Journal of Neuroimmunology, 37 (1992) 99-103 fc3 1902 Elsevier Science Publishers B.V. All rights reserved 0165-5728/92/$05.00
99
JNI 02141
Sympathectomy augments adoptively transferred experimental allergic encephalomyelitis E w a C h e l m i c k a - S c h o r r a,c,d, Michael N. Kwasniewski ~ a n d R o b e r t L. W o l l m a n n ~,b Departments of ~ Neurology, b Pathology, and c Pediatrics, and the d Brain Research Institute, Unicersity of Chicago, Chicago, IL 60637, USA
(Received 2 August 1991) (Revised, received 15 October 1991) (Accepted 15 October 1991)
Key words: Experimental allergic encephalomyelitis; Passive transfer; Sympathetic nervous system; Sympathectomy
Summary Adoptively transferred experimental allergic encephalomyelitis (EAE) was significantly augmented in Lewis rats with ablated sympathetic nervous system. Sympathectomy was obtained by treatment of newborn rats with 6-hydroxydopamine. Sham-injected rats were used as a control. EAE was elicited in 7-8-week-old donor Lewis rats by immunization with a suspension of guinea pig (GP) brain and spinal cord in complete Freund's adjuvant. Successful transfer of EAE was accomplished with 50 × 106 lymph node cells ( L N C ) / r a t , incubated for 72 h with GP myelin basic protein. LNC were obtained from draining lymph nodes, 9 days after immunization for EAE. The severity of passively transferred EAE was significantly augmented when donor LNC obtained from normal Lewis rats immunized for EAE were injected into sympathectomized rats as compared to sham-injected rats. When LNC were obtained from sympathectomized or sham-injected donors, the disease was significantly more severe in recipients of cells from sympathectomized animals. The severity of histological lesions in the brain and spinal cord was greater in rats with passively transferred EAE which received LNC from sympathectomized donors.
Introduction The sympathetic nervous system (SNS) modulates immune responses. SNS innervation has been documented in lymph nodes, spleen, thymus and Peyer's patches (Reilly et al., 1976; Giron et al., 1980; Williams et al., 1981). Neuropharmaco-
Correspondence to: Ewa Chelmicka-Schorr, M.D., Department of Neurology, MC2030, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA.
logical studies on the effects of adrenergic ligands on lymphocytes give further evidence supporting an interaction between the SNS and immune system (Felten et al., 1984a, b). We studied the influence of the SNS on immune responses and have shown in past work that chemically induced sympathectomy of newborn mice or rats is followed by enhancement of many immune responses including an augmented severity of autoimmune diseases (Miles et al., 1981, 1984a, b; Miles, 1984; Agius et ai., 1987; Chelmicka-Schorr et al., 1988). We have pre-
llJCp
scnted data that experimental allergic cnccphalomyelitis (EAE), which serves as a modcl for multiplc sclerosis, is significantly augmentcd in adult rats sympathcctomized by treatment with 6-hydroxydopamine as ncwborns (ChelmickaSchorr, 1988). Wc now present data on the influence of sympathectomy on passively transfcrred EAE.
Materials and methods
Sympathectomy To achieve sympathectomy, newborn Lewis rats wcre injected i.p. daily for 10 days with 6-hydroxydopamine ( 6 - O H - D A ) (Sigma Chemical Corp., St. I_x~uis, MO, USA) at a dosc of 150 ~ g of body weight pcr day. 6 - O H - D A was dissolved in 0.8% NaCI containing 0.1 m g / m i ascorbic acid (Miles, 1984). Control rats were injected with 0.8% NaCI containing 0.1 m g / m l ascorbic acid (sham injected).
Immunization protocol Six- to 8-week-old sympathectomized, sham-injected, or normal Lewis rats were immunized with 20% crude guinea pig (GP) brain and spinal cord emulsified in complete Freund's adjuvant (CFA). CFA was made with three parts of Marcol, one part of Arlacel A and with heat-killed Mycobacterium tuberculosis 3 m g / m l . Rats were immunized by inoculation in one hind footpad with 0.1 ml of GP brain and spinal cord in CFA.
Passit'e transfer of cells Ten days after immunization with GP brain and spinal cord in CFA. popliteal and inguinal draining lymph nodes from sympathectomized, sham-injected or normal rats were removed and pressed through stainless steel screens. Lymph node cells (LNC) were then washed twice and suspended at 2 × I0 ~ cells/ml in Dulbecco's modified Eagle's medium ( D M E M ) containing 5% fetal bovine serum, 100 U / m l penicillin, 100 m g / m l streptomycin and 10 g g / m l of GP myelin basic protein and incubated for 72 h in 5% CO 2 at 37°C. GP basic protein was prepared in our laboratories using the method described by Deibler et al. (1972). Cells were then washed
twice, viability checked with trypan blue, suspended in phosphate-buffered saline (PBS) at 25 × 106 cclls/ml and injected intraperitoncaIly (i.p.) into sympathectomized, sham-injected or normal Lewis rat recipients at a dose of 50 × 1W' L N C / r a t (see Experimental design, Expt. A and Expt. B). The 50 × 10 ~' L N C / r a t produced mod~ erately severe EAE. The first symptoms of E A E were seen between days 5 and 6 after passive transfer. Recipient rats were evaluated every day for clinical disease, which was assessed as ( 0 ) = no illness; 1 ( + ) = tail weakness; 2 ( + + ) = paraparesis; and 3 ( + + + ) = tetraparesis or death. Rats were sacrificed between 10 and 11 days after passive transfer. For histological examination, brains and spinal cords were fixed in formalin and paraffin sections stained with hematoxylin and eosin. Coronal sections of the brain and transverse sections of the spinal cord were examined at multiple levels. Histology was read by one of us blindly and lesions were scored as: (0) if no lesions were seen; 1 ( + ) if one lesion per section was seen; 2 ( + + ) if there were 2 - 6 lesions per section; and 3 ( + + + ) if there were more than six lesions per section. Lesions consisted of perivascular mononuclear inflammatory cell infiltrates within the parenchyma with disruption of the underlying neuropil. Peak severity of illness in each animal (scale 0-3) was used to calculate results of treatment. In most animals, the most severe E A E was seen between days 6 and 7. Statistical analyses were done using Student's t-test. The experiments were designed as follows.
Experimental design Experiment A. Donor LNC were obtained from normal Lewis rats immunized for E A E and transferred to sympathectomized or sham-injected Lewis rat recipients. Severity of the illness was compared between sympathectomized and sham-injected rats with adoptively transferred EAE. Experiment B. Donor LNC were obtained from sympathectomized or sham-injected Lewis rats immunized for E A E and transferred to normal Lewis rats. Severity of the illness was compared between recipients of LNC from sympa-
101
thectomized donors and recipients of LNC from sham-injected donors with an intact SNS.
Results
Experiment A When donor LNC from normal Lewis rats immunized for EAE were injected into sympathectomized or sham-injected recipients clinical illness was significantly more severe in sympathectomized recipients. The peak of the illness in both groups was between day 6 and 7 after passive transfer of cells. Six out of ten sympathectomized recipients (60%) developed severe ( + + ) EAE; none of the ten sham-injected recipients developed severe disease (Table 1). The difference was statistically significant at p = 0.03 (three experiments).
Experiment B When donor LNC from sympathectomized or sham-injected rats immunized for EAE were injected into normal Lewis rat recipients, the severity of clinical disease was significantly augmented in recipients of cells from sympathectomized animals. The peak of the illness in both groups was between day 6 and 7 after passive transfer of
cells. Seven out of 12 recipients of cells from sympathectomized rats developed severe ( + + ) EAE (58.3%) versus two out of 14 recipients of cells from sham-injected rats (14.2%) (Table 1). The difference was statistically significant at p = 0.003 (three experiments). The severity of histological lesions was augmented in rats which received LNC from sympathectomized donors but did not differ between sympathectomized and control recipients (Table 1).
Discussion
These experiments demonstrate that severity of passively transferred EAE is augmented in sympathectomized recipients as compared to sham-injected recipients. The severity of EAE is also augmented in rats with passively transferred LNC obtained from sympathectomized donors as compared to rats which received LNC from sham-injected donors. We have shown in earlier work that chemical sympathectomy of newborn mice or sympathetic axotomy of adult mice is followed by a significant increase in the antibody response of spleen cells to thymus-independent antigens, a decrease in
TABLE 1 T H E E F F E C T O F SYMPATHEC~FOMY ON PASSIVELY T R A N S F E R R E D E A E Group
Clinical disease
Mean _+ SEM
Significance
p=0.003
0
1.3 + 0 . 3 ~ ) 0.5 + 1.71
0
1.5 + 0.2 ]
0
+
++
+++
Sympathectomized recipients a Sham-injected recipients b
3
1
6
0
5
5
0
Recipients of cells from sympathectomized donors ~
1
4
7
Recipients of cells from sham-injected donors ~
8
p =0.003 4
2
0
0.57 + 0.2]
0
Histological disease +
++
+++
Mean 5: SEM
B SC B SC
6 3 6 6
2 0 0 0
1 7 3 3
1 0 1 1
0.7 1.4 0.9 0.9
B
2
0
4
1
SC
0
0
6
1
1.57 _+ 0.4} 2.14 + 0.1
B SC
5 2
2 1
1 3
0 2
Sympathectomized recipients were injected i.p. with LNC from normal Lewis rats immunized for EAE. b Sham-injected recipients were injected i.p. with LNC from normal Lewis rats immunized for EAE. ¢ Normal recipients were injected i.p. with LNC from sympathectomized donors immunized for EAE. c Normal recipients were injected i.p. with LNC from sham-injected donors immunized for EAE. B = brain; SC = spinal cord.
Significance
:t: 0.3 -t- 0.3 _+ 0.4 5: 0.4
0.5 + 0.3 1.62 5: 0.4
p = 0.048
I02
the population of splenic B cells, a decreased number ot" suppressor [ymphocytes within the spleen and an increase in the number of ~-adrenergic receptors on lymphocytes (Miles et al., 1981, 1984a, b; Miles, 1984). We also found that severity of two autoimmunc diseases, EAE and experimental autoimmune myasthenia gravis (EAMG), is augmented in sympathcctomized animals (Agius et al., 1987; Chelmicka-Schorr et al., 1988). Wc have data that macrophagc function as measured by tumor necrosis factor (TNF) secretion is augmented in sympathectomized animals (Chelmicka-Schorr et aI., 1991). EAE is a T-cell-mediated autoimmunc disease which serves as an experimental model for multiple sclerosis (Paterson, 1966, 1976). The central nervous system (CNS) lesions in EAE consist of perivascular infiltration by T-cells, macrophages and to a lesser extent by B-cells (Sobel et al., 1984). It was shown in mouse EAE that the sequence of events leading to lesion formation is infiltration of CNS by T-cells in the early stages of EAE followed by infiltration of B-cells and Ig " macrophages shortly before onset of clinical signs. In mouse EAE la" cells were found only in small numbers (Traugott el al., 1985). EAE can be passively transferred to syngeneic recipients (Stone, 1961). T-helper cells are required for induction and transfer of the disease (Pettinelli and McFar[in, 1981). Monoclonal antibodies specific for helper cells inhibit passively transferred EAE (Swanborg, 1983). Our study indicates that sympathectomy augments Thelper cell function as shown by augmented EAE in recipients of cells from sympathectomized animals. Macrophages have a major role in tissue damage in EAE, and in myelin damage in multiple sclerosis. It was shown in in vitro studies that the activated macrophage product T N F causes demyelination and is toxic to oligodendrocytes (Brosnan eta[., 1988; Selmaj and Raine, 1988). It was also reported that treatment of recipient mice with antibodies that neutralize T N F - a / B prevents transfer of EAE and that elimination of macrophages in animals immunized for EAE several days before development of clinical signs of EAE suppressed clinical disease significantly (Huitinga ct al., 1990; Ruddle et al., 1990). Significantly augmented adoptively transferred EAE in
sympathcctomizcd recipients as compared to control recipients indicates augmented function o1 macrophagcs in rats with ablated SNS in vivo. This observation is in accordance with our in vitro studies in which we showed that macrophage function is augmented in sympathectomized animals. The basis for immune augmentation in animals with ablated SNS is not fully understood. One possibility is that catecholamines, normally secreted by the SNS, bind to ~-adrenergic receptors on lymphocytes and macrophages, increase intracellular cAMP and thereby downregulatc immune responses (Hadden, 1975; Williams et al., 1976; Loveland et al., 1981; Abrass, 1985). In the absence of norepinephrine in sympathectomized animals levels of cAMP are lowered and immune responses are augmented. We have shown previously that treatment of animals with EAE with /3-adrenergic agonists which increase cAMP production suppresses EAE significantly and that treatment of macrophages with a /3adrenergic agonist in vitro suppresses macrophagc function (Chelmicka-Schorr ctal., 1989, 1991). It was also reported by others that agents that elevate intracellular cAMP inhibit adoptive transfer of EAE (Richert eta[., 1983). This study indicates that previously observed augmentation of EAE in sympathcctomized rats depends on augmented function of T helper cells and macrophage function and adds further evidence for the role of the SNS in immune diseases.
Acknowledgments This work was supported by grants NIH 2ROl NS18413-07A1 and National Multiple Sclerosis Society RG:2175-A01 (to E.C.-S.).
References Abrass, CK., O'Connor, S.W., Scarpacc, P.I. and Abrass, I.B. (1985) Characterization of the /3-adrenergic receptor of the rat peritoneal macrophage. J. Immunol. 135, 13381352. Agius, M.A., Checinski, M.E., Richman, D.P. and C h e f micka-Schorr. E. (1987) Sympathectomy enhances the severity of experimental autoimmunc myasthenia gravis (EAMG). J. Neuroimmunol. 16. 11 -12.
103 Brosnan, C.F., Selmaj, K. and Raine, C.S. (1988) Hypothesis: a role for tumor necrosis factor in immune-mediated demyelination and its relevance to multiple sclerosis. J. Neuroimmunol. 18, 87-94. Chelmicka-Schorr, E., Checinski, M.E. and Arnason, B.G.W. (1988) Chemical sympathectomy augments the severity of experimental allergic encephalomyelitis. J. Neuroimmunol. 17, 347-350. Chelmicka-Schorr, E., Kwasniewski, M.N., Thomas, B.E. and Arnason, B.G.W. (1989) The /3-adrenergic agonist isoproterenol suppresses experimental allergic encephalomyelitis in Lewis rats. J. Neuroimmunol. 25, 203-207. Chelmicka-Schorr, E., Kwasniewski, M.N. and Czlonkowska, A. (1991) Sympathetic nervous system and macrophage function. Ann. NY Acad. Sci. (in press). Deibler, G.E., Martenson, R.E. and Kies, M.W. (1972) Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Prep. Biochem. 2, 139-165. Felten, D.L., Ackerman, K.D., Wiegand, S.J. and Felten, S.Y. (1987a) Noradrenergic sympathetic innervation of the spleen. I. Nerve fibers associate with lymphocytes and macrophages in specific compartments of the splenic white pulp. J. Neurosci. Res. 18, 28-36. Felten, D.L., Felten, S.Y., Bellinger, D.L., Carlson, S.L., Ackerman, K.D., Madden, K.S., Olschowski, J.A. and Livnat, S. (1987b) Noradrenergic sympathetic neural interactions with the immune system: structure and function. lmmunol. Rev. 100, 225-259. Giron. L.T., Crutcher, K.A. and Davis, J.N. (1980) Lymph nodes - - a possible site for sympathetic neuronal regulation of immune responses. Ann. Neurol. 8, 520-525. Hadden, J.W. (1975) Cyclic nucleotides in lymphocyte function. NY Acad. Sci. 256, 352-363. Huitinga, I., van Rooijen, N., de Groot, C.J.A., Uitdehaag, B.M.J. and Dijkstra, C.D. (1990) Suppression of experimental allergic encephalomyelitis in Lewis rats after elimination of macrophages. J. Exp. Med. 172, 1025-1033. Loveland, B.E., Jarrott, B. and McKenzie, I.F.C. (1981) The detection of /3-adrenoreceptors on murine lymphocytes. Int. J. Immunopharmacol. 3, 45-55. Miles, K. (1984) The Sympathetic Nervous System and the Immune Response in Mice, Ph.D. Dissertation, University of Chicago, Chicago, IL. Miles, K., Quintans, J., Chelmicka-Schorr, E. and Arnason, B.G.W. (1981) The sympathetic nervous system modulates antibody response to thymus-independent antigens. J. Neuroimmunol. 1, 101-105. Miles, K., Chelmicka-Schorr, E., Otten, G. and Arnason, B.G.W. (1984a) Sympathetic nervous system and immune response in mice. Neurology 34 (Suppl. 1), 259. Miles, K., Atweh, S., Otten, G., Arnason, B.G.W. and Chelmicka-Schorr, E. (1984b) /3-Adrenergic receptors on splenic lymphocytes from axotomized mice. Int. J. Immunopharmacol. 6, 171-177.
Paterson, P.Y. (1966) Experimental allergic encephalomyelitis and autoimmune disease. Adv. Immunol. 5, 131-208. Paterson, P.Y. (1976) Experimental autoimmune encephalomyelitis: induction, pathogenesis and suppression. In: P.A. Mieocher and H.J. Muller-Eberhard (Eds.), Textbook of Immunopathology, Vol. 1, Grune and Stratton, New York, NY, pp. 179-213. Pettinelli, C.B. and McFarlin, D.E. (1981) Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1 - 2 - T lymphocytes. J. lmmunol. 127, 1420-1423. Rcilly, F.D., McCuskey, R.S. and Meineke, H.A. (1976) Studies of the hemopoietic microenvironment. VIII. Adrenergic and cholinergic innervation of the murinc spleen. Anat. Rec. 105, 100. Richert, J.R., Kies, M.W. and Alvord, Jr., E.C. (1983) Adoptive transfer of experimental allergic encephalomyelitis: inhibition of agents that elevate intracellular cyclic AMP levels. Neurochem. Pathol. 1, 81-90. Rudd[e, N.tt., Bergman, C.M., McGrath, K.M., Lingenheld, E.G., Grunnet, M.L., Padula, S.J. and Clark, R.B. (1990) An antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis. J. Exp. Med. 172, 1193-1200. Selmaj, K.W. and Raine, C.S. (1988) Tumor necrosis factor mediates myelin and oligodendroeyte damage in vitro. Ann. Neurol. 23, 339-346. Sobel, R.A., Blanchette, B.W., Bhan, A.K. and Colvin, R.B. (1984) The immunopathology of experimental allergic encephalomyelitis. 1. Quantitative analysis of inflammatory cells in situ. J. Immunol. 132, 2393-2401. Stone, S.H. (1961) Transfer of allergic encephalomyelitis by lymph node cells in inbred guinea pigs. Science 134, 619620. Swanborg, R.H. (1983) Autoimmune effector cells. V. A monoclonal antibody specific for rat helper T lymphocytes inhibits adoptive transfer of autoimmune encephalomyelitis. J. lmmunol. 130, 1503-1505. Traugott, U., Raine, C.S. and McFarlin, D.E. (1985) Acute experimental allergic encephalomyelitis in the mouse: immunopathology of the developing lesion. Cell. lmmunol. 91,240-254. Williams, J.M., Peterson, R.G., Shca, P.A., Schedtje, J.F., Bauer, D.C. and Felten, D.L. (1981) Sympathetic innervation of murine thymus and spleen: evidence for a functional link between the nervous and immune systems. Brain Res. Bull. 6, 83-94. Williams, L.T., Snyderman, R. and Lefkowitz, R.J. (1976) Identification of/3-adrenergic receptors in human lymphocytes by (-)3H-alprenolol binding. J. Clin. Invest. 57, 149-155.
99
JNI 02141
Sympathectomy augments adoptively transferred experimental allergic encephalomyelitis E w a C h e l m i c k a - S c h o r r a,c,d, Michael N. Kwasniewski ~ a n d R o b e r t L. W o l l m a n n ~,b Departments of ~ Neurology, b Pathology, and c Pediatrics, and the d Brain Research Institute, Unicersity of Chicago, Chicago, IL 60637, USA
(Received 2 August 1991) (Revised, received 15 October 1991) (Accepted 15 October 1991)
Key words: Experimental allergic encephalomyelitis; Passive transfer; Sympathetic nervous system; Sympathectomy
Summary Adoptively transferred experimental allergic encephalomyelitis (EAE) was significantly augmented in Lewis rats with ablated sympathetic nervous system. Sympathectomy was obtained by treatment of newborn rats with 6-hydroxydopamine. Sham-injected rats were used as a control. EAE was elicited in 7-8-week-old donor Lewis rats by immunization with a suspension of guinea pig (GP) brain and spinal cord in complete Freund's adjuvant. Successful transfer of EAE was accomplished with 50 × 106 lymph node cells ( L N C ) / r a t , incubated for 72 h with GP myelin basic protein. LNC were obtained from draining lymph nodes, 9 days after immunization for EAE. The severity of passively transferred EAE was significantly augmented when donor LNC obtained from normal Lewis rats immunized for EAE were injected into sympathectomized rats as compared to sham-injected rats. When LNC were obtained from sympathectomized or sham-injected donors, the disease was significantly more severe in recipients of cells from sympathectomized animals. The severity of histological lesions in the brain and spinal cord was greater in rats with passively transferred EAE which received LNC from sympathectomized donors.
Introduction The sympathetic nervous system (SNS) modulates immune responses. SNS innervation has been documented in lymph nodes, spleen, thymus and Peyer's patches (Reilly et al., 1976; Giron et al., 1980; Williams et al., 1981). Neuropharmaco-
Correspondence to: Ewa Chelmicka-Schorr, M.D., Department of Neurology, MC2030, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA.
logical studies on the effects of adrenergic ligands on lymphocytes give further evidence supporting an interaction between the SNS and immune system (Felten et al., 1984a, b). We studied the influence of the SNS on immune responses and have shown in past work that chemically induced sympathectomy of newborn mice or rats is followed by enhancement of many immune responses including an augmented severity of autoimmune diseases (Miles et al., 1981, 1984a, b; Miles, 1984; Agius et ai., 1987; Chelmicka-Schorr et al., 1988). We have pre-
llJCp
scnted data that experimental allergic cnccphalomyelitis (EAE), which serves as a modcl for multiplc sclerosis, is significantly augmentcd in adult rats sympathcctomized by treatment with 6-hydroxydopamine as ncwborns (ChelmickaSchorr, 1988). Wc now present data on the influence of sympathectomy on passively transfcrred EAE.
Materials and methods
Sympathectomy To achieve sympathectomy, newborn Lewis rats wcre injected i.p. daily for 10 days with 6-hydroxydopamine ( 6 - O H - D A ) (Sigma Chemical Corp., St. I_x~uis, MO, USA) at a dosc of 150 ~ g of body weight pcr day. 6 - O H - D A was dissolved in 0.8% NaCI containing 0.1 m g / m i ascorbic acid (Miles, 1984). Control rats were injected with 0.8% NaCI containing 0.1 m g / m l ascorbic acid (sham injected).
Immunization protocol Six- to 8-week-old sympathectomized, sham-injected, or normal Lewis rats were immunized with 20% crude guinea pig (GP) brain and spinal cord emulsified in complete Freund's adjuvant (CFA). CFA was made with three parts of Marcol, one part of Arlacel A and with heat-killed Mycobacterium tuberculosis 3 m g / m l . Rats were immunized by inoculation in one hind footpad with 0.1 ml of GP brain and spinal cord in CFA.
Passit'e transfer of cells Ten days after immunization with GP brain and spinal cord in CFA. popliteal and inguinal draining lymph nodes from sympathectomized, sham-injected or normal rats were removed and pressed through stainless steel screens. Lymph node cells (LNC) were then washed twice and suspended at 2 × I0 ~ cells/ml in Dulbecco's modified Eagle's medium ( D M E M ) containing 5% fetal bovine serum, 100 U / m l penicillin, 100 m g / m l streptomycin and 10 g g / m l of GP myelin basic protein and incubated for 72 h in 5% CO 2 at 37°C. GP basic protein was prepared in our laboratories using the method described by Deibler et al. (1972). Cells were then washed
twice, viability checked with trypan blue, suspended in phosphate-buffered saline (PBS) at 25 × 106 cclls/ml and injected intraperitoncaIly (i.p.) into sympathectomized, sham-injected or normal Lewis rat recipients at a dose of 50 × 1W' L N C / r a t (see Experimental design, Expt. A and Expt. B). The 50 × 10 ~' L N C / r a t produced mod~ erately severe EAE. The first symptoms of E A E were seen between days 5 and 6 after passive transfer. Recipient rats were evaluated every day for clinical disease, which was assessed as ( 0 ) = no illness; 1 ( + ) = tail weakness; 2 ( + + ) = paraparesis; and 3 ( + + + ) = tetraparesis or death. Rats were sacrificed between 10 and 11 days after passive transfer. For histological examination, brains and spinal cords were fixed in formalin and paraffin sections stained with hematoxylin and eosin. Coronal sections of the brain and transverse sections of the spinal cord were examined at multiple levels. Histology was read by one of us blindly and lesions were scored as: (0) if no lesions were seen; 1 ( + ) if one lesion per section was seen; 2 ( + + ) if there were 2 - 6 lesions per section; and 3 ( + + + ) if there were more than six lesions per section. Lesions consisted of perivascular mononuclear inflammatory cell infiltrates within the parenchyma with disruption of the underlying neuropil. Peak severity of illness in each animal (scale 0-3) was used to calculate results of treatment. In most animals, the most severe E A E was seen between days 6 and 7. Statistical analyses were done using Student's t-test. The experiments were designed as follows.
Experimental design Experiment A. Donor LNC were obtained from normal Lewis rats immunized for E A E and transferred to sympathectomized or sham-injected Lewis rat recipients. Severity of the illness was compared between sympathectomized and sham-injected rats with adoptively transferred EAE. Experiment B. Donor LNC were obtained from sympathectomized or sham-injected Lewis rats immunized for E A E and transferred to normal Lewis rats. Severity of the illness was compared between recipients of LNC from sympa-
101
thectomized donors and recipients of LNC from sham-injected donors with an intact SNS.
Results
Experiment A When donor LNC from normal Lewis rats immunized for EAE were injected into sympathectomized or sham-injected recipients clinical illness was significantly more severe in sympathectomized recipients. The peak of the illness in both groups was between day 6 and 7 after passive transfer of cells. Six out of ten sympathectomized recipients (60%) developed severe ( + + ) EAE; none of the ten sham-injected recipients developed severe disease (Table 1). The difference was statistically significant at p = 0.03 (three experiments).
Experiment B When donor LNC from sympathectomized or sham-injected rats immunized for EAE were injected into normal Lewis rat recipients, the severity of clinical disease was significantly augmented in recipients of cells from sympathectomized animals. The peak of the illness in both groups was between day 6 and 7 after passive transfer of
cells. Seven out of 12 recipients of cells from sympathectomized rats developed severe ( + + ) EAE (58.3%) versus two out of 14 recipients of cells from sham-injected rats (14.2%) (Table 1). The difference was statistically significant at p = 0.003 (three experiments). The severity of histological lesions was augmented in rats which received LNC from sympathectomized donors but did not differ between sympathectomized and control recipients (Table 1).
Discussion
These experiments demonstrate that severity of passively transferred EAE is augmented in sympathectomized recipients as compared to sham-injected recipients. The severity of EAE is also augmented in rats with passively transferred LNC obtained from sympathectomized donors as compared to rats which received LNC from sham-injected donors. We have shown in earlier work that chemical sympathectomy of newborn mice or sympathetic axotomy of adult mice is followed by a significant increase in the antibody response of spleen cells to thymus-independent antigens, a decrease in
TABLE 1 T H E E F F E C T O F SYMPATHEC~FOMY ON PASSIVELY T R A N S F E R R E D E A E Group
Clinical disease
Mean _+ SEM
Significance
p=0.003
0
1.3 + 0 . 3 ~ ) 0.5 + 1.71
0
1.5 + 0.2 ]
0
+
++
+++
Sympathectomized recipients a Sham-injected recipients b
3
1
6
0
5
5
0
Recipients of cells from sympathectomized donors ~
1
4
7
Recipients of cells from sham-injected donors ~
8
p =0.003 4
2
0
0.57 + 0.2]
0
Histological disease +
++
+++
Mean 5: SEM
B SC B SC
6 3 6 6
2 0 0 0
1 7 3 3
1 0 1 1
0.7 1.4 0.9 0.9
B
2
0
4
1
SC
0
0
6
1
1.57 _+ 0.4} 2.14 + 0.1
B SC
5 2
2 1
1 3
0 2
Sympathectomized recipients were injected i.p. with LNC from normal Lewis rats immunized for EAE. b Sham-injected recipients were injected i.p. with LNC from normal Lewis rats immunized for EAE. ¢ Normal recipients were injected i.p. with LNC from sympathectomized donors immunized for EAE. c Normal recipients were injected i.p. with LNC from sham-injected donors immunized for EAE. B = brain; SC = spinal cord.
Significance
:t: 0.3 -t- 0.3 _+ 0.4 5: 0.4
0.5 + 0.3 1.62 5: 0.4
p = 0.048
I02
the population of splenic B cells, a decreased number ot" suppressor [ymphocytes within the spleen and an increase in the number of ~-adrenergic receptors on lymphocytes (Miles et al., 1981, 1984a, b; Miles, 1984). We also found that severity of two autoimmunc diseases, EAE and experimental autoimmune myasthenia gravis (EAMG), is augmented in sympathcctomized animals (Agius et al., 1987; Chelmicka-Schorr et al., 1988). Wc have data that macrophagc function as measured by tumor necrosis factor (TNF) secretion is augmented in sympathectomized animals (Chelmicka-Schorr et aI., 1991). EAE is a T-cell-mediated autoimmunc disease which serves as an experimental model for multiple sclerosis (Paterson, 1966, 1976). The central nervous system (CNS) lesions in EAE consist of perivascular infiltration by T-cells, macrophages and to a lesser extent by B-cells (Sobel et al., 1984). It was shown in mouse EAE that the sequence of events leading to lesion formation is infiltration of CNS by T-cells in the early stages of EAE followed by infiltration of B-cells and Ig " macrophages shortly before onset of clinical signs. In mouse EAE la" cells were found only in small numbers (Traugott el al., 1985). EAE can be passively transferred to syngeneic recipients (Stone, 1961). T-helper cells are required for induction and transfer of the disease (Pettinelli and McFar[in, 1981). Monoclonal antibodies specific for helper cells inhibit passively transferred EAE (Swanborg, 1983). Our study indicates that sympathectomy augments Thelper cell function as shown by augmented EAE in recipients of cells from sympathectomized animals. Macrophages have a major role in tissue damage in EAE, and in myelin damage in multiple sclerosis. It was shown in in vitro studies that the activated macrophage product T N F causes demyelination and is toxic to oligodendrocytes (Brosnan eta[., 1988; Selmaj and Raine, 1988). It was also reported that treatment of recipient mice with antibodies that neutralize T N F - a / B prevents transfer of EAE and that elimination of macrophages in animals immunized for EAE several days before development of clinical signs of EAE suppressed clinical disease significantly (Huitinga ct al., 1990; Ruddle et al., 1990). Significantly augmented adoptively transferred EAE in
sympathcctomizcd recipients as compared to control recipients indicates augmented function o1 macrophagcs in rats with ablated SNS in vivo. This observation is in accordance with our in vitro studies in which we showed that macrophage function is augmented in sympathectomized animals. The basis for immune augmentation in animals with ablated SNS is not fully understood. One possibility is that catecholamines, normally secreted by the SNS, bind to ~-adrenergic receptors on lymphocytes and macrophages, increase intracellular cAMP and thereby downregulatc immune responses (Hadden, 1975; Williams et al., 1976; Loveland et al., 1981; Abrass, 1985). In the absence of norepinephrine in sympathectomized animals levels of cAMP are lowered and immune responses are augmented. We have shown previously that treatment of animals with EAE with /3-adrenergic agonists which increase cAMP production suppresses EAE significantly and that treatment of macrophages with a /3adrenergic agonist in vitro suppresses macrophagc function (Chelmicka-Schorr ctal., 1989, 1991). It was also reported by others that agents that elevate intracellular cAMP inhibit adoptive transfer of EAE (Richert eta[., 1983). This study indicates that previously observed augmentation of EAE in sympathcctomized rats depends on augmented function of T helper cells and macrophage function and adds further evidence for the role of the SNS in immune diseases.
Acknowledgments This work was supported by grants NIH 2ROl NS18413-07A1 and National Multiple Sclerosis Society RG:2175-A01 (to E.C.-S.).
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