatment of ankylosing spondylitis in a twenty-six-week placebo controlled clinical trial. Arthr. Rheum. 31, 1111-1116. Offner, H., Hashim, G.A., Celnik, B. et al. (1989) T cell determinants of myelin basic protein include a unique encephalitogenic I-E-restricted epitope for Lewis rats. J. Exp. Med. 170, 355-367. Olsson, T., Holmdahl, R., Klareskog, L. and Forsum, U.
(1983) Ia-expressing cells and T lymphocytes of different subsets in peripheral nerve tissue during experimental allergic neuritis in Lewis rats. Scand. J. Immunol. 18, 339-343. Olsson, T., Maehlen, J., L6ve, A. et al. (1987) Induction of class i and class II transplantation antigens in rat brain during fatal and non-fatal measles virus infection. J. Neuroimmunol. 16, 215-224. Olsson, T., Wang, W.-Z., H6jeberg, B. et al. (1990) Autoreactive T lymphocytes in multiple sclerosis determined by antigen induced secretion of interferon-gamma. J. Clin. Invest. 86, 981-985. Oppenheimer-Marks, N. and Ziff, M. (1988) Migration of lymphocytes through endothelial cell monolayers: augmentation by interferon. Cell. Immunol. 114, 307-323. Panitch, H.S., Hirsch, R.L., Schindler, J. and Johnsson, K.P. (1987) Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system. Neurology 37, 1097-1102. Peppercorn, M.A. (1984) Sulfasalazine. Pharmacology, clinical use, toxicity and related new drug development. Ann. Intern. Med. 101,377-386. Prosiegel, M., Neu, I., Ruhenstroth-Bauer, G., Vogl, S. et al. (1989) Suppression of experimental autoimmune encephalomyelitis by sulfasalazine. New Engl. J. Med. 312, 545. Prosiegel, M., Neu, I., Vogl, S. et al. (1990) Suppression of experimental autoimmune encephalomyelitis by sulfasalazine. Acta Neurol. Scand. 81,237-238. Pullar, T., Hunter, J.A. and Capell, H.A. (1983) Sulphasalazine in rheumatoid arthritis: a double blind comparison of sulphasalazine with placebo and sodium aurothiomalate. Br. Med. J. 287, 1102-1104. Sakai, K., Sinka, H., Mitchell, D.J. et al. (1988) Involvement of distinct murine T-cell receptors in the autoimmune encephalitogenic response to nested epitopes of myelin basic protein. Proc. Natl. Acad. Sci. U.S.A. 85, 8608-8612. Sedgwick, J., Brostoff, J. and Mason, D. (1987) Experimental allergic encephalomyelitis in the absence of a classical delayed-type hypersensitivity reaction. J. Exp. Med. 165, 1058-1075. Sedgwick, J.D., MacPhee, J.H.M. and Puklawec, M. (1989) Isolation of encephalitogenic CD4 ÷ T cell clones in the rat. Cloning methodology and interferon-7 secretion. J. Immunol. Methods 121, 185-196. Situnayake, R.D., Grindulis, K.A. and Mcconkey, B. (1987) Long term treatment of rheumatoid arthritis with sulfasalazine, gold or penicillamine: a comparison using lifetable methods. Ann. Rheum. Dis. 46, 177-183. Skoskiewicz, M.J., Calvin, R.B., Schneeberger, E.E. and Russel, P.S. (1985) Widespread and selective induction of major histocompatibility complex determined antigens in vivo by gamma-interferon. J. Exp. Med. 162, 1645-1664. Steiniger, B. and Van der Meide, P.H. (1988) Rat ependyma and microglia cells express class II MHC antigens after intravenous infusion of recombinant gamma interferon. J. Neuroimmunol. 19, 111-118. Strig~rd, K., Brismar, T., Olsson, T. et al. (1987) T-lymphocyte
120 subsets, functional deficits, and morphology in sciatic nerves during experimental allergic neuritis. Muscle Nerve 10, 329-337. Strig~rd, K., Holmdahl, R., Van der Meide, P.H. et al. (1989) In vivo treatment of rats with monoclonal antibodies against gamma interferon: effects on experimental allergic neuritis. Acta Neurol. Scand. 80, 201-207. Vandenbark, A.A., Offner, H., Reshef, T., Fritz, R., Jen Chou, C.-H. and Cohen, I.R. (1985) Specificity of Tlymphocyte lines for peptides of myelin basic protein. J. Immunol. 135, 229-233. Van der Heijde, D.M.F.M., van Riel, P.L.C.M., Nuver-Zwart, I.H. and van de Putte, L.B.A. (1990) Sulphasalazine versus hydroxichloroquine in rheumatoid arthritis: 3 year followup. Lancet i, 539. Van der Meide, P.H., Dubbeld, M., Vijverberg, K. et al. (1986) The purification of rat gamma interferon by use of two monoclonal antibodies. J. Gen. Virol. 67, 1054-1071. Vercelli, D., Jabara, H.H., Lauener, R.P. and Geha, R.S. (1990) IL-4 inhibits the synthesis of IFN-y and induces the
synthesis of IgE in human mixed lymphocyte cultures. J. Immunol. 144, 570 573. Voorthuis, J.A.C., Vitdehaag, B.M.J., Groop, C.J.A. et al. (1990) Suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon gamma in Lewis rats. Clin. Exp. Immunol. 81, 183 188. Williams, A.F., Gallfre, G. and Milstein, C. (1987) Analysis of cell surfaces by zenogeneic myeloma hybrid antibodies: differentiation antigens of rat lymphocytes. Cell 12, 663673. Woolett, G.R., Barclay, A.N., Puklavec, M. and Williams, A.F. (1985) Molecular and antigenic heterogeneity of the rat leucocyte common antigen from thymocytes and T and B lymphocytes. Eur. J. lmmunol. 15, 168-173. Zachau, A., Strig~rd, K., Baig, S. et al. (1989) Distribution of plasma cells secreting antibodies against nervous tissue antigens during experimental allergic encephalomyelitis enumerated by a nitrocellulose immunospot assay. J. Neurol. Sci. 91,323-336.
109
© 1991 ElsevierScience Publishers B.V. All rights reserved 0165-5728/91/$03.50 JNI 02053
Sulfasalazine aggravates experimental autoimmune encephalomyelitis and causes an increase in the number of autoreactive T cells Jorge Correale 1, Tomas Olsson 1, Jakob Bj6rk 2 G 6 r a n Smedeg~.rd and Hans Link 1
2 Bo H6jeberg
1
1Department of Neurology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden, and 2 Department of Inflammation Research, Kabi Pharmacia Therapeutics, Uppsala, Sweden
(Received 1 April 1991) (Revised, received16 May 1991) (Accepted 17 May 1991)
Key words: Interferon-y;Myelinbasic protein; Immunopharmacology
Summary Sulfasalazine (SASP; 5-(p-(2-pyridylsulfamoyl)phenylazo)salicylic acid) has beneficial effects on certain inflammatory diseases and has been proposed for clinical trials in multiple sclerosis (MS). We have explored the effects of SASP on actively induced experimental autoimmune encephalomyelitis (EAE) in Lewis rats. SASP was given orally at three different doses from the day of immunization to day 40 post-immunization (p.i.). All doses led to a clinically more protracted disease, increased numbers of T cells infiltrating into the central nervous system (CNS) and to increased numbers of interferon-y-secreting cells (IFN-y-sc) in the CNS. The effects of SASP treatment on T cell-mediated autoimmunity against CNS myelin and peptides of myelin basic protein (MBP) were measured by IFN-y secretion and proliferation by lymph node mononuclear cells in response to these antigens. In SASP-treated rats, increased numbers of IFN-y-sc appeared in response to myelin antigens, while the proliferative responses were decreased. We suggest that monitoring cell-mediated immunity with the IFN-y-sc method may be relevant for the evaluation of new immunotherapeutic strategies in inflammatory demyelinating diseases. Furthermore, our results demand caution as to clinical trials with SASP in MS.
Introduction It is important to find effective and relatively safe therapies for inflammatory demyelinating
Address for correspondence: Dr. Tomas Olsson, Department of Neurology,KarolinskaInstitute,HuddingeUniversity Hospital, S-141 86 Huddinge,Stockholm,Sweden.
diseases such as multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (EAE) can be used to evaluate various therapeutic strategies a n d / o r specific immunomodulating drugs with potential, usefulness in MS. Sulfasalazine (5-p-(2pyridylsulfamoyl)phenylazo)salicylic acid; SASP) represents one immunomodulating drug that is attractive in this respect, mainly due to its application in other chronic inflammatory diseases and
110
low frequency of serious adverse effects. The use of SASP is thus well documented in the treatment of inflammatory bowel diseases (Peppercorn, 1984). In addition, it is known that SASP acts as a disease-modifying agent in rheumatoid arthritis (Pullar et al., 1983; Situnayake et al., 1987; van der Heijde et al., 1990), and recently beneficial clinical effects have also been reported in other diseases with suspected autoimmune pathogenesis such as ankylosing spondylitis and psoriasis (Nissil~i et al., 1988). SASP was recently shown to have beneficial effects both on the clinical course and the histopathological severity of acute EAE in guinea pigs (Prosiegel et al., 1990). On the basis of these results clinical trials with SASP for treatment of MS have been suggested (Prosiegel et al., 1989). We have now used EAE in Lewis rats to further study the effects of SASP on the clinical course, and the infiltration of immunocompetent cells into the central nervous system (CNS). Since T cells recognizing myelin autoantigens hold a key role in the pathogenesis of EAE (Ben-Nun et al., 1981), we also examined the T cell response to myelin components. This is mostly measured by lymphocyte proliferation in response to antigen. However, lymphocytes are also triggered to secrete cytokines such as interferon-7 (IFN-y) after recognition of antigen in context with the major histocompatibility complex (MHC) (Hecht et al., 1983). The number of IFN-y secreting cells (IFN-y-sc) in response to presented antigen is an alternative measure of specific T cell immunity (Kabilan et al., 1990; Olsson et al., 1990; Mustafa et al., 1991). We measured both proliferation and the number of IFN-y-sc among lymph node mononuclear cells in response to myelin antigens in SASP-treated and untreated EAE rats.
Material and methods
Induction of EAE, assessment of clinical signs and sulfasalazine treatment Lewis rats (150-250 g, locally bred, originally obtained from Alab, Stockholm, Sweden) were injected into each hind foot pad with 100 ~1 of inoculum containing 25 mg of guinea pig spinal
cord, 1 mg Mycobacterium tuberculosis (strain H37RA, Difco, Detroit, MI, U.S.A.), 50/~l saline and 50/zl Freund's incomplete adjuvant (Difco). Body weights and clinical signs were assessed every second day. The clinical grading was as follows: 0 = no illness, 1 = flaccid tail, 2 = moderate paraparesis, 3 = severe paraparesis, 4 = moribund state or death. SASP (Pharmacia, Uppsala, Sweden) was given via the drinking water and initiated on day 0. The following groups were studied; I: 25 m g / k g / d a y (9 rats); II: 100 m g / k g / d a y (19 rats); III: 200 m g / k g / d a y (10 rats); IV: immunized controls, no treatment (19 rats). These rats were killed at day 40 post-immunization (p.i.). For studies of T cell responses, three additional groups of eight animals each were treated with SASP 25 m g / k g / d a y or 100 m g / k g / d a y or water only until killing at day 21 p.i.
Immunohistochemistry Segments of the lumbar spinal cord were snap-frozen in liquid nitrogen at sacrifice on day 40 p.i. (numbers as shown in Table 1). Cryostat sections (8 ~m) were exposed to appropriate dilutions of the following mouse monoclonal antibodies (Mabs): Ox19 = anti-rat CD5, pan T cells (Dallman et al., 1984); Ox8 = anti-rat CD8, T cytotoxic/suppressor cells (Brideau et al., 1980); W3/25 = anti-rat CD4, T helper cells and macrophages (Williams et al., 1987); Ox39 = antirat interleukin 2 (IL-2) receptor (Sedgwick et al., 1987); Ox6 = anti-rat MHC class II (McMaster et al., 1979); Ox18 = anti-rat M H C class I (Fukumoto et al., 1982); Ox33 = anti-rat B cells (Woolett et al., 1985). The antibodies were purified from culture supernatants (Holmdahl et al., 1985) of hybridomas originally obtained from Dr. Allan Williams (Oxford, U.K.), except for Ox33, Ox39 and Ox18 that were purchased from Seralab (Crawley-Down, U.K.). The avidin-biotin peroxidase method was used for final staining, applying the ABC Vectastain Elite Kit (Vector Lab., Burlingame, CA, U.S.A.). Peroxidase staining was performed according to Kaplow (1974). Omission of the primary antibody served as a negative control. Specificity of the staining was also controlled on sections of peripheral lymphoid organs. Ox6, Ox8, Ox18, Ox33 and W3/25
111 stained cells were semiquantitatively scored on coded sections using an arbitrary scale from 0 = no stained cells, to + + + = more than 30 stained cells per visual field at X 100. Ten randomly selected areas of x 100 magnification were evaluated for each antibody in each rat. The numbers of cells stained with Ox19 and Ox39 were counted in ten random visual fields at x 200 magnification and the mean per visual field was calculated.
Antigens Myelin (Kadlubowski et al., 1981) from Lewis rats' CNS and guinea pig myelin basic protein (GP-MBP) (Deibler et al., 1972) were isolated. MBP peptides were synthesized (Olsson et al., 1990). Sequences were chosen to encompass previously reported T cell immunodominant regions in Lewis rats and other species as follows: MBP(63-88) (Lewis rat; Vandenbark et al., 1985), MBP(89-101) (SJL/J mouse; Sakai et al., 1988), MBP(107-124) (strain 13 guinea pig; Ben-Nun et al., 1981), MBP(148-165) (rhesus monkey; Karkhanis et al., 1975). Purified protein derivate (PPD) was purchased from Statens Seruminstitut (Copenhagen, Denmark) and phytohemagglutinin (PHA) from Difco (Detroit, MI, U.S.A.).
Isolation of mononuclear cells from lymph nodes and CNS Rats were killed at day 21 p.i. and mononuclear cells (MNC) from popliteal and inguinal lymph nodes were prepared by standard techniques. MNC from brain and spinal cords were isolated by washing their surfaces as described (Zachau et al., 1989; Mustafa et al., 1991). Both lymph node and CNS MNC suspensions were then washed twice and resuspended in complete Dulbecco's modified Eagle's medium (CDME) (Flow Lab., Irvine, U.K.) containing 1% (v/v) minimum essential medium (Flow), 50 I U / m l penicillin, 60 ~ g / m l streptomycin (Gibco, Paisley, U.K.), 2 mM glutamine (Flow), 5 x 10 -5 M 2-mercaptoethanol (Merck, Darmstadt, F.R.G.) and 5% (v/v) heat-inactivated fetal calf serum (Gibco). Lymph node cells were adjusted to 2 x 106 viable cells/ml. From the CNS wash fluid, between 5 x 10 4 and 10 × 10 4 cells were recovered that were resuspended in 100/zl CDME.
Assay for single cells secreting IFN-7 An immunospot assay that enables detection of single cells secreting IFN-7 (Czerkinsky et al., 1988) was adopted to enumerate rat IFN-y-sc (Mustafa et al., 1991). In principle, nitrocellulosebottomed microtiter plates (Millititer-HAM, Millipore Co., Bedford, MA, U.S.A.) were coated with Mab directed against rat IFN-7 (DB1, a generous gift from Dr. Peter van der Meide, TNO Primate Center, Rijswijk, The Netherlands) (Van der Meide et al., 1986). Then, duplicate aliquots (100 /xl) containing 2 x 105 MNC from lymph nodes were added. CNS wash fluid MNC were sufficient for plating into one well per rat. After 24 h of culture, cells were discarded, and secreted and bound IFN-7 visualized by secondary immunochemicals, resulting in stained spots corresponding to single cells that have secreted the cytokine. The numbers of spots were enumerated and standardized to number per 106 mononuclear cells.
T cell-mediated immunity measured by antigen-induced IFN-7 secretion Lymph node cell suspensions (200/zl aliquots), at a cell concentration of 2 x 106/ml in CDME, were cultured in round-bottomed polystyrene 96well microtiter plates (Nunc, Copenhagen, Denmark). Antigens were added to duplicates to obtain final concentrations as follows: PPD 50 /zg/ml, rat CNS myelin 40 /zg/ml, GP-MBP, MBP(107-124), MBP(148-165), MBP(89-101) and MBP(63-88) 10/zg/ml. These antigen concentrations were found optimal in preliminary experiments both for this assay and the proliferation assay described below. Other duplicate wells received no exogenous antigen to serve as background controls. After 48 h culture at 37°C, and 7% CO 2 in humid atmosphere, the cells were washed and transferred to microtiter plates for detection of IFN-y-sc, as described above. In some experiments, SASP (Pharmacia) was added to obtain final concentrations of 25-100 ~ g / m l .
T cell-mediated immunity measured by antigen-induced lymphocyte proliferation Aliquots in triplicate (200 /.d) of lymph node MNC suspensions were applied in round-bottomed 96-well microtiter plates (Nunc) at a cell
112 3"
density of 2 × 106 cells/ml CDME. The different antigens were added as described above. PHA was used as positive control mitogen at a final dilution of 1:1000 (v/v). In some experiments SASP was added to obtain final concentrations of 25 or 100 /zg/ml. After 60 h of incubation the cells were pulsed for 10 h with [3H]methylthymidine (10 ~1, 100/zCi/ml; Amersham, Little Chalfont, U.K.). Cells were harvested and thymidine incorporation was counted in a /3-scintillation counter.
o AA ~
2"
•
CONTROL 25 mg
E
10
20
30
40
i 50 Days PI
Statistical methods Statistical evaluation was performed with the non-parametric Mann-Whitney's U-test. 2'
Results
I
Clinical course of EAE A survey of these data is presented in Fig. 1. The non-treated E A E rats showed an acute monophasic disease. Rats treated with 100 or 200 mg S A S P / k g / d a y showed neurological deficits similar to the untreated controls until day 22 p.i. Thereafter they were, however, more severely affected (Fig. lb and c). Around day 26 p.i. and onwards clinical scores were significantly higher in SASP-treated rats ( p < 0.02) as compared to the controls. The rats given 25 mg S A S P / k g / d a y exhibited a delay in onset of E A E with less severe clinical signs than controls on days 10 and 12 p.i. (Fig. la). However, during the ensuing days this group showed more severe neurological deficits with a maximum at day 18 p.i. After a slight improvement the rats once again deteriorated and remained diseased until sacrifice at day 40. CNS immunopathology At day 40 p.i. CD5 + T cells in perivascular inflammatory infiltrates were conspicuously more numerous in SASP-treated rats than in controls (Table 1; Fig. 2a and b). The same was true for IL-2 receptor expressing cells (Fig. 2c and d). The CD4 antigen is well known to be expressed both on round lymphocyte-like cells and on irregularly shaped macrophage-like cells in organspecific autoimmune diseases (Klareskog et al.,
CONTROL
1'
100rag
i
10
20
30
40
50
Days PI
3
o~ CONTROL
200 rng
] -
O" 10 C
20
30
40
50 Days
PI
Fig. 1. Clinical scores (mean values) during the course of EAE in untreated animals (filled circles) and rats treated with SASP (open circles). Dose of SASP in m g / k g body weight/day as indicated. Statistics comparing controls and treated rats are indicated: • p < 0.05; • • p < 0.02; * p < 0.002; * * p < 0.001.
1983; Olsson et al., 1983). This was the case also presently and we did not attempt to numerically differentiate these two stained cell types. The overall number of CD4-stained ceils did not appear to differ between controls and SASP-treated rats. CD8 may as well appear on non-lymphocytic cells in local inflammation (Olsson et al., 1987; Strig~rd et al., 1987). Presently, CD8 staining
113
appeared both on lymphocyte-like cells and irregularly shaped cells, although in this case the number of CD8 + cells was higher in the SASP-
treated rats than in the controls. Also B cells were more numerous in treated rats than in controis. Both treated and control animals showed
Fig. 2. Immunostained spinal cord cryosections from a SASP-treated (a, c, e) and an untreated EAE rat (b, d, f ) on day 40 p.i. Panels (a) and (b) show infiltrating Oxl9-1abelled T cells, (c) and (d) IL-2 receptor-expressing Ox39-1abelled cells, while (e) and ( f ) show MHC class II-expressing cells detected by the Ox6 antibody. Note more conspicuous infiltration of T cells and IL-2 receptor-expressing cells in the treated rat, while the MHC class II immunoreactivity is approximately similar in the treated and untreated rat.
114
similar large numbers of cells stained for MHC class I and MHC class II (Fig. 2e and f).
4001 300
Spontaneous and antigen-induced IFN-y-secreting cells To enumerate cells that produce IFN-y or are triggered to this production already in vivo, lymph node or CNS wash fluid MNC from SASP-treated and control animals obtained at day 21 p.i. were analyzed without preculture by direct application into nitrocellulose plates. IFN-y-sc were significantly more numerous among CNS wash fluid MNC from SASP-treated animals than among CNS MNC from the untreated EAE rats (Fig. 3). The number of IFN-y-sc in the regional lymph nodes also tended to be higher in SASP-treated animals as compared to controls (Fig. 3). In order to quantitate the number of primed T
TABLE
~
:~:~
• Lymph o~gans [] CNS
11111-
0
CONTROL
25 mg
100 mg
Fig. 3. N u m b e r o f I F N - y - s e c r e t i n g cells a m o n g M N C recovered from lymph nodes (lymph organs) and CNS from unt r e a t e d E A E r a t s ( c o n t r o l ) a n d S A S P - t r e a t e d r a t s (25 a n d 100 mg/kg/day) o n d a y 21 p.i. m e a s u r e d by t h e i m m u n o s p o t assay. C o l u m n s a n d b a r s r e p r e s e n t m e a n v a l u e s a n d S E M , respectively. N o t e s i g n i f i c a n t i n c r e a s e in n u m b e r o f IFN-3,s e c r e t i n g cells a m o n g C N S M N C in t r e a t e d rats, * p < 0.01; • * p < 0.001.
1
ESTIMATION OF IMMUNOHISTOCHEMICALLY DETECTED LYMPHOCYTES SPINAL CORDS OF NON-TREATED EAE CONTROLS AND SULFOSALAZINE
AND MHC ANTIGEN EXPRESSION (SASP)-TREATED EAE RATS
1N
E a c h r o w s h o w s o b s e r v a t i o n f r o m o n e a n i m a l . In t h e m o r e d e t a i l e d q u a n t i t a t i v e a n a l y s i s ( O x 1 9 a n d O x 3 9 ) cells w e r e c o u n t e d at a m a g n i f i c a t i o n o f × 2 0 0 . T h e a r b i t r a r y s c a l i n g w a s as follows: 0 = n o s t a i n e d cells; + = 1 - 4 cells; + + = 5 - 3 0 cells; + + + = m o r e t h a n 30 s t a i n e d cells. R e s u l t s a r e e x p r e s s e d as t h e a v e r a g e n u m b e r o f s t a i n e d cells p e r visual field at a m a g n i f i c a t i o n o f × 100. Monoclonal antibodies and labelled antigens Group
Controls
Animal number
2 5 6 8 9
Mean ± SD 25 m g S A S P / k g / d a y
Mean ± SD
43.3 _+ 27.7 6 . 0 + 1.2 14.4 + 12.2 11.6 ± 17.1 16.1 + 11.6 18.3 _+ 14.5
Ox39 IL-2 rec
5.2 0.0 1.3 3.5 4.2 2.8
± 4.6 ± ± ± ±
2.5 2.8 1.5 1.5
W3/25 CD4
+ + + + +
+ + + + + + +
Ox33 B cells
Ox18 MHC
Ox6 MHC
class 1
class II
0 0 0 0 0
+ + ++ + + + + + +
+ + + ++ + + + + + + + +
+ + + + + + +
+ + + + + +++ + + + + + +
+ + + + ++ + + + +
+ + + + + + + + +++
+ + + + +
+ + + +
10 13 14 17 18
23.0 _+ 27.8 93.7 ± 47.5 24.1±10.3 29.7 ± 18.5 34.5 ± 15.4 41.0 + 29.8 *
0.0 10.0 ± 5.7 6.3±2.0 10.3 ± 5.3 9.6 + 4.7 7.4 ± 4.5 *
+ + + + +++ + + + + + +
0 + + + + +
+ + + + +++ + + + +
+ +
19 20 25 26 28
68.3 ± 22.7 56.5 ± 35.3 30.9 ± 11.1 55.9 ± 43.0 35.5±32.3 49.4 ± 15.7 * *
11.4 ± 8.8 9.5 ± 4.1 2.4 ± 2.2 4.7 ± 4.4 1.6±1.7 5.9 ± 4.3 ~
+ + + + + + + + + + + +++
+ 0 0 + + 0
+ + + + + + + + +++
+ + + +
Mean + SD 100 m g S A S P / k g / d a y
Ox19 CD5
# N o t s i g n i f i c a n t ; * p < 0.005; * * p < 0.01.
+ +
Ox8 CD8
+ + + +
+ + + + + +
115
500400-
180
•
CONTROL
• CONTROL [ ] 25mg [ ] 100mg
E
ml~ 25rng tOOmg
so
,.= 100 -
o Ag 0
r-CNS
GP-MBP
MBP
o
107-124 MBP §3-1~8
$~o
Fig. 4. Numbers of primed T cells that in response to antigen secrete IFN-3,, determined on regional lymph node MNC on day 21 p.i. in untreated EAE rats (control) or SASP-treated rats (25 and 100 mg/kg/day). Immunospots were determined after 48 h preculture in vitro without antigen (Ag 0) or with different antigens added: r-CNS = rat CNS myelin, GP-MBP = guinea pig myelin basic protein, MBP 107-124 and MBP 63-88 = synthetic MBP peptides. Columns and bars represent mean values and SEM, respectively. Note dramatic increase in number of IFN-,/-sc after stimulation with all antigens in treated rats.
r-CNS
G~-M~p MBp 107-124MBp 63-85
PHA
Fig. 5. Cell-mediated immunity measured by [3H]thymidine incorporation in response to myelin antigen of lymph node MNC from untreated EAE rats (control) and SASP-treated rats (25 and 100 mg/kg/day) 21 days p.i. Background in unstimulated cultures (Ag 0) is also indicated. Columns and bars represent mean values and SEM, respectively. PHA = phytohemagglutinin; other abbreviations as in Fig. 4. Note decreased proliferative activity in treated rats both in nonstimulated cultures and in most antigen- or lectin-stimulated cultures.
Spontaneous and antigen-induced lymphocyte proliferation cells among lymphoid cells that produced IFN-~/ in response to different antigens, these cells were precultured for 48 h to allow for antigen uptake; processing, presentation and T cell triggering were then counted. The number of spots obtained in antigen-exposed cultures exceeding zero antigen, background control cultures, gives an estimate of the number of antigen-specific T cells. The SASP-treated animals showed approximately 2to 6-fold higher numbers of IFN-y-sc in response to GP-MBP or the different MBP peptides as compared to the control animals (Fig. 4; Table 2).
As shown in Fig. 5, the proliferation of cells from treated rats was less pronounced as compared to those obtained from control animals. In the group treated with 25 m g / k g / d a y a clear decrease was seen in the spontaneous as well as the syngeneic rat CNS myelin and PHA-induced proliferation, while no difference was seen after stimulation with GP MBP and the different peptides of MBP. A still more conspicuous decrease in lymphocyte proliferation, now encompassing all antigens was observed in the group of animals treated with 100 m g / k g / d a y (Fig. 5).
TABLE 2 NUMBERS OF IMMUNOSPOTS PER 106 LYMPH NODE MONONUCLEAR CELLS IN UNTREATED AND SASPTREATED EAE RATS, AFTER STIMULATION WITH DIFFERENT NERVOUS TISSUE AUTOANTIGENS Ag 0 = no antigen added. GP-MBP: guinea pig myelin basic protein; r-CNS: rat central nervous system myelin; MBP: myelin basic protein; PPD = purified protein derivate. Ag 0 Control (n = 8) 25 mg S A S P / k g / day (n = 7) 100 mg S A S P / k g / day (n = 8)
9 5:5
GP-MBP 60+32
r-CNS
MBP (63-88)
61+46
110+ 53
MBP (89-101)
MBP (107-124)
MBP (148-165)
33+15
22+ 7
23+17
PPD 27+13
34 +- 15
200+77 #
154+_64
248+- 81
199+-50"*
126+47 #
154+42 **
40+15
17 _+4
110_+67
128+-54
331+-110 **
138+-47 *
114_+48
125+55 ##
102+-42
# p < 0.03; ## p < 0.02; *p < 0.01; * * p < 0.002.
116 120 •
Discussion ~
80"
~
.
_
.
_
~
Ag 0 PPD MBP 107-124 MBP63.S8
40"
PHA
z. i 0
i 25
i 50
i 100
Suffasallzlnl
concintratlon
(microgr,/mJ)
~o
180
-
E =
80-
~
~
.o E
:~
~
-20
.
, 20
=
.
, 40
-~-e-~
0
.
MBP107-124 MBP63-88
=
, 60
.
, 80
.
, 100
.
, Sulfaza[azlnl
concentration
(rnlcrogrJml)
Fig. 6. Effects in vitro of sulfasalazine at different concentrations on the proliferative activity of (a) lymph node cells from EAE rats measured by [3H]thymidine incorporation, and (b) number of IFN-y-secreting cells in unstimulated cultures (Ag 0), and cultures stimulated with different antigens. For [3H]thymidine incorporation, results are expressed as percent of the incorporation obtained without drug added. Abbreviations are as in Fig. 4. Note dose-dependent decrease of [3H]thymidine incorporation, while there are constant levels of IFN-y-secreting cells both in unstimulated and antigenstimulated cultures.
Effects of sulfasalazine in vitro on antigen-induced IFN-y secretion and lymphocyte proliferation Unstimulated and antigen-stimulated lymph node MNC from control EAE animals were exposed to different concentrations of SASP and the number of IFN-y-sc was analyzed. The number of IFN-y-sc remained unaffected among both unstimulated and antigen-stimulated cells (Fig. 6b). In contrast, a dose-dependent inhibition of spontaneous and antigen-induced lymphocyte proliferation was observed (Fig. 6a).
We here show that SASP treatment intensifies and prolongs EAE in Lewis rats, both clinically and histologically. The number of T cells and interleukin-2 receptor-expressing cells was increased in the CNS tissue. In parallel, the number of IFN-y-sc among mononuclear cells in CNS wash fluid was more elevated in treated rats than in non-treated counterparts. Furthermore, the number of primed 'memory' T cells from regional lymph nodes that produce IFN-y in response to myelin autoantigens was increased in SASPtreated rats. IFN-y shows a number of activities that are important in inflammation, e.g. (i) activation of macrophages (Adams and Hamilton, 1987; Goldberg et al., 1990), (ii) induction of MHC antigens on many cell types (Skoskiewicz et al., 1985) including MHC class II on astrocytes (Fontana et al., 1984), and microglia (Steininger and Van der Meide, 1988), and (iii) induction of T cell homing (Duijvestijn et al., 1986; Issekutz et al., 1988; Oppenheimer-Marks and Ziff, 1988). In vivo blocking of IFN-y with monoclonal antibodies leads to decreased clinical signs of experimental allergic neuritis (Strig~rd et al., 1989; Hartung et al., 1990). IFN-y is produced by activated T ceils that passively transfer EAE (Ando et al., 1989; Karpus and Swanborg, 1989; Sedgwick et al., 1989). Systemic administration of IFN-y leads to exacerbations of multiple sclerosis (Panitch et al., 1987). We detected approximately 10-fold more cells among peripheral blood mononuclear cells that produced IFN-y in response to myelin antigens in multiple sclerosis patients compared to controls (Olsson et al., 1990). Furthermore, we recently observed that IFN-y-producing T cells appeared in the CNS shortly preceding onset of clinical signs of EAE (Mustafa et al., 1991). On the other hand, although these data are compatible with a deleterious effect of IFN-y on EAE, Voorthuis et al. (1990) suppressed EAE by injecting high doses of the cytokine intrathecally. Also, in vivo blocking of IFN-y during EAE with antibodies was found to enhance disease (Billiau et al., 1988). Furthermore, high endogeneous levels of IFN-y that are produced during graft versus host disease (Klimpel et al., 1990) and trypanoso-
117
miasis (Bakhiet et al., 1990) are associated with immunosuppression. Thus, the net effect of IFN-7 in a certain disease might be difficult to predict, and effects may be dependent on time, site and level of production. We speculate that the increased secretion of IFN-y up to a certain level enhances the local inflammatory response, while even higher levels might depress it. Such a mechanism might also explain the drug effects seen presently that did not follow a simple dose-response curve (Fig. 1). It is anyhow tempting to suggest that during SASP treatment the increased numbers of IFN-y-sc, especially within the CNS, may be causally related to the worsened course of EAE. An in vivo expansion of T cells recognizing myelin antigens was indicated by our observation that SASP-treated EAE rats had increased number of lymph node T cells that in response to such antigens produced IFN-y in vitro. At the same time, the proliferative response to these antigens was decreased. SASP is known to inhibit signals involved in cellular activation (Carlin et al., 1989) and to reduce proliferation in vitro (Ali et al., 1982). Since the number of memory T cells secreting IFN-y was expanded in the SASPtreated rats, this would imply a preferential drug effect in vivo on proliferation of cells other than those with potential capacity to produce IFN-y. In mice, T helper cell subsets are defined, where TH1 cells characteristically produce IFN-y and TH2 cells produce IL-4 and IL-5 (Mosmann et al., 1986). Surface marker analysis suggests that a similar distribution may exist also in the rat (Arthur and Mason, 1986). Recently it has been shown that the TH1 and TH2 subsets use different T cell receptor signal transduction mechanisms for the induction of lymphokine gene expression (Gajewski et al., 1990), which could relate to a preferential effect of SASP on different T cell subsets. It will be of interest to determine if SASP inhibits proliferation or function of TH2 cells that normally secrete IL-4, since this cytokine downregulates IFN-y production by TH1 cells (Chr6tien et al., 1990; Vercelli et al., 1990). Our observed increase in the number of T cells responding with IFN-y secretion in response to
MBP peptides during SASP treatment was not restricted to the peptide previously defined encephalitogenic in Lewis rats (Vandenbark et al., 1985). Another region, MBP(89-99) was recently shown encephalitogenic (Offner et al., 1989) and still another MBP peptide was shown to promote EAE in Lewis rats during measles infection (Liebert et al., 1990). We have shown that cellular responses to all MBP peptides used here did indeed occur, and developed late in the course of EAE (Mustafa et al., 1991). Thus, it is possible that any of the MBP peptide responses increased by SASP may contribute to the deterioration of EAE. The observed augmentation of EAE in Lewis rats shown here contradicts previous results on EAE in guinea pigs, where SASP was shown to reduce severity of disease (Prosiegel et al., 1989). Important differences between the two studies include rate of treatment as well as observation times. We observed the animals for 40 days and found no augmentation of clinical signs until day 22 p.i. Prosiegel et al. (1990) observed their animals for only 21 days and a relapse could well occur later also in this model. One must also point to the difference in species between the two studies. It may even be speculated that the strain of rats used could be of importance. A parallel can be drawn to the effect of SASP in experimental models of arthritis where contrasting effects are obtained depending on species, strain and immunization protocol (Klareskog et al., 1987; Bj6rk et al., 1989; Fenfer et al., 1990). However, the present observations as well as a recent case report on onset of MS during treatment with SASP (Gold et al., 1990) demand caution with regard to further applications of this drug in clinical trials on MS. Preferably, before such trials, a variety of treatment protocols should be tested on different EAE models with a chronic course. Finally, our observations strengthen the hypothetical role of IFN-y as a deleterious effector molecule in EAE, and suggest that effects on T cell cytokine secretion may be relevant in the evaluation of new immunotherapeutic strategies in demyelinating diseases.
118
Acknowledgements We greatly appreciate the excellent technical assistance of Mrs. Anita Gustafsson, and we thank Ms. Yvonne Nilsson for secretarial help in preparing the manuscript. This study was supported by grants of the Swedish Medical Research Council (project 7488), the Swedish Multiple Sclerosis Society (NHR) and Karolinska Institutet.
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