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T T-A/T-T D'ssertation vJ l V l l Information Service
University Microfilms International A Bell & Howell Information Company 300 N. Zeeb Road, Ann Arbor, Michigan 48106 8618788
Jackson, Kelly Michael
GANGLIOSIDES SUPPRESS THE PROLIFERATION OF AUTOREACTIVE CELLS IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: THE EFFECTS OF GANGLIOSIDES ON INTERLEUKIN 2 ACTIVITY
The Ohio Stale University Ph.D. 1986
University Microfilms
International300 N. Zeeb Road, Ann Arbor, Ml 48106
Copyright 1986
by Jackson, Kelly Michael All Rights Reserved GANGLIOSIDES SUPPRESS THE PROLIFERATION OF AUTOREACTIVE CELLS IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: THE EFFECTS OF GANGLIOSIDES ON INTERLEUKIN 2 ACTIVITY
DISSERTATION
Presented In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In the Graduate School of the Ohio State University
By
Kelly Michael Jackson, B.S.
* * * * *
The Ohio State University
1986
Dissertation Committee: Approved by
Caroline C. Whitacre, Ph.D.
Raymond Lang, Ph.D.
Frank Kapral, Ph.D. P auli. 'nJJ d WA l& o U' Dennis Pollack, Ph.D. Advisor Department of Medical Microbiology and ImmunologyAllan Yates, M.D., Ph.D. and ImmunologyAllan Copyright by Kelly Michael Jackson 1986 DEDICATION
To the memory of my mother, Mrs. Laura T. Jackson
- i i - ACKNOWLEDGMENTS
I would like to express my sincere appreciation and gratitude to my research advisor, Dr. Caroline Whitacre, for her guidance, support, and especially for her endurance throughout the course of this study. I also thank the other members of Dr. Whitacre’s laboratory, Ingrid Gienapp, Clara
Pelfry, Kathy Fuller, and Dina Bitar, for their moral support and friendship.
I offer my sincere thanks to my advisory committee, Dr. Raymond Lang,
Dr. Dennis Pollack, Dr. Frank Kapral, and Dr. Allan Yates, for their helpful suggestions. Dr. Yates and the members of his laboratory deserve special thanks for training that I received, and for the use of their laboratory space and equipment. I also wish to extend special thanks to Nancy Zinn and
Dr. Charles Orosz for their assistance and ideas.
I would also like to express my deepest appreciation to my father,
Dr. Aubrey Jackson, and to my brothers, Aubrey and Carl, for their support and encouragement throughout this experience.
- i i i - VITA
February 21, 1958...... Born - Fairmont, West Virginia
198 0 ...... B.5., Marshall University, Huntington, West Virginia
1 9 8 1 -1 9 8 5 ...... C.I.C. Minority Student Fellow, The Ohio State University, Columbus, Ohio
1981 - 1986 ...... Graduate Research Associate, The Ohio State University, Columbus, Ohio
BIBLIOGRAPHY
Jackson, K. M., A. J. Yates, N. Zinn, C. Orosz, C. C. Whitacre. Experimental Allergic Encephalomyelitis (EAE): The Suppressive Effect of Gangliosides on In Vitro Proliferative Responses. Fed. Proc., 44; 3176, 1985. (Abstract)
FIELD OF STUDY
Major fields of study: Medical Microbiology and Immunology TABLE OF CONTENTS
Page
DEDICATION...... ii
ACKNOWLEDGMENTS...... iii
VITA ...... iv
LIST OF TABLES...... vi
LIST OF FIGURES vii, viti
LIST OF PLATES...... ix
ABBREVIATIONS...... x, xi
CELL LINES...... xii
CHAPTER
I. INTRODUCTION...... I
II. MATERIALS AND METHODS...... 36
III. RESULTS...... 48
IV. DISCUSSION...... 91
V. REFERENCES...... 112
-V- LIST OF TABLES
Table Page
1 Effects of gangliosides on antigen and mitogen-induced LNC proliferation ...... 53
2 Effects of gangliosides on Con A-induced proliferation of normal unsensitized rat L N C ...... 56
3 Effects of gangliosides, asialo GMi, and sulfatides on MBP and Con A-induced LNC proliferation ...... 57
A Assessment of ganglioside-mediated suppression of MBP-induced LNC proliferation, in the presence of supraoptimal MBP concentrations ...... 61
5 Proliferative responses of an MBP-reactive rat T-cell l i n e ...... 65
6 Proliferative responses of an MBP-reative encephalito- genic T-cell line: Effects of gangliosides on MBP- induced proliferation ...... 67
7 Effects of gangliosides on the in vitro activation of LNC to transfer E A E ...... 68
8 Assessment of LNC transfer of EAE in recipients injected with gangliosides ...... 70
9 Direct CTLL-20 indicator cell addition assay for the detection of IL 2 in MBP-activated LNC cultures .... 79
10 Effects of gangliosides on the expression of IL 2 receptors on Con A-activated L N C ...... 87
11 Effects of excess IL 2 on ganglioside-mediated suppression of Con A-induced LNC proliferative responses ...... 89
-v i- LIST OF FIGURES
Figure Page
1 Effects of rat and bovine brain gangliosides on the viability of rat and human lymphoid c e l l s ...... 50
2 Effects of rat brain gangliosides on the viability of rat lymphoid cells and human PBM C ...... 52
3 Effects of gangliosides on the antigen and mitogen-induced proliferation of rat L N C ...... 55
4 Kinetics of ganglioside-mediated suppression of MBP- induced LNC proliferative responses ...... 59
5 Effects of supraoptimal MBP concentrations on ganglioside- mediated suppression of LNC proliferative responses . . . 62
6 Differential suppressive effects of individual gangliosides on the MBP-induced proliferative responses of LNC .... 64
7 Sialic acid concentrations in the sera from unsensitized rats and in the sera from rats with E A E ...... 72
8 Detection of IL 2 activity in supernatants derived from Con A or MBP-activated cells: Effects of gangliosides on IL 2 ac tivity ...... 76
9 Effects of gangliosides on IL 2-induced CTLL-20 cell proliferation ...... 80
10 Effects of various concentrations of gangliosides on CTLL-20 cell proliferation and viability ...... 83
-v ii- 11 Assessment of IL 2-induced CTLL-20 cell proliferation: Effects of ganglioside-IL 2 preincubation ...... 84
12 Effects of gangliosides on the cel 1-surface binding of a monoclonal antibody directed against the rat IL 2 receptor ...... 86
- v ii i- LIST OF PLATES
P late Page
I TLC pattern of purified rat brain gangliosides ...... 40
II TLC pattern of gangliosides extracted from the sera of unsensitized and MBP-CFA-sensitlzed rats .... 73
-ix - ABBREVIATIONS
AER Active E-rosette
ALS Anti-lymphocyte serum
ATS Anti-thymocyte serum
CFA Complete Freund's adjuvant
CNS Central nervous system
Con A Concanavalin A
DTH Delayed-type hypersensitivity
EAE Experimental allergic encephalomyelitis
FITC Fluorescein Isothiocyanate
HBSS Hank's balanced salts solution
IFA Incomplete Freund's adjuvant
IL 2 Interleukin 2
LNC Lymph node cells
MBP Myelin basic protein
MS Multiple sclerosis NLNC Normal lymph node cells
PBMC Peripheral blood mononuclear cells
PHA Phytohemagglutinin
PPD Purified protein derivative
RPBMC Rat peripheral blood mononuclear cells
SPC Spleen cells
TLC Thin-layer chromatography
-x i- CELL LINES
IL 2-dependent murine T-cell line
MBP-reactive Lewis rat T-cell line INTRODUCTION
Multiple sclerosis (MS), first described by Cruveilheir (1829 - 1842) and Carswell (1838), and later by Charcot (1868), is a demyelinating disease that affects the central nervous system (CNS) in humans. MS is usually seen in young adults beginning between the second and fifth decades, although It can occur in both younger and older Individuals
(McFarlin and McFarland, 1982). Geographically, MS Is most prevalent In northern Europe, the northern United States and southern Canada, as well as
In parts of Australia and New Zealand. Genetic factors, such as genes within the major histocompatibility complex (MHC), may play some role in predisposition for the development of MS (Batchelor, et al., 1978).
Particularly, MS has been associated with the occurrence of the HLA haplotypes A3, B7, Dw2, and DRw2 in Caucasians. Persons with MS commonly display sensory, visual, and motor dysfunction, and the disease can present with an acute, chronic, or exacerbating-remitting clinical course. The primary pathology in MS is restricted to the CNS, where widely disseminated macroscopic plaques of demyelination are found.
Microscopically, the characteristic feature is the breakdown of the myelin sheath with relative sparing of the underlying axons (Adams and Sidman,
1968). Demyelinated lesions are usually perivascular in distribution and early lesions contain infiltrating macrophages, lymphocytes, and plasma cells (Prineas and Wright, 1978).
i 2
Presently, the etiology of MS Is unknown, but Investigations have focused mainly on three hypotheses: 1) viral infection of the CNS 2) auto- immunity directed against CNS tissue 3) an antecedent viral infection which results in autoimmunity to CNS tissue (McFarlln and McFarland,
1982). The suggestion that MS might result from an autoimmune reaction comes largely from similarities between MS and the animal model, experimental allergic encephalomyelitis (EAE), an experimentally induced inflammatory demyelinating disease (Paterson, 1979).
1. EAE: Characteristics of Disease
EAE is an autoimmune disease that affects the CNS of susceptible animals. It can be readily induced by a single injection of brain or spinal cord homogenate, purified myelin basic protein (MBP), or encephalitogenic peptide fragments of MBP, In combination with an immunologic adjuvant
(Paterson, 1976a; Paterson et al., 1970). EAE can be induced in all common laboratory animal species, the most frequently used being inbred strains of rats, mice and guinea pigs (Paterson, 1976a). Injection of isogeneic, allogeneic, or xenogeneic CNS tissue, emulsified with adjuvant, produces the characteristic manifestations of EAE.
Early descriptions of EAE and the disease-inducing ability of nervous system tissue can be traced back to the 1880‘s and 1890‘s, during the development of rabies virus vaccine by Pasteur (Paterson, 1976). Some individuals who received attenuated rabies virus, which had been passaged in rabbit CNS tissue, developed severe and sometimes fatal "paralytic accidents", consequences which were later shown to be due to the injected brain tissue and not the virus. The encephalitogenic activity of normal 3 uninfected brain tissue for laboratory animals was demonstrated in the mfd-1930's in monkeys (Rivers et al., 1933; Rivers and Schwentker, 1935).
In these studies, repeated injections of brain extracts over a period of several months produced an acute disseminated encephalomyelitis.
Accelerated EAE was observed in the 1940’s with the development and use of Freund-type immunologic adjuvants, which are composed of a light paraffin or mineral oil, an emulsifying agent, and killed mycobacteria
(Morgan, 1947 and Freund et a l, 1947). The potentiating effect of the adjuvant allowed the production of EAE following a single injection of neuroantigen-adjuvant emulsion.
The primary encephalitogenic component of mammalian CNS tissue was shown by Kies and Alvord (1965, 1970), and Einstein (1966a, 1966b) to be myelin basic protein (MBP), one of the major protein constituents of myelin, comprising approximately 308 of the total myelin protein. MBP is a small cationic protein of approximately 18,300 daltons in molecular weight and consists of 169 amino acids, with a high lysine, arginine and histidine content. MBP is heat and acid stable, has no sulfhydryl groups, and lacks tertiary structure in solution (Braun and Brostoff, 1977). Small peptide regions within the MBP molecule have been shown to have differential encephalitogenicity for various animal species. For example, the amino acid sequences 65 - 74 of guinea pig basic protein induce EAE in the rabbit, 114 - 122 cause disease in the guinea pig, and 68 - 88 are encephalitogenic in the Lewis rat (Hashim, 1978 and Chou, 1977).
Other myelin constituents, such as polar lipids and lipoproteins, have been shown to contribute to the development of EAE. Moore et al. (1984) reported that demyelination in guinea pigs was more severe following 4 sensitization with MBP in combination with the myelin lipids galactocerebroside, sulfatide, and ethanolamine phospho-glyceride.
Chronic or relapsing models of EAE have been induced in Lewis rats
(McFarlin et al., 1974 and Panitch and Ciccone, 1981) or mice (Fritz et al.,
1983), by immunization with MBP alone or with spinal cord homogenate
(Raine and Stone, 1977; Wisniewski and Keith, 1977). Myelin proteolipid protein plus adjuvant was shown to produce EAE in mice and guinea pigs
(Olitsky and Tal, 1952; Goldstein et al., 1953) and more recently, chronic
EAE was produced in rabbits by sensitization with myelin proteolipid protein (Williams et al., 1982 and Sobel et al., 1986).
The inbred Lewis rat is highly susceptible to EAE and is the prototype species used presently in EAE research. Ten to fourteen days following sensitization with neuroantigen in complete Freund’s adjuvant (CFA), Lewis rats display characteristic clinical signs of EAE, which may include weight loss, loss of tail tonus, ataxic gait, hindlfmb paresis, and total hindlimb paralysis, usually accompanied by urinary retention and fecal impaction
(Paterson, 1966 and Paterson, 1976a). The histopathologic changes of EAE begin with perivascular deposition of fibrin (Oldstone and Dixon, 1968;
Paterson, 1976b), perhaps an early sign of an increase in CNS vascular permeability. Perivascular mononuclear cell infiltration, consisting of macrophages, lymphocytes, and plasma cells, with a predominance of macrophages, is observed to occur, particularly in the white matter. Late in the disease course, often following recovery, demyelination is seen in the vicinity of the mononuclear cell infiltrates (Paterson, 1976a).
Strong evidence for the role of cellular immunity In the development of
EAE was provided by Paterson (I9 6 0 ) and Stone (1961) with the demonstration of the passive transfer of EAE using lymphoid ceils. Lymph node cells (LNC) derived from donors which had received neuroantigen and adjuvant, transferred clinical and histopathological manifestations of EAE to naive recipients. The early experiments of Paterson (I9 6 0 ) were performed with outbred Wistar rats, which made it necessary to first tolerize the recipients to donor alloantigens by the injection of donor rat spleen cells into neonatal recipients. Levine et al. (1967) demonstrated the cellular transfer of disease using inbred rat strains. EAE cellular transfer can be markedly enhanced by the in vitro cultivation of sensitized donor spleen cells in the presence of concanavalln A (Con A) (Panitch and
McFarlln, 1977), or MBP (Rlchert et al., 1979). This modification of the adoptive transfer of EAE produces severe clinical and histopathological effects, but requires relatively few transferred cells (10 - 40 x 106 cells as compared with a direct transfer which requires 100 - 500 x 106 cells).
Similar attempts to transfer EAE with serum from sensitized donors have proven unsuccessful (Chase, 1959; Paterson and Harwin, 1963).
T-lymphocytes have been shown to be important in the development of
EAE. Immunofluorescence studies in guinea pigs have shown that
T-lymphocytes infiltrate the CNS early In the course of EAE (Traugott et al., 1982). Rats depleted of T cells by thymectomy and total body irradiation were observed to be refractory to subsequent EAE induction
(Gonatas and Howard, 1974). Further evidence for the role of T-lymphocytes in EAE was obtained by the pretreatment of rat lymphoid cells with anti-thymocyte serum (ATS) and complement prior to the use of these cells to reconstitute thymectomized, irradiated recipients (Ortiz-Ortiz et al., 1976). In this study, rats that were reconstituted with ATS-treated 6 cells, did not develop EAE upon MBP-CFA sensitization. In another study, monoclonal antibody W3/25, which recognizes a determinant on the surface of the rat T-helper cell, significantly accelerated recovery from EAE, when injected parenterally into Lewis rats that had been sensitized with
MBP-CFA (Brostoff and Mason, 1984). Similarly, SJL/J mice were immunized with mouse spinal cord homogenate plus CFA and pertussis vaccine to induce EAE. Intraperitoneal injection of monoclonal antibody
GK 1.5, which recognizes the L3T4 marker present on murine T-helper lymphocytes, prevented development of disease when administered before the onset of EAE and also facilitated early recovery from disease when administered shortly after the first clinical signs appeared (Waldor et al.,
1985).
Further evidence for the importance of T cells in EAE was provided with the development of long-term encephalitogenic T cell lines. Such cell lines have been derived from the Lewis rat (Ben-nun, et al., 1981; Ben-nun and
Cohen, 1982; Vandenbark et al., 1985) and from the SJL/J mouse (Richert at al., 1985), and these cells mediate EAE when restimulated with MBP and injected back into syngeneic hosts. Encephalitogenic lines derived from
Lewis rats by in vitro propagation with guinea pig MBP, have been shown to have the T-lymphocyte phenotype and to undergo a vigorous proliferative response in vitro specifically to the selecting antigen, MBP (Ben-nun and
Cohen, 1982). This antigenic specificity extends to the peptide level since encephalitogenic cell lines selected with guinea pig MBP responded well to guinea pig and rat MBP, which differ in the encephalitogenic region of the
MBP molecule by only one amino acid, while the T line cells responded only moderately or not at all to human and bovine MBP, which have greater amino acid differences (Vandenbark et al., 1985). Activation of these line cells in vitro was shown to require MHC-syngeneic or semi-syngeneic LNC, splenocytes (Ben-nun and Cohen, 1982) or thymocytes (Holoshitz et al.,
1983) as accessory cells. MHC-restriction was also demonstrated when
CNS-derived astrocytes were used as accessory cells (Fontana et al., 1984).
In this study, Lewis and (Lewis x Brown Norway)Fi rat astrocytes presented antigen to MBP-specific line cells in v itro, whereas,
MHC-congenic L.BN astrocytes were ineffective. In addition to their ability to mediate EAE, MBP-reactive T cell lines have been shown to vaccinate rats against the subsequent induction of EAE by MBP-CFA sensitization
(Ben-nun and Cohen, 1981). The line cells were first attenuated by irradiation or mitomycin-C treatment, then 107 cells were injected intravenously into recipient rats prior to sensitization with MBP-CFA or transfer of encephalitogenic T cell lines 15 days later. Vaccination with attenuated cells inhibited MBP-CFA induction of EAE, but was ineffective in preventing transfer of disease by the intact cell line.
Some reports have suggested that antibody or antibody-producing cells may play some role in EAE (Gausas et al., 1982; Willenborg and Prowse,
1983). In both of these studies, treatment of neonatal Lewis rats with anti-IgM antibody rendered the animals resistant to the subsequent induction of EAE. Willenborg and Prowse (1983) also demonstrated that this treatment did not adversely affect T-lymphocyte responses to PHA, or the ability to reject tissue allografts.
Circulating antibody, directed against CNS antigens, has been reported to play some role in EAE (Appel, 1964). In this study, rat and mouse cerebellar myelinated tissue cultures were exposed to serum derived from 8 rabbits with EAE. EAE serum was markedly myelinotoxic in the presence of complement. Other investigators have found a cell-free factor In the supernatant of a 1 hour culture of LNC obtained from spinal cord plus
CFA-sensitized Lewis rats. This EAE-supemate transfer activity
(EAE-STA) was shown to transfer histological but not clinical signs of EAE in the Lewis rat (Whitacre and Paterson, 1977; 1980; Whitacre et al.,
1981). EAE-STA was found to have a molecular weight in excess of
100,000 daltons, and its activity was not attributable to antigen-carryover or to antibody.
II. Inhibition and Treatment of EAE
The suppression of EAE has been an important goal, not only from the standpoint of establishing therapeutic approaches to MS, but also because such work might enhance our understanding of the underlying pathogenesis of inflammatory CNS diseases (reviewed by Paterson, 1976a). Due to the immunologic nature of EAE, various immunosuppressive agents have been tested for their ability to suppress EAE (e.g. cyclophosphamide, cyclosporine, corticosteroids, and niridazole). Cyclophosphamide has been shown to be effective at Inhibiting EAE in the Lewis rat when given before the onset of clinical signs (Paterson, 1971). However, recrudescence of paralysis was observed in these rats as early as four days after the termination of cyclophosphamide treatment. Paterson and Drobish (1969) treated Lewis rats with intraperitoneal injections of cyclophosphamide after the onset of clinical signs and observed reduced mortality and significant recovery in the treated group.
Cyclosporine, a fungal metabolite known to suppress DNA synthesis in 9
T-lymphocytes, was shown to Inhibit paralysis significantly in Lewis rats
when administered orally (Borel et al., 1976). In another study,
cyclosporine treatment of rats delayed the onset of EAE without preventing
overt disease (Cammisuli et al., 1984). Dunkln-Hartley guinea pigs
immunized with homologous spinal cord plus CFA, displayed suppressed EAE
clinical signs when given prophylactic and therapeutic treatment with
cyclosporine ( Bolton and Cuzner, 1980). Treatment was most effective,
however, when cyclosporine was administered beginning from the day of
sensitization.
Many studies have examined the suppressive effects of steroids on EAE,
since steroids are well known for their anti-inflammatory activity
(reviewed by Komarek and Dietrich, 1971). In two early approaches, the
development of EAE was assessed following the use of stress to increase endogenous steroid production (Levine et al., 1962a) and the use of adrenalectomy to reduce endogenous steroid levels (Levine et al., 1962b).
In the first approach, Sprague-Dawley rats were stressed by restraining
all four limbs prior to EAE induction. This manipulation resulted in decreased EAE clinical and histopathological signs (Levine et al., 1962a).
In the latter study, adrenalectomy of female Sprague-Dawley rats 2 days prior to EAE induction increased the severity of EAE clinical signs, an effect that was reversed by the daily intramuscular administration of cortisone (Levine et al., 1962b). Treatment of EAE with high doses of corticosteroids is generally suppressive when given early after sensitization, but has little curative effect (Komarek and Dietrich, 1971).
Other workers have reported suppression of EAE by treatment with melengestrol acetate and hydrocortisone acetate (Grieg et al., 1970). In 10 this study, both steroids were shown to prevent EAE development and also to reverse established disease in the Wistar rat.
Niridazole, an immunosuppressive drug used for the therapy of schistosomiasis in man, was tested for suppressive effects in EAE
(Paterson et ai., 1977). Daily doses of 25 to 100 mg of niridazole/kg of body weight markedly suppressed clinical signs and reduced histopathological changes in the treated Lewis rats. However, significant neurotoxic effects were observed in the rats given the highest dose (100 mg/kg).
In addition to the use of immunosuppressive drugs, irradiation treatment has been explored for its effects on EAE. Total body x-irradiation (400 rads), administered prior to CNS antigen plus adjuvant sensitization, was shown to decrease EAE in Wistar and Fisher 344 rats
(Paterson and Beisaw, 1963). In the same study, irradiating the rats 18 hours after sensitization produced no inhibitory effect.
It has been suggested that destruction of myelin proteins may be an early event in the development of CNS demyelination, and indeed, protease activity has been shown to be elevated in the vicinity of both MS and EAE
CNS lesions (reviewed by Smith, 1980). Therefore, protease inhibitors have been tested for their suppressive effects in EAE. Three inhibitors of plasminogen activator, trans-aminomethylcyclohexane carboxylic acid, epsllon-amino caproic acid, and p-nitrophenylguanidinobenzoate, were effective in reducing weight loss and incidence of paralysis in Lewis rats
(Smith, 1980 and Brosnan et al., 1979). The acid protease inhibitor, pepstatin, was shown to cause slightly reduced EAE clinical signs, while neutral protease inhibitors, leupeptin and antipain, were ineffective. 11
A number of studies have examined the inhibitory effects of specific antisera on the development of EAE. Waksman et al. (19 61) reported that injection of anti-lymphocyte serum (ALS) could inhibit EAE development in guinea pigs. Anti serum used in this study was raised in rabbits by immunization w ith guinea pig lymph nodes and adjuvant. Leibowitz and associates (1968a, 1968b) also reported the preventive and therapeutic value in guinea pigs of antiserum raised against thymocytes. In these studies, injection of the anti-thymocyte serum (ATS) on alternate days following sensitization resulted in complete inhibition of EAE.
Additionally, ATS treatment of guinea pigs, that were already displaying clinical signs, resulted in survival of some of the paralyzed animals, whereas all nontreated animals died. As mentioned previously, monoclonal antibodies have been used successfully to suppress EAE. Administration of monoclonal antibody directed against determinants on mouse (Waldor et al.,
1985) and rat (Brostoff and Mason, 1984) T-helper lymphocytes, were effective measures to inhibit EAE. Additionally, Steinman et al. (1983) reported that monoclonal antibody specific for a MHC l-A gene product of the rat, was able to prevent EAE and reduced the number of CNS antigen-reactive cells which Infiltrated into the brain and spinal cord.
A great deal of work in this area has focused on suppression of EAE using antigen-specific means (reviewed by Paterson, 1976a). Ferraro and
Cazzullo (1949) were the first to report that repeated injections of monkey brain homogenates emulsified with incomplete Freund's adjuvant (IFA) into guinea pigs resulted in a decreased incidence of EAE following subsequent encephalitogenic challenge. In another approach, neonatal injection of spinal cord homogenate was used to induce a state of immunological 12 tolerance to CNS tissue in outbred Wistar rats (Paterson, 1958). In this study, the rats were pretreated 1,9, 14, or 28 days after birth with guinea pig spinal cord In saline, then challenged with guinea pig spinal cord-CFA at 8 to 10 weeks of age. The most dramatic inhibitory effect was seen when animals were treated one day after birth, since only 30% of the rats subsequently developed EAE, as compared with 888 of untreated controls.
Generally, treatment of rats I or 9 days after birth resulted in protection, but, if the rats were treated at 14 or 28 days of age, there was no protection from the subsequent induction of EAE.
Purified MBP, emulsified in IFA, is not encephalitogenlc, but, when injected into animals, inhibits subsequent EAE induction by MBP-CFA challenge (Alvord, 1965; Cunningham and Field, 1965). Swanborg (1972) confirmed the suppressive effect of MBP-IFA emulsions in EAE, and extended these observations to include the use of a non-encephalitogenic modified MBP to suppress EAE in the guinea pig. In these experiments, MBP was modified by the addition of 2-hydroxy-5-nitrobenzyl bromide
(benzyl-MBP) to the tryptophan residue occupying position 118. This tryptophan residue is known to be critical for the encephalitogenlc activity of MBP in guinea pigs (Swanborg, 1973). Animals sensitized with whole
MBP-CFA, then given the benzyl-MBP in IFA, showed a decreased incidence of EAE. Intraderma! injection of benzyl-MBP plus IFA prior to encephalitogenlc challenge resulted in the complete prevention of EAE clinical signs, and only moderate histologic lesions. Swanborg later presented evidence to suggest, for the first time, that different sites on the MBP molecule could be responsible for disease induction and inhibition of EAE (Swanborg, 1973). In this study, guinea pigs were rendered 13
refractory to EAE by the prior injection of either guinea pig MBP or the
small component of rat myelin basic protein (which has a 40 amino acid
deletion including a region necessary for encephalitogenicity in the guinea
pig) in combination with IFA. Both treatments significantly inhibited the
subsequent Induction of EAE.
Intravenous injection of MBP has also been shown to exert suppressive
effects in Lewis rats with EAE (Levine et al., 1972). Intravenous
administration of MBP after the onset of clinical signs produced less
progressive disease and earlier regression, than in control rats treated
with saline or calf thymus histone. In a comparative study by Levine and
Sowinski (1984), intravenous administration of MBP was found to be more
suppressive of EAE than either intramuscular or subcutaneous injection, as
assessed by histological scores. Using a different approach, Lewis rats
were pretreated with MBP conjugated to syngeneic spleen cells (MBP-SPC)
or red blood cells (MBP-RBC), a procedure which was shown to suppress
subsequent EAE induction (McKenna et al., 1983). The Intraperitoneal or
Intravenous Injection of MBP-SPC or MBP-RBC did not change the day of
onset of EAE, but did significantly decrease clinical severity,
histopathologic changes, and in vitro proliferative responses to MBP.
Antibody responses to MBP were not affected. LNC derived from rats that
were pretreated with MBP-RBC, then challenged with MBP-CFA, produced much less severe EAE when transferred into naive recipients, in comparison
to control LNC derived from rats injected with MBP-CFA only. Suppression
of EAE, generated by the injection of MBP-coupled cells, may be
attributable to suppressor T cells, since the suppressive effect can be
transferred from MBP-RBC pretreated animals by LNC. (McKenna et al., 1984). Additionally, cyclophosphamide treatment, two days prior to the administration of MBP-coupled syngeneic spleen cells, abolished the suppressive effect on EAE.
Liposomes have been reported as an effective vehicle to deliver MBP treatment to animals with EAE (Strejan et al., 1981). MBP was entrapped in phosphatidyl-serine liposomes, and then injected via the intracardiac route into Hartley guinea pigs. Animals given 1 or 2 doses of MBP- liposomes, prior to challenge with MBP-CFA, showed significant protection from EAE development, comparable to the protective effect of MBP-IFA treatment.
Administration of adjuvant alone has been shown to be able to suppress
EAE in guinea pigs (Kies et al., 1958) and rats (Englert and Hempel, 1981).
In the latter study, Lewis rats were pretreated with CFA at varying times prior to challenge with an encephalitogenic dose of MBP-CFA. Pretreatment in this manner 14-37 days before challenge prevented development of any clinical signs of EAE. Additionally, LNC derived from CFA pretreated rats could transfer the protective effect. However, conclusions from these studies are difficult to draw due to the small number of animals tested in each group.
III. Recovery from Acute EAE
EAE induced in the Lewis rat by MBP-CFA sensitization, is an acute, monophasic disease with spontaneous recovery from clinical signs approximately 17-20 days post-sensitization. Convalescent rats are resistant to the reinduction of EAE. Much research has focused on this disease recovery and unresponsive state since an understanding of the underlying mechanisms involved might lead to some insight into the relapsing-remitting course observed in most MS patients. Evidence exists which argues against the clonal deletion of autoreactive cells during recovery from EAE. CNS antigen-reactive cells were demonstrated in convalescent rats by the ability of these lymphocytes to proliferate in vitro, in response to MBP (Willenborg, 1979). Additionally, suppressed
EAE-effector cells were recovered from convalescent rats and propagated in vitro as long-term cell lines capable of mediating EAE (Ben-nun and
Cohen, 1982). The latter data must, however, be interpreted in light of a recent report in which an MBP-reactive, encephalitogenic cell line was derived from normal Lewis rats (Schluesener and Wekerle, 1985). Current hypotheses concerning the mechanism of recovery from acute EAE include:
1) suppressor T cell activity 2) finite life-span of EAE-effector cells, and 3) generation of suppressor factors, possibly in the blood.
Suppressor lymphocytes have been implicated both in the recovery phase of EAE and In the acquired resistance to induction of EAE in MBP-IFA tolerized animals (Swierkosz and Swanborg, 1975, Welch, etal., 1980;
Killen and Swanborg, 1982). Adda et al. (1977) transferred 108 thymus, spleen, or non-draining LNC from Lewis rats that had been previously sensitized with MBP-CFA and Bordete/iapertussis, into recipient rats, that were given an encephalitogenic challenge. Spleen cells or thymocytes obtained 15 days after sensitization of the donors (at the height of clinical disease) and non-draining LNC obtained 17 days later (at the onset of recovery), suppressed EAE induction in the recipients. These same cell populations harvested on days 4 or 8 did not inhibit EAE induction in the cell recipients. In a report by Ben-nun and Cohen (1982), suppressor cell 16 activity was demonstrated in vitro on the antigen-specific proliferative responses of an liBP-reactive cell line (Zla line). Lymphoid cells were taken from rats 35 days post sensitization, after recovery from EAE clinical signs, and were used as accessory cells in a proliferative assay for the Zla cell line. In general, suppressor lymphocytes demonstrable both after giving a tolerizing regimen of MBP-IFA, or at the time of convalescence, have been shown to inhibit the denovo induction of EAE, but not the transfer of EAE with preformed effector cells (Willenborg, 1979 and Swierkosz and Swanborg, 1977).
EAE in the Lewis rat, passively induced by cellular transfer, also runs a self-lim iting clinical course, with subsequent recovery. Willenborg et al.
(1986) recently presented evidence against a role for suppressor cells in this recovery process. Lewis rats were sensitized with MBP-CFA, then, at the height of disease (the time at which suppressor cells would begin to appear), these animals received Con A-stimulated spleen cells obtained from MBP-CFA-sensitized rats. This transfer system was used to assess whether activated cells could alter the normal convalescence of Lewis rats at a time during which putative suppressor cells would be active. The transfer activity of these sensitized spleen cells was not suppressed upon injection into diseased animals, as evidenced by prolongation of the clinical course or secondary disease episodes in the recipients. Willenborg
(1986) offered speculation that the recovery from EAE is due to a finite life span of the EAE effector cells, since preformed effector cells were not suppressed.
Another possible mechanism operative in the recovery from EAE is the activity of a serum suppressor factor. Attempts to lim it disease induction 17
or severity in a naive animal by the administration of serum, derived from
an animal recovered from EAE, have been somewhat successful. Paterson
and Harwin (1963) demonstrated the protective effect of EAE immune
serum on disease development in the Wistar rat. Donor animals were
injected with guinea pig spinal cord and adjuvant, then bled 1 to 6 weeks
later, to obtain sera before and after the development of clinical EAE.
These sera were then administered intravenously to spinal cord-adjuvant-
sensitized rats on the day of sensitization and on alternate days thereafter.
Serum obtained at the time of convalescence inhibited EAE Induction in
recipients, whereas serum obtained from donors prior to the development
of clinical signs had no effect. This protection was suggested to be due to
the production of serum complement-fixing (CF) antibrain antibody. Nakao
and Roboz-Einstein (1965) demonstrated similar suppressive effects of
convalescent serum, but were unable to correlate this protective effect
with CF antibody titers. This lack of correlation between protection from
EAE and CF anti-brain antibody was confirmed by Hughes (1974) using
immune serum from guinea pig spinal cord-adjuvant-sensitized Wistar rats
to lim it EAE development in the inbred AS rat. Hughes (1974) found that
hemagglutinating antibody to guinea pig MBP was not present in
suppressive serum, and protection did not correlate with CF antibodies to
galactocerebroside, the major antigen against which antibrain antibodies are directed (Rapport et al., 1967). Convalescent serum was also reported
to suppress in vitro mitogenic responses of CNS antigen-primed
lymphocytes (Vandenbark et al., 1974). This serum was shown to have
increased levels of alpha globulin, and it was suggested by the authors that protein associated with the alpha globulin fraction could be a factor in this 18 suppressive effect. Additionally, elevated corticosteroids in convalescent serum have been implicated as a factor in the recovery from EAE, as suggested by Levine and Sowinski (1980).
There is as yet no convincing evidence that any one or a combination of the factors mentioned above actually are responsible for recovery from
EAE. My efforts have been directed toward the investigation of another possible inhibitory substance which may contribute to recovery from EAE in the Lewis rat, namely, a class of glycolipfds called gangliosides.
IV. Oangliosldes
Gangliosides are a group of complex acidic glycosphingolipids found predominantly in the outer leaflet of cell plasma membranes, where they may influence surface properties and cellular Interactions (Reviewed by
Ledeen, 1983). The first extensive purification and study of gangliosides was performed in the 1930‘s and early 1940’s by Klenk (1935 and 1942), who also proposed the name ‘‘ganglioside" to indicate their concentration in the ganglienzellen (neurons). Early advances in the identification of gangliosides occurred in the study of brain tissue, where gangliosides are found in greatest abundance. Yamakawa and Suzuki (1951) first identified an extraneural ganglioside, GM3, in erythrocyte membranes. Work progressed in the 1960‘s on the biochemistry, metabolism, and the cellular and subcellular distribution of gangliosides. Contemporary studies have concentrated on attempts to define this class of glycolipid functionally.
Chemically, gangliosides are composed of both hydrophobic and hydrophilic moieties. The hydrophobic portion called ceramide, contains a long-chain base (sphingosine), linked through an amide bond to a fatty acid. 19
Attached to the 1 -hydroxyl group of sphingosine are one or more neutral carbohydrates, primarily glucose, galactose, and their derivatives.
Fucose-containing gangliosides have also been identified (Ghidoni et al.,
1976). The occurrence of one or more residues of sialic acid, attached to galactose or another sialic acid residue, distinguishes gangliosides from other glycosphingoltpids. Sialic acid is the common name given to derivatives of the 9-carbon amino sugar, neuraminic acid. The n-acetylated form of sialic acid predominates in mammalian CNS, and n-glycolyl- neuraminic acid has been found commonly in extraneurai tissue (Nagai and
Iwamori, 1980). The presence of sialic acids gives gangliosides a net negative charge at physiological pH. Structurally, the gangliosides are very diverse and this variation arises from several factors: the numbers, types, and arrangements of the carbohydrates, the presence of fucose, the numbers and linkages of sialic acid residues, and the substitution of glycolyl for acetyl groups on neuraminic acid. Variation also occurs in the length of the fatty acid moieties and in the type of long-chain base in the ceramide portion of the molecule. The nomenclature commonly used is that proposed by Svennerholm (1963), but the identification of more complex glycolipid structures has prompted the use of a more chemically descriptive nomenclature proposed by a commission of the International
Union of Pure and Applied Chemistry (IUPAC-IUB commission, 1978).
Although gangliosides have been identified in almost all tissues tested, they are found in highest concentration in the brain, predominantly in the gray matter where concentrations of 2800 - 3500 nanomoles of lipid-bound sialic acid per gram of fresh tissue have been reported (Ledeen and Yu,
1982). CNS myelin has a ganglioside content and pattern comparable to 20
that found In the myelin-producing oligodendrogliai cell, with a predominance of GMi (Suzuki et al., 1967). The presence of ganglioside GM4 has been reported to be specific for myelin (Ledeen et al., 1973).
y-:J M 9 i . i P5,i,de5,in EAE and m s In light of the variation in EAE histopathological changes observed (e.g. the extent of demyelination) depending upon whether sensitization is performed with MBP-CFA or spinal cord-brain homogenate plus CFA, studies have been conducted to examine the influence of other CNS components on the encephalitogenicity of MBP. In one study, Hartley strain guinea pigs were immunized with either purified myelin, MBP, or MBP complexed with acidic Iipids, in combination with CFA (Koh et al., 1981). The MBP-acidic lipid complex consisted of sulfatide, phosphatidyl serine, gangliosides, and
MBP in a molar ratio of approximately 6:3:1:1 respectively. This
MBP-acidic lipid inoculum Induced only mild EAE in the guinea pigs tested, with delayed onset of disease and lower mortality, when compared with
MBP-CFA sensitization. Brinkman et al., (1983) found that challenging guinea pigs with a mixture of MBP and gangliosides or cerebrosides plus
CFA, prevented the development of EAE, yet the animals developed an intense delayed-type hypersensitivity (DTH) skin reaction to MBP.
Similarly, guinea pigs inoculated with a mixture of MBP, CFA, and the myelin-specific ganglioside GM4. had less severe EAE clinical and histopathological signs as compared with controls given MBP-CFA (Mullin et al., 1984). Collectively, these data suggest that gangliosides, and possibly other myelin lipids, can alter the encephalitogenic potential of
MBP, an idea which leads to speculation as to a possible role of 21 gangliosides in altering the encephalitogenicity of MBP in the normal myelin membrane.
Immune responses to gangliosides have been measured In animals with
EAE. Anti-ganglioside antibody was detected by the ELISA method in guinea pigs with chronic relapsing EAE (Tabira and Endoh, 1985). Recently, DTH responses to gangliosides were measured in Lewis rats with EAE (Offner et al., 1985). In this study, sensitization with guinea pig spinal cord homogenate plus CFA elicited significant ear swelling responses following the intradermal injection of ganglioside-mBSA complexes. Neither gangliosides alone, nor mBSA alone induced the DTH response. This DTH reaction to gangliosides correlated with the onset of EAE clinical signs, but was not elicited in rats sensitized to MBP-CFA. In accord with classical DTH reactions, spleen cells from spinal cord-adjuvant-sensitized rats adoptively transferred specific DTH responses to recipient rats.
Recently, a mixture of bovine brain gangliosides or purified individual gangliosides was shown to inhibit the phenotypic and functional properties of an MBP-reactive encephalitogenic Lewis rat T cell line (Offner and
Vandenbark, 1985). Exogenous gangliosides, when incubated with the T line cells for 1 hour prior to intravenous injection, were shown to impair the ability of these cells to transfer EAE to recipient rats.
In contrast to the report of ganglioside suppression of EAE, Cohen et al.
(1981) have reported that immunization of outbred rabbits with gangliosides, conjugated noncovalentiy with mBSA, plus CFA resulted in clinical signs and neurological lesions similar to those found in MS and EAE.
Approximately 50% of the inoculated rabbits became paralyzed. The CNS white matter of these animals showed plaques of demyelination as well as 22 perivascular mononuclear cell Infiltration.
Ganglioside abnormalities have also been studied in patients with MS.
Examination of MS plaques revealed decreased ganglioside levels, relative to normal appearing white matter, and a complete absence of GM* ganglioside, as demonstrated by gas chromatography and TLC analyses (Yu et al., 1974). In another study, pooled sera from 20 MS patients were compared with pooled sera from healthy donors for their respective levels of gangliosides (Sela et al., 1982). MS sera showed a 34% increase in ganglioside sialic acid over controls. However, the TLC patterns of MS and control serum gangliosides were not different. Peripheral blood lymphocyte-associated gangliosides were also compared yielding similar results (i.e. MS patients' lymphocytes had 39% higher ganglioside levels).
In searching for clues to the etiology of MS, many studies have focused on the specificity of antibody elicited in MS patients. Sera from MS patients were shown to react with GMi ganglioside incorporated into liposomes, but sera from some healthy controls also reacted with the
GMi-liposomes (Mullin et al., 1980). This reactivity was measured by the release of HC-glucose from multilamellar liposomes containing GMi in the lipid bilayer, and was shown not to involve complement activity. This test was shown, by Endo et al. (1984), to be more sensitive than the ELISA in detecting antibody to these glycolipids. in this study, antibody to GMi or asialo GMi was found in the serum of over 50% of MS patients and patients with systemic lupus erythematosus. Sera from healthy controls did not react significantly with the GMi-liposomes.
A number of studies have examined cellular reactivity to gangliosides in
MS patients (reviewed by Baumhefner and Tourtellotte, 1985). One 23 technique that has been employed to assess this cellular response to gangliosides is the active E-rosette assay (AER), which is believed to be an in vitro correlate to DTH in vivo (Felsburg and Edelman, 1977). This test detects the presence of high affinity receptors for sheep red blood cells on sensitized T-lymphocytes after re-exposure of the T-cells to specific antigen. T-lymphocytes from 94% of MS patients tested showed a positive
AER to ganglioside (Offner and Konat, 1980). This response was later shown to be directed against the highly sialylated gangliosides GTi and
GQib (Offner et al., 1981). Significant increases in MBP or ganglfoside- induced AER were demonstrated in MS patients with progressive disease or those patients in acute relapse. However, patients with MS in remission showed no such sensitivity (Ilyas and Davison, 1983). This cellular response was shown to be more MS-specific using gangliosides as specific antigen rather than with MBP, since patients with other neurological diseases showed sensitivity to MBP as well.
VI. MnfllJfistde Function In spite of many years of investigation, little is known about ganglioside biological function. The primary areas of investigation include:
1) the role of gangliosides in the normal function of the nervous system
2) the role of gangliosides in cellular differentiation, neurite formation, and nerve regeneration 3) the role of gangliosides as markers for cellular transformation 4) the role of gangliosides as cellular receptors for various biologically active agents, and 5) ganglioside immunogenfcity and immunomodulatory properties (reviewed by Ledeen, 1983 and Marcus,
1984). 24
Within the CNS, gangliosides are distributed throughout neuronal membranes particularly at synaptic terminals and may function in synaptic transmission (Ledeen, 1978). It has been suggested that membrane gangliosides could affect signal conduction through interaction with calcium ions (Rahmann, 1976). Calcium ion flux is known to be important in maintaining membrane potential in general, and in neurons, calcium influx plays a role in the release of neurotransmitters and in the activity of many membrane enzymes (Hille, 1981). The formation of calcium- ganglioside complexes has been reported (Behr and Lehn, 1973). In another study, exogenous polysialogangliosides were shown to promote the release of dopamine from synaptosomes in the presence of calcium (Cumar et al.,
1978). Leon et al. (1981) reported that the addition of gangliosides to cultures of rat brain neuronal membranes produced enhancement of
(Na*,K+)ATPase activity. Gangliosides have also been shown to affect the activity of adenylate cyclase and phosphodiesterase, the enzymes Involved in the formation and degradation of cyclic AMP (Daly, 19 8 1). In this study, brain gangliosides caused a marked activation of adenylate cyclase and stimulated the activity of both a calcium-dependent and a calcium- independent phosphodiesterase in rat cerebral cortex membrane preparations.
Gangliosides have been studied for their potential role in cellular differentiation, as suggested by the observation of membrane ganglioside changes accompanying cell differentiation, and the evidence of ganglioside-induced differentiation (Ledeen, 1983). For example, exposure of cultured Hela cells to butyrate, a differentiation inducing agent, produced morphological changes, the extension of cell processes, and a 3 to 25
5-fold Increase In GM3 ganglioside (Simmons et al., 1975). A number of studies have shown that exogenously added gangliosides Induce sprouting of neurltes In neuromuscular preparations and cultured neuroblastoma cells
(Gorloetal., 1981; Roisen, 1981; Faccl et al., 1984). In the latter study,
GMi ganglioside promoted neurltogenesls upon addition to cultures of the mouse neuroblastoma cell line, neuro-2a (Faccl et al., 1984). Ganglioslde-
Induced differentiation has been shown to be accompanied by increased cellular cyclic AMP (Dimpfel et al., 1981), elongation of the G1 phase of the cell cycle, and decreased [3H]-thymidlne Incorporation (Leon et al., 1982), in murine neuroblastoma cells. The effects of brain gangliosides in the promotion of neurite outgrowth in vitro and the stimulation of nerve regeneration (Caccia et al., 1979) have led to the use of gangliosides in clinical trials to treat human nerve disorders. In one such study, insulin-dependent diabetics, who displayed abnormal nerve conduction, were treated daily with 20 mg of a mixture of gangliosides (Pozza et al.,
1981). Significant improvement was observed in measurements of nerve action potential and conduction velocity of the ganglioside-treated patients. Brain gangliosides have also been used to treat patients with amyotrophic lateral sclerosis (Harrington et al., 1984). In a 6-month double-blind study, daily intramuscular injection of 40 mg of a mixture of gangliosides produced no significant beneficial effects.
In the area of cancer research, aberrant expression of both gangliosides and neutral glycolipids have been reported to occur in several neoplastic conditions (reviewed by Hakomort and Kannagi, 1983). For example, some melanomas have been shown to produce large amounts of the gangliosides
GM2, GMs, GD2, and GD3 (Puke! et al., 1982, Tai et al., 1985). A monoclonal 26 antibody directed against GDa ganglioside was recently used in the treatment of patients with malignant melanoma, resulting in tumor regression in one-fourth of the patients (Houghton et al., 1985).
Ganglioside GD 2 levels have been shown to be elevated in neuroblastoma patients as demonstrated by biochemical analysis (Yates et al., 1979) and by the use of monoclonal antibody directed against this ganglioside (Schulz et al., 1984).
Gangliosides have been studied as possible cellular receptors for bacterial toxins, glycoprotein hormones, Sendai virus, and lymphoktnes.
There is strong evidence that GMi ganglioside serves as the receptor for cholera toxin (Cuatrecasas, 1973; Ledeen, 1983). A high affinity association exists between the receptor-binding (B) subunit of the toxin and GMi. Addition of exogenous GMi was shown to restore the response to choleragen in a mouse fibroblast cell line which had previously been rendered unresponsive by neuraminadase treatment (Moss et al., 1976).
Gangliosides and tetanus toxin have also been reported to interact, although with a much lower affinity than the interaction observed between GMi and cholera toxin (Ledeen, 1983). It has also been suggested that thyroid-stimulating hormone (TSH) binds to target cells via a ganglioside receptor (Mullin et al., 1976), although this contention is in question
(Beckner et al., 1981). Some data suggest that gangliosides may serve as cell surface receptors for Sendai virus. Plastic-adsorbed gangliosides were found to bind the virus in one study (Holmgren et al., 1980), and, in another report, the incubation of Sendai virus-resistant bovine kidney cells with specific gangliosides restored their susceptibility to infection by this virus (Markwell et al., 1981). In addition to toxin, hormone, and virus 27 interactions, affinity binding and inhibition studies have suggested that gangliosides may act as cell surface receptors for the lymphokines macrophage migration inhibitory factor (MIF) (Poste et al., 1979; Liu et al.,
1980), macrophage activation factor (MAF) (Poste et al., 1979), interferon
(Besancon and Ankel, 1974), and T-cell growth factor (Merritt et al., 1984;
Parker et al., 1984). In particular, there have been recent reports examining the interaction between gangliosides and T-cell growth factor
(IL 2), which w ill be discussed in greater detail below.
Gangliosides in general are weakly immunogenic. Polyclonal antibody directed against gangliosides can be raised in rabbits by coupling these glycolipids to protein carriers such as human serum albumin or methylated bovine serum albumin (Kundu et al., 1980), incorporation of the gangliosides into liposomes (Nagai and Ohsawa, 1974), or by immunization with whole cells (Tai, et al., 1985). Monoclonal anti-ganglioside antibody has been produced using the latter approach and has been used in cancer research and therapy ( Pukel et al., 1982; Tai et al., 1984; Schulz et al.,
1984; Houghton et al., 1985). In the latter study, an lgG3 mouse monoclonal antibody, which recognizes GD3, was administered intravenously to 12 patients with metastatic melanoma. Partial tumor regression was observed in 3 of the 12 patients.
A large amount of evidence has accumulated regarding the role of cell surface gangliosides in the functioning of the immune system. Nagai and
Iwamorl (1980) have reported great molecular diversity and species specificity for gangliosides in the thymus. Analysis of purified gangliosides from calf, rabbit, human, and rat thymus revealed a large amount of n-glycolylneuraminic acid in each and distinctly different 28
thin-layer chromatography (TLC) patterns when comparing between species.
Gangliosides have been shown to modulate various Immmunologlcal responses in vitro (Reviewed by Marcus, 1984). In a series of reports,
Esselman and Miller examined the effects of gangliosides on B lymphocyte responses (Miller and Esselman, 1975; Esselman and Miller, 1977; and
Miller et al., 1982). Ganglioside GMi, incorporated into cholesterol- leclthin liposomes, was found to reduce anti-sheep red blood cell hemolytic plaque-forming responses (SRBC-PFC) in vitnq an effect that was inhibited by absorbing GMi-liposomes with antibody directed against Thy-1.2 antigen
(Miller and Esselman, 1975). These authors conducted additional experiments in which conditioned media, from cultures of helper or suppressor T-cells, when added to SRBC-PFC assays, either enhanced or suppressed the responses respectively (Esselman and Miller, 1977).
Pretreatment of the T-suppressor cell medium (Ts-M) with anti-Thy-1.2 or anti-GMt sera abrogated its inhibitory effect, indicating that the suppressor activity contained determinants in common with Thy-1.2 and
GMi ganglioside. Suppressor activity was isolated from the Ts-M which migrated on TLC similarly to GMi ganglioside. In other experiments, Miller et al. (1982) examined the effect of gangliosides on the induction of tolerance in B cells. Disialogangliosides were shown to abrogate the induction of tolerance to dlnitrophenyl coupled to carrier protein in splenic fragment cultures. The authors concluded from this series of studies that antigen-stimulated T-cells could release membrane components including the Thy-1 antigen and gangliosides, which inhibit differentiation of B cells into antibody-producing cells, and that gangliosides can prevent the 29
Induction of tolerance in immature B cells.
Several laboratories have reported ganglioside-mediated suppression of lymphocyte proliferative responses in vitro (reviewed by Marcus, 1984).
Lengle et al. (1979) reported that exogenous gangliosides inhibited tritiated thymidine incorporation by mouse thymocytes cultured with
Con A. Inhibition was not due to cytotoxic effects of gangliosides and could be reversed after 4 hours by washing out unbound gangliosides.
Whisler and Yates (I9 6 0 ) reported similar suppressive activity when brain gangliosides were added to cultures of human peripheral blood mononuclear cells (PBMC). In these experiments, exogenous gangliosides suppressed the proliferative responses of PBMC to Con A and allogeneic cells. Suppression was enhanced when gangliosides were first incorporated into cholesterol- lecithin liposomes and then cultured with PBMC. The ganglioside suppressive effect was reversible if the cells were preincubated with gangliosides for 18 hours, then washed and stimulated, but not when cells were pretreated with gangliosides for 72 hours. Yates et al. (1980) reported that GM2, GDib, and GTib gangliosides showed the greatest inhibitory effects. Similarly, Lengle et al. (1979) had found that GTi and
GM2 were more suppressive than other gangliosides tested.
In terms of the site of action for gangliosides, Ladisch et al. (1984) reported that gangliosides acted principally upon the adherent accessory cell subpopulation within human PBMC. In this study, either plastic adherent (>95% esterase positive) or nonadherent cell subpopulations, were preincubated separately with exogenous gangliosides for 72 hours.
The cells were then washed, recombined, and stimulated in vitro with streptoktnase-streptodomase. Ganglioside pretreatment of the adherent 30
cells resulted In reduced proliferative responses, an effect not seen with
ganglioside preincubation of nonadherent cells.
Homogeneous populations of T-lymphocytes can also be regulated by
gangliosides as demonstrated in a recent report by Offner and Vandenbark
(1985), describing the effects of exogenous gangliosides on an
EAE-inducing MBP-reactive T-lymphocyte line. These investigators found
that bovine brain gangliosides suppressed the MBP-induced proliferation,
the IL 2-dependent expansion, and the transfer capacity of this T-cell line.
Exogenous gangliosides also preferentially inhibited the fluorescent staining of the T-helper cell epitope (W3/25), while not similarly affecting the staining of cells with monoclonal antibodies W3/13 (total T cells),
OX-8 (T-non-helper), or OX-6 (la antigen).
Ganglioside immunosuppression has also been examined in relationship to cancer. Ladisch et al. (1983) and Gonwa et al. (1984) have reported that both murine and human tumor cells shed gangliosides that can subsequently inhibit lymphocyte proliferative responses in vitro. Ladisch et al. (1983) quantitated the gangliosides shed by the murine lymphoma cell line, Yac-I,
in vitro and in the ascites fluid in vivo. Gangliosides, isolated from Yac-1 cells, caused a 90% reduction in [3H]-thymidine incorporation by Con A- stimulated mouse splenocytes. These findings, along with the increased serum ganglioside levels found in some cancer patients (Katopodis et al.,
1982 and Lo et al., 1980), have prompted the suggestion that released tumor cell gangliosides may adversely affect Immune surveillance thus protecting tumor cells from destruction, and possibly contributing to the general immunosuppression seen in some cancer patients
(Lengle et al., 1979). 31
VII. Gangliosides and IL2
Evidence has recently emerged to suggest that the ganglioside-medlated suppression of lymphocyte proliferative responses may involve interference with interleukin 2 (IL 2)-dependent processes (Merritt et al.,
1984; Parker et al., 1984; and Robb, 1986). IL 2, originally termed T-cell growth factor (TCGF) because of its ability to maintain long-term cultures of T-lymphocytes, is a glycoprotein molecule produced by activated
T-lymphocytes (reviewed by Smith, 1980 and Robb, 1984). IL 2 derived from human, rat, and mouse is markedly hydrophobic and appears to be quite similar in size (approximately 15,000 daltons by SDS-PAGE), although isoelectric points vary (6.5 for human, 5.5 for rat, and 4.3 -4.9 for mouse)
(Smith, 1980). Charge heterogeneity has also been demonstrated within human IL 2, which is most likely attributable to variation in post- translational glycosylation, particularly involving sialic acid residues
(Robb and Smith, 1981).
Current Immunological dogma states that IL 2 is secreted from antigen or mitogen-activated T-lymphocytes in response to two signals (Smith,
1980). First, T-cells must recognize antigen in the context of a class II
MHC product on the surface of an antigen-presenting accessory cell, such as the macrophage or dendritic cell. The second signal is a soluble macrophage-derived product, interleukin I (IL I). Once secreted, IL 2 acts only on cells that have been previously activated by antigen or mitogen and are thus displaying specific receptors for IL 2. Therefore, the specificity of the lymphocyte response to a particular antigen is controlled at the level of antigen presentation, resulting in the expression of IL 2 receptors on the appropriate antigen-specific clones, and is followed by the 32 nonspecific amplification and clonal expansion induced by IL 2 (Robb,
1984).
Part of the mode of action of IL 2 is through its binding to cell-surface receptors, much like the action of hormones and other growth factors
(Robb, 1984). It has been shown that resting cells have few if any IL 2 receptors (Robb et al., 1981). The events that occur following the binding of IL 2 to its receptor are not yet known, but the net effect is to cause progression from the G1 phase into the 5 phase of the cell cycle (Klaus and
Hawrylowicz, 1984).
In EAE, IL 2 was shown to be required to activate cells in vitro for adoptive transfer of disease (Ortiz-Ortiz and Weigle, 1982). A previous report had shown that Con A-activation of spleen cells but not LNC in vitro could potentiate the transfer of EAE (Panitch and McFarlin, 1977).
Ortiz-Ortiz and Weigle (1982) demonstrated that optimal stimulation of
LNC, thus enhancing IL 2 production, could also result in enhanced EAE transfer. Additionally, supernatants from cultures of Con A-activated normal spleen cells (containing IL 2) or purified exogenous IL 2, could replace Con A in the in vitro activation of both spleen and LNC to transfer
EAE.
Recently, gangliosides have been reported to interfere with IL 2-induced proliferative responses (Parker et al., 1984; Merritt et al., 1984a; and
Robb, 1986). Parker et al. (1984) reported that preincubation of IL 2 with gangliosides, covalently attached to poly-l-lysine-agarose beads, resulted in decreased IL 2 activity as measured by the proliferation of an IL 2- dependent cloned T-cell line, AKIL-1.E8. The more complex gangliosides,
GT ib and GDib, were the most effective of those tested. The direct binding 33 of gangliosides to IL 2 was examined by passing IL 2-containing solutions over a column of ganglioside-polylysine-agarose beads. Virtually all of the
IL 2 activity was retained on the column, whereas, 738 of the IL 2 activity did not bind to a control sodium acetate column.
Merritt et al., (1984a) reported that exogenous bovine brain gangliosides suppressed the IL 2-induced proliferation of the murine IL 2-dependent cell line, CT-6. In accord with previous reports, the most suppressive gangliosides tested were GM 2, GDia, and GTib. The suppressive effect of gangliosides was shown to be reversible for up to 24 hours by washing the
CT-6 cells free of unbound ganglioside and then restimulating the CT-6 cells with IL 2. Merritt et al. (1984b) have also reported that exogenous gangliosides could restore IL 2 responsiveness to the CT-6 murine cell line at a time during which the cells were unresponsive to IL 2. Based on these observations, the authors suggested that exogenous gangliosides may function as IL 2 receptors.
In a very recent study by Robb (1986), gangliosides GMi and GT ib were shown to suppress the PHA-induced stimulation of human PBMC, the IL 2- dependent proliferation of 5-day PHA-stimulated human PBMC blasts, and the IL 2-induced expansion of the murine IL 2-dependent cell line HT-2.
The data presented showed a dichotomy of ganglioside suppression. The inhibition of IL 2-dependent growth could be reversed by the addition of excess IL 2, but ganglioside-mediated suppression of PHA-stimulated fresh
PBMC was not reversible by excess IL 2. While gangliosides suppressed the
PHA-induced proliferation of PBMC, IL 2 production by these cells was not affected. However, fluorescence analysis did show a 358 reduction in the expression of IL 2 receptors on these cells. Whether or not such a 34 reduction in receptor staining has any physiologic relevance is unknown. In the same study, gangliosides were shown to associate directly with IL 2 by the following results: 1) The inhibitory effect of GT ib on the association of IL 2 with its receptor was greatest when the ganglioside and IL 2 were preincubated together before measurement of IL 2-receptor binding 2)
Preincubation of gangliosides with [3H)-IL 2 for 30 minutes reduced the subsequent binding of radio-labeled IL 2 to monoclonal anti-IL 2 antibodies.
The ability of the Lewis rat to recover spontaneously from acute EAE may be relevant to understanding the exacerbating and remitting clinical course in most MS patients. The purpose of the present study was to examine whether gangliosides, known to be potent suppressors of lymphoid cell activation, might play a role in the recovery from EAE by down-regulating EAE-effector cells. The first portion of this study addressed the questions:
1) Do gangliosides suppress the in vitro proliferation of lymph node cells
(LNC) from rats sensitized with MBP-CFA? 2) Linder what conditions is this suppression optimal? 3) Do gangliosides inhibit the LNC transfer of
EAE? 4) Are serum ganglioside levels elevated in rats with EAE?
In the second portion of this study, I investigated whether the ganglioside-mediated suppressive effects on the proliferative responses of encephalitogenic cells involved some effect on IL 2 activity. Specifically:
1) Do gangliosides affect the antigen or mitogen-stimulated production of
IL 2? 2) Do gangliosides interfere with the IL 2-induced proliferation of an !L 2-dependent cell line which continuously expresses 1L 2 receptors? 3)
Do gangliosides alter the expression of mitogen-induced IL 2 receptors on the cell surface? and A) Will excess IL 2 abrogate the suppressive effect of gangliosides on proliferative responses? MATERIALS AND METHODS
Animals Male Lewis rats, 175-300 grams (Harlan Sprague Dawley Inc.,
Indianapolis, IN), were used for all experiments. They were provided with food and water ad libitum and hand-watered during paralytic episodes. Antioens.and Mitogens Phytohemagglutinin (PHA) and Concanavalin A (Con A) were purchased from Miles Scientific Co., Naperville, II. The purified protein derivative
(PPD) of Mycobacteria was obtained from Parke-Davis and Co., Rochester,
Ml. Guinea pig spinal cords were purchased frozen from Rockland Inc.,
Gilbertsville, PA.
Preparation of Myelin Basic Protein
Myelin basic protein (MBP) was prepared from whole CNS tissue by the method of Swanborg et al. (1974). Guinea pig spinal cords were homogenized in a Waring blendor using chloroform-methanol (CM 2:1, v/v).
The residue was re-extracted with CM 2:1, washed with distilled water, and acid extracted using 0.01 N HC1. The acid extract was precipitated using 50% saturated ammonium sulfate, then resuspended in 0.01 N HC1, dialyzed against deionized water and then lyophilized. The MBP preparation was further purified by passage over a Sephadex G-50 superfine column
(Pharmacia, Uppsala, Sweden) in 0.01 N HC1. The individual protein- containing fractions were monitored by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE), using a 15% running
36 37 gel, to test for the presence of the major MBP band (18,300 dal tons). Based on the gel analysis, appropriate fractions were pooled, dialyzed against water and lyophilized to obtain column-purified MBP. Ganglloslde Purification Gangliosides were extracted from normal rat brains by the method of
Suzuki (1965). Three gram samples of brain were homogenized in 20 volumes CM 2:1 and vacuum filtered through sclntered glass funnels. This procedure was repeated once with CM 2:1 and again with CM 1:2 containing
5% water (CMW 1:2:5%). The combined filtrates were dried on a rotary evaporator and resuspended in CM 2:1. Gangliosides were partitioned into the upper aqueous phase with the addition of 0. IM KC1. Following a 10 minute centrifugation at 150 x g, the upper phase was removed and the remaining lower phase was partitioned once with theoretical upper phase
(TUP), CMW containing 0. IM KC1 (3:48:47), and once more using TUP without
KC1. All upper phases were combined, taken to dryness, then treated with
0.6N NaOH in methanol for one hour at room temperature. After neutralization with concentrated HCI, gangliosides were again dried completely, resuspended in a small volume of distilled water, and dialyzed against 3 changes of distilled water. Following dialysis, the ganglioslde mixture was taken to dryness and resuspended in CMW 5:5:1, acidified with
40 pi of 6N HCI, and then applied to a 16 x 686 mm column of Sephadex
LH-20 (Pharmacia Fine Chemicals, Piscataway, N.J.) to remove peptide contaminants. Gangliosides were then eluted from the column with
40-50 ml of CMW 5:5:1 as described by Byrne et al. (1985). Final purification was by column chromatography on a one gram silicic acid column (Unisil, Clarkson Chemical Co., Williamsport, PA) which was eluted 38 successively with CM 4 1 , CM I ; I, and methanol. The latter two ganglioside-containing fractions were pooled, dried completely, resuspended in CM 2:1 and stored at -20 *C.
Ganglioside sialic acid was quantitated by the resorcinol method of
Svennerholm (1957) as modified by Miettinen and Takki-Luikkainen (1959).
Samples to be tested were dried down in duplicate in glass tubes. One m illilite r each of 5N HCI and the resorcinol-HCl reagent (prepared with
0.2% resorcinol in 5N HCI with 0.25 mM CuS04) were added to each tube, and the tubes were stoppered, mixed by vortexing, and placed in a boiling water bath for 15 minutes. The tubes were then placed in ice water to cool. Each sample then received I ml butanol-butyl acetate (15.85, v/v) and was mixed. The upper alcohol phase was removed, and the absorbance determined at 470 nm, 580 nm, and 620 nm for each sample. The sialic acid-resorcinol complex has a maximum absorbance at 580 nm. The 470 nm reading was used with the 580 nm reading in the following formula to generate a correction factor, which adjusted for other sugars which absorbed at 580 nm.
(1) Correction Factor (CF) * 1.073 - [ Absorbance at 470nm x 0.265 ] ( Absorbance at 580nm ]
The 620 nm reading is used in the event that CF is less than 0.700.
Purity was assessed by thin-layer chromatography (TLC) on 10 x 10 cm silica gel precoated high performance TLC glass plates (E. Merck,
Darmstadt, FR6) and developed in CMW 55:45:10 containing 0.2% CaCl 2 or in
CMW 65:25:4 to test for phospholipid or neutral glycolipid contaminants, which were visualized with molybdenum blue and diphenylamine sprays 39 respectively (Dlttmer and Lester, 1964; Harris and MacWi 11 lam, 1954).
Gangliosides were visualized with resorcinol-HC! spray (Svennerholm,
1957). A typical rat brain ganglioside preparation contained approximately
14% GMi, 13% GD3, 16 %GDi«, 18%GD2, 12%GDiband 10%GT as the major gangliosides present, along with some minor species, as determined by TLC and scanning densitometry at 580 nm, using a Shimadzu model CS-910
Dual- Wavelength TLC Scanner (Shimadzu, Columbia, MD) and a Hewlett
Packard 3385A Automation System Integrator (Hewlett Packard,
Naperville, IL). Plate I shows a typical TLC pattern for purified rat brain gangliosides. A mixture of bovine brain gangliosides as well as individual purified bovine gangliosides GMi, GDu, GDtb, and GTib were kindly provided by Dr. Silvana Lorenzi of Fidia Research Laboratories, Abano Terme, Italy.
Bovine sulfatides and asialo-GMi were purchased from Supelco (Bellefonte,
PA). All organic solvents used were of reagent grade and redistilled in glass prior to use.
For addition to culture, gangliosides were dried under nitrogen, then resuspended in HBSS with sonication and vortex mixing. Ganglioside suspensions were then filter sterilized through 0.2 pm syringe-top filters
(Gelman Sciences, Inc., Ann Arbor, Michigan).
Serum Ganglioside Extractions
Serum was obtained at the time of sacrifice from either normal Lewis rats or rats sensitized with MBP-CFA. Gangliosides were extracted from 2 or 3 ml of serum by the addition of CM 1:1, then the samples were centrifuged to pellet the insoluble material. The pellet was re-extracted with CM 1:2 containing 5% water. Combined extracts were dried, Plate I. TLC pattern of Purified Rat Brain Gangliosides
GM2 GMI « r \ T <■»*• n m » * »
GDla GD2 GDIb GTIb GQ
1 2 3 4 5 6
1. Standards GM 2, GMi, GDia
2. Normal human cerebral cortex gangliosides
3. Rat brain gangliosides, preparation *1
4. Rat brain gangliosides, preparation * 2
5. Rat brain gangliosides, preparation *3
6. GMi ganglioside 41 resuspended In CM 2:1 and partitioned Into 2 phases with the addition of
0.1 M KCI which contained 12.5 mM EDTA The EDTA was added to prevent the calcium-dependent partitioning of gangliosides into the lower chloroform- rich phase (Lo et al., 1980). The upper aqueous phase containing gangliosides was removed, and the lower phase was partitioned twice more with theoretical upper phase (CMW 3:48:47). All upper phases were combined, taken to dryness, and then desalted either by dialysis against distilled water, or by the use of Cie bonded phase columns
(Analytichem International, Harbor City, California). Gangliosides were added to the columns in 0 .1 M KCI solution, the columns were desalted under vacuum with distilled water, then the gangliosides were eluted with
CM 1:1. Ganglioside sialic acid was then quantitated using the resorcinol assay as described earlier.
EAE Induction
Lewis rats were sensitized by hind footpad injection of 0 .1ml inoculum containing 10-50 pg MBP emulsified in complete Freund's adjuvant (CFA), which contained 200 pg Mycobacterium tuberculosis, Jamaica strain.
Alternatively, rats received 0.1 ml of guinea pig spinal cord homogenate
(33% in water) emulsified in CFA. All rats were monitored for the development of EAE clinical signs and scored as follows: no clinical signs
(0); limpness of the distal portion of the tail (♦ I ); complete loss of tail tonus (+2); hind limb weakness or ataxia (+3); complete hind limb paralysis or death (+4). Rats were sacrificed under ether anesthesia by exsanguination on various days postsensitization.
EAE Adoptive Transfer with Amplification in vitro
An adaptation of the procedures described by Panitch and McFarlin 42
(1977), Rfchert et al. (1979), and Ovadia and Paterson (1981) was used.
Lewis rats were sensitized either with guinea pig spinal cord-CFA or
MBP-CFA (10 - 50 pg MBP), then sacrificed 9 -12 days later. Lymph nodes draining the site of footpad injection were excised and processed into a single cell suspension. LNC were cultured (2-6 x lOVml) with 5-50 pg/ml
MBP and/or 1 pg/ml Con A in the presence or absence of partially purified bovine brain gangliosides (8-250 pg/ml), or rat brain gangliosides (26 pM), in 25 mM HEPES-buffered RPMI 1640, supplemented with 2 mM L-glutamine,
50 units/ml penicillin, 50 pg/ml streptomycin, 5 x 10"5 M
2-mercaptoethanol and 5% fetal bovine serum (complete medium). Cells were incubated for 72 hours at 37*C and 5% C02 in 25 cm2 tissue culture flasks (Coming Glass Works, Coming, New York). After incubation, cells were washed 3x in Hank’s balanced salt solution (HBSS), counted, and adjusted to the appropriate concentration for injection. From 10-40 x 106 cultured LNC were then injected i.v. into naive syngeneic recipient rats. In one experiment, LNC were cultured for 3 days with MBP, then incubated for
1 hour at 37’ C with 20 pM rat brain gangliosides prior to i.v. injection. Cell Preparation To obtain spleen cells (SPC), normal unsensitized rats were sacrificed, their spleens removed, minced, and expressed through a stainless steel mesh screeen. Erythrocytes were lysed upon the addition of 0.17 M Tris
(hydroxymethyl) aminomethane and 0 .16M NH4CI (1:9 v/v). SPC were then washed twice in HBSS, and adjusted to the proper concentration for culture.
To obtain human peripheral blood mononuclear cells, blood was drawn into a syringe containing saline and 5% EDTA. Collected blood was layered on Lymphocyte Separation Medium (Litton Bionetics, Kensington, MD) ( 6.25? 43 f icoll, 9.4% sodium diatrizoate), and centrifuged 25 minutes at 240 x g.
The mononuclear cell layer was collected, washed 3x in HBSS, then suspended at 2 x 106 cells/ml for culture Production of MBP-Beactlve CelLLlne An MBP-reactive T-cell line (Tmbp-i ) was established as described previously (Ben-nun et al., 1981 and Vandenbark et al., 1985). Draining LNC were obtained from Lewis rats 9 days after MBP-CFA sensitization. These
LNC (7 x 10®/ml) were incubated in 6-cm petri dishes in RPMI 1640 (M.A.
Bioproducts, Walkersville, MD) containing 50 units/ml penicillin, 50 pg/ml streptomycin, 2-mercaptoethanol (5 x 10_5M), 2 mM L-glutamine, 25 mM
HEPES, 2% fresh autologous rat serum, and stimulated with 33 pg/ml MBP.
Three days later, lymphoblasts were obtained by centrifugation using
Lymphocyte Separating Medium, at 240 x g, for 25 minutes. The cells were washed and expanded in medium containing 108 fetal bovine serum and
10% (v/v) rat growth factor (48 hour supernatant from Con A-stimulated rat spleen cells). After 4-8 days of culture in 10 cm petri dishes, the cells were restimulated with MBP (10-60 pg/ml) in the presence of syngeneic gamma irradiated (3300 R) thymocytes ( 107/m l) as a source of accessory cells, for 3 days. The cells were then alternately expanded in rat growth factor or restimulated with antigen and accessory cells. Proliferation assays in vitro revealed significant (3H]-thymidine incorporation in response to MBP but not to PPD.
Lymphocyte Proliferation Assay
The source of lymphoid cells for assay were either draining LNC obtained from Lewis rats 12 days after sensitization with MBP-CFA, or
Tmbp-i cells. The lymph nodes (popliteal, inguinal, and periaortic) from 44
MBP-CFA sensitized rats were excised, expressed through a stainless steel mesh screen, and the LNC washed in HBSS. The LNC were adjusted to
2 x loVml in 25 mM HEPES-buffered RPM! 1640 supplemented with 2 mM
L-glutamine, 50 units/ml penicillin, 50 pg/ml streptomycin, 5 x 10‘5 M
2-mercaptoethanol and 5% fetal bovine serum. Viability was assessed by trypan blue dye exclusion. Cells were cultured at 2 xlO5 LNC/well in
96-well flatbottom plates (Costar, Cambridge, MA) with either MBP, 3.1 pg/ml, PPD, lOOpg/ml or Con A, 0.5-3.1 pg/ml in the presence or absence of various concentrations of purified rat brain gangliosides. Cultures were incubated for a total of 66-72 hours in 5.4% C02 at 37*C in a humidified atmosphere, the final 6-1 8 hours of which included a pulse with l3H]-thymidine at I pCi/well (Amersham Corp., Arlington Heights, IL., specific activity 5 Ci/mmole). Cells were harvested with a multiple automated sample harvester (MASH II, M. A. Bioproducts) onto glass fiber filters and counted in a Beckman Model LS 9000 liquid scintillation counter.
Tmdp- i cells (104/w e ll) were cultured with 106 irradiated thymocytes,
3.1 pg/ml MBP, with or without various concentrations of gangliosides and incubated as described above.
Some of the proliferation data (l3H]-thymidine incorporation) is expressed as the stimulation index (S.I.), which ts calculated as follows:
(2) S I. « Mean CPM in the presence of stimulant
Mean CPM in the absence of stimulant
Quantitation of IL 2 Activity in Culture Supernatants
LNC were cultured in 96-w ell flatbottom plates as described above with
Con A, in the presence or absence of various concentrations of gangliosides. 45
After 24 hours, lOOpl of culture supernatant was aspirated from each of triplicate wells, and placed in the wells of another 96-w ell plate. Serial two-fold dilutions of the supernatants (1:2 - 1:2048 dilutions) were made across the plate and then I0 4 CTLL-20 cells were added to each well.
These cultures were then incubated for a total of 24 hours, the final 6 to
12 hours included a pulse of [3H]-thymidine (1 pCi/well).
CTLL-20 cells, an IL 2-dependent murine T-cell line, were maintained by weekly transfer of I04 viable cells into 10 ml Dulbecco's modified Eagle’s medium (DMEM) containing 2% FCS and 10% supernatant from Con
A-stimulated rat spleen cells as a source of IL 2.
IL 2-containing supernatants were obtained from cultures of Lewis rat spleen cells (2 x lOVm l) incubated with 2.5 pg/ml Con A at 37*C in 25 cm2 tissue culture flasks (Coming, Coming, NY). After 24 hours, the cells were pelleted, the supernatant was collected, filtered through 0.2 pm filters, and stored at -20*C until use.
IL 2 Quantitation: Direct indicator Cell Addition Assay
The method of Clouse et al. (1984) was used to test for small quantities of IL 2 in antigen-stimulated cultures. LNC were obtained from rats 12 days following sensitization with MBP-CFA, and cultured in a 96-well flat-bottomed plate with 3.1 pg/ml guinea pig MBP or 100 pg/ml PPD, with or without exogenous gangliosides (3 - 23 pM ganglioside sialic acid).
After 24 hours, the entire culture plate was irradiated (2000 rad from a
,37Cesium source). Finally, 104 CTLL-20 cells were added to each well and incubated for 24 hours at 37*C and 5% C02, the final 6 to 8 hours including a [3H)-thymidine pulse (1 pCi/well). 46
Detection of IL 2 Receptors
LNC from MBP-CFA-sensitized rats were cultured as described above
(2 x 105 cells/well in 96-well flatbottom plates with 3.1 pg/ml
Con A ♦ 5 -10 pM rat brain gangliosides). Cultures were incubated for 48 hours then harvested. LNC were layered on Lymphocyte Separation Medium and processed as described above. The mononuclear cell layer was collected and washed 3x in HBSS. After washing, 106 cells were suspended in 50 pi of a 1:4 dilution of monoclonal antibody ART-18, diluted inRPMI containing 5% FCS and 0.01 % sodium azide. Incubation with monoclonal antibody was carried out on ice for 30 minutes. Mouse monoclonal antibody
ART-18, directed against a determinant on the rat cellular IL 2 receptor
(Diamantstein et al., 1983), was kindly provided by Dr. Mohan Sopori from the University of Kentucky, Lexington, Kentucky. Control Con A-actlvated cells were incubated in diluent only. Following incubation, cells were washed twice with PBS containing 0.0 IX azide, and resuspended in 200 pi of a 1:50 dilution of FITC-labeled F(ab )’2 goat anti-mouse immunoglobulin and incubated for 30 minutes on ice. Cells were then washed with
PBS-azide, and analyzed using an Ortho Cytof luorograf System 50 H fluorescence-activated cell sorter (Ortho Instruments). The instrument was standardized daily by maximizing all signals during the analysis of standard 2 micron latex beads. The excitation wavelength used was
488 nm, and green fluorescence was analyzed between 515 and 530 nm.
For each test, data were collected from 10,000 cells whose forward and
90* light scatter were characteristic of lymphocytes and Con A-stimulated lymphocytes. Positive and negative fluorescence was determined using the control cells (Con A-activated LNC reacted with the FITC-labeled second 47 antibody only), by establishing the negative flourescence region as that in which i 95% of these control cells was found.
Statistical Analysis
The statistical significance of the effects of gangliosides on cell viability was evaluated with a linear regression model, using the percent viable cells as the response variables, and the log of the ganglioside concentrations as the predictor variables. Comparisons (viability) between different cell or ganglioside types were made by the analysis of covariance.
The statistical significance of the effects of gangliosides or IL 2 on l3H]-thymidine incorporation was analyzed by one of two methods. Where
Indicated, Student's t-test was used, with stringent criteria for rejection of the null hypothesis (p<0.005). Alternatively, the analysis of variance was used, along with Tukey’s post-hoc multiple comparison test, with a
0.05 confidence level. The mean day of onset of EAE clinical signs was compared with Student's t-test. RESULTS
Since lymph node cells (LNC) from rats sensitized with neuroanttgen- adjuvant are known to transfer EAE (Paterson, I960; Levine etal., 1967), they serve as an effector cell population which I have utilized in the investigation of the role of gangliosides in EAE. The first question that was addressed was whether gangliosides could affect the proliferative responses of LNC in v itra Antigen or mitogen-stimulated LNC, which were obtained from Lewis rats with EAE, were incubated in the presence or absence of various concentrations of gangliosides, approximating the effective range of gangliosides used in the previous study of Whisler and
Yates (1980). In preliminary experiments, antigen and mitogen-induced
[3H]-thymidine incorporation by the LNC was suppressed by the addition of gangliosides. However, significant cytotoxicity, as assessed by trypan blue dye exclusion, was also observed in the cultures containing high ganglioside concentrations. Therefore, it was necessary to investigate whether there were ganglioside levels that were not directly toxic to the rat cells, yet were able to suppress the proliferative responses.
Additionally, since most of the studies describing the effects of gangliosides on proliferation in the immunological literature used bovine brain gangliosides, I also tested whether gangliosides of bovine origin produced similar cytotoxic activity as rat brain gangliosides.
48 49
LNC derived from naive rats (NLNC) or MBP-CFA sensitized rats (SLNC),
were incubated with 0, 13, 26, 52, or 104 pM gangliosides for 72 hours.
Figure 1 shows a decrease in rat cell viability with increasing ganglioside
concentration, regardless of whether normal or sensitized cells were
tested. At the highest ganglioside concentration tested (104 pM
ganglioside sialic acid), normal rat LNC viability was reduced from 61 to
14% with rat gangliosides and from 77 to 30% with bovine gangliosides.
Similarly, the viability of LNC from an immunized rat was decreased from
64% to 20% by rat gangliosides and from 53 to 32 % with the addition of bovine gangliosides.
In light of the previous reports (Lengle et al. 1979; Whisler and Yates,
1980) in which murine thymocytes and human PBMC respectively had decreased proliferative responses in the presence of gangliosides without demonstrable cytotoxicity, I examined whether purified rat brain gangliosides would display differential toxicity for rat as opposed to human cells. Figure 1 shows data from one representative experiment in which the concentrations of rat or bovine gangliosides which were highly toxic for rat cells, had no effect o/> the viability of human PBMC. This finding was observed repeatedly using mononuclear cells from several human blood donors.
Next, I tested the possibility that rat LNC might be particularly susceptible to ganglioside toxicity, when compared with other rat lymphoid populations. Lymphoid cells derived from rat lymph nodes, spleen, and peripheral blood were incubated with 13-104 jiM gangliosides. The 50
100 ft ■■ — •1 -§~—ii ft
♦ - NLNC+ Rat ganglioside
* ' SLNC-t-Ral ganglioside
* HPBHC+ Rat ganglioside
NLNC+ Bovine ganglioside
^ SLNC+ Bovine ganglioside
HPBMC+ Bovine ganglioside
Q « * * - » ‘ 0 13 26 52 104
Ganglioside Concentration (uM)
Figure 1. Effects of rat and bovine brain gangliosides on the viability of rat and human lymphoid cells
Lymph node cells from naive Lewis rats (NLNC), from rats sensitized 12 days previously with 10 pg MBP-CFA (SLNC), or human peripheral blood mononuclear cells (HPBMC) were incubated at 2 x 105 cells/well for 72 hours with or without exogenous rat or bovine brain gangliosides (13-104 pM). At the end of culture, viability of the cells was assessed by trypan blue dye exclusion. Each data point represents the mean percentage of viable cells from triplicate wells. Both rat and bovine gangliosides showed significant effects (p<0.005) on SLNC and NLNC viability. There was an overall statistical difference between the effects of bovine versus rat gangliosides. Both rat cell types (SLNC and NLNC) differed significantly (p<0.005) from HPBMC, in their susceptibility to ganglioside toxicity. There was a marginal difference (p<0.05) between SLNC and NLNC in their percent viable cells, at the various ganglioside concentrations. 51
glycoliplds in this case were found to be toxic for all rat cell populations
tested, while not affecting human PBMC viability (Figure 2). In the absence of gangliosides, the three rat cell populations showed 68, 63, and 80% viable cells respectively at the end of the three day culture. Gangliosides
(104 pM) reduced this viability to 14, 39, and 42% respectively.
In light of these results, in all subsequent experiments, rat brain gangliosides were used at a dose no greater than 26 pM sialic acid, and viability determinations were performed at the termination of each in vitro culture. Experiments where gangliosides reduced viability by 25% were disregarded (i.e., in cultures where the addition of gangliosides resulted in cell viability less than 75% of the viability in control cultures containing no gangliosides).
Effect of Gangliosides on Cellular Proliferation
To analyze the effect of gangliosides on proliferative responses of an autoreactive lymphoid cell population in v itro, draining LNC were obtained from Lewis rats 12 days after sensitization with MBP-CFA and cultured with either 3.1 pg/ml MBP, 100 pg/ml PPD, or 3.1 pg/ml Con A.
[3H]-thymidine incorporation was measured at the end of a 3 day incubation.
Table 1 shows that the addition of rat brain gangliosides to these cultures significantly (p<0.05) reduced the proliferative responses to antigens and
Con A. The results from eight representative animals tested 12 days after
MBP-CFA sensitization show that 20-26 pM gangliosides suppressed
MBP-lnduced proliferation by 62-79%. Similarly, Con A, and PPD-induced proliferation was inhibited by as much as 79% and 67% respectively. The most pronounced inhibition of [3H)-thymidine incorporation occurred at 52
Q ■ 0 Gangliosides CO + 1 13 13 |iM Gangliosides CO I 26 pH Gangliosides (1) c_> EH 52 pM Gangliosides D 104 pM 6angliosides J3 CTJ •H > S'S
LNC SPC RPBMCHPBMC
Figure 2. Effects of rat brain gangliosides on the viability of rat lymphoid cells and human PBMC
Human PBMC and lymphoid cells derived from naive rat lymph nodes (LNC), spleen (SPC), or peripheral blood (RPBMC) were incubated at 2 x 105 cells/w ell with or without rat brain gangliosides (13-104 pM), for 72 hours. Viability was assessed by trypan blue dye exclusion. Each bar represents the mean percentage of viable cells ♦ S.D. from triplicate wells. Gangliosides were found to significantly (p<0.005) affect the viability of rat LNC and PBMC, and to marginally (p<0.05) affect the viability of rat SPC, while having no effect on HPBMC. The effect of gangliosides on cell viability was significantly different (p<0.005) when comparing rat LNC versus HPBMC, and also between rat PBMC and HPBMC. 53
Table 1. Effects of gangliosides on the antigen and mitogen-induced proliferation of LNC®
Ganglioside concentration, pM sialic acid Antigen 0 5-7 10-13 20-26
Mean CPMx 10-3 + 5. D. (percent inhibition)
12+1.5 9.0+0.4 (26) 8.0+0.9 (34)b 2.6+0.4 (79)b MBP 11 + 1.1 11+0.6 (-4) 8.2+1.1 (24) 3.1 + 1.1 (72)b 19+1.0 17+0.9(12) 13+0.9 (34)b 7.4+1.0 (62)b
129±1.1 119+5.0 (8) 99+7.5 (24)b 77+7.7 (41 )b ConA 318+13 258+5.3 (19)b 125+34 (61 )b 68+14 (79)b 117+11 119+5.9 (-2) 107+11(9) 98+7.9(16)
9.4+0.9 12+1.1 (-24) 8.6+1.2 (8) 3.3+0.3 (63)b PPD 41+3.2 31+2.0 (25)b 23+2.5 (45)b 14+1.4 (67)h 26+2.6 21 + 1.0 (21) 14+1.8 (46)b 8.7+1.9 (67)b
a LNC were obtained 12 days after MBP-CFA sensitization and cultured with 3.1 pg/ml MBP, 0.5 or 3.1 pg/ml ConA, or 100 pg/ml PPD + rat brain gangliosides at the indicated concentrations. Each determination was done in quadruplicate. The percent inhibition (in parenthesis) is calculated from the amount of tritiated thymidine incorporation (in CPM) in the presence of gangliosides, compared with the CPM obtained in the absence of gangliosides. b Mean values, with added gangliosides, differed significantly from controls, without gangliosides, p<0.005, by Student's t-test analysis. 54 most points for 20-26 pM and 10-13 pM gangliosides (p<0.005). The mitogen phytohemagglutinin (PHA) was also used for LNC stimulation. As shown in Figure 3, PHA-stimulated proliferative responses were similarly suppressed by the addition of gangliosides. The data represented in Figure
3 are pooled from five experiments, including data from Table 1, and it clearly shows the ganglioside dose-related Inhibition of proliferative responses, regardless of the stimulating agent. These results (Table 1 and
Figure 3) demonstrate that exogenously added gangliosides can Inhibit antigen- specific as well as mitogen-induced proliferative responses of
LNC derived from Lewis 12 days after sensitization with MBP-CFA. Similar suppressive effects were seen in experiments where LNC were obtained 9,
15, or 22 days after immunization with MBP-CFA.
In order to test whether prior in vivo sensitization with MBP-CFA influenced the observed ganglioside-mediated suppression, LNC derived from normal unsensitized Lewis rats were cultured with Con A in the presence or absence of various concentrations of gangliosides. Table 2 shows that gangliosides suppressed [3H]-thymidine incorporation by 63, 93, and 89% at the highest ganglioside concentration tested.
In the next series of experiments, MBP or Con A-stimulated LNC were
Incubated either with gangliosides or with one of two control glycolipids.
Asialo GMi is a neutral glycosphlngolipid with the same carbohydrate structure as GMi ganglioside except that it lacks sialic acid. The other glycolipid used was bovine brain sulfatide, a galactosylceramide containing a sulfate group at the 3 position of galactose, which gives the molecule a negative charge. Table 3 shows data from one of two experiments. In every 55
70 w w 60 + ! T d 50 o ■ Ganglioside 3 ptt XI 40 •H 0 Ganglioside 7 pM
a EH Ganglioside 13 pM 30 S'S B Ganglioside 26 pM 20
10
0 A m MBP ConA PHA PPD
Figure 3. Effects of gangliosides on the antigen and mitogen-induced proliferation of rat LNC
LNC were obtained from rats 12 days after sensitization with MBP-CFA, and cultured for 3 days with 3.1 pg/ml MBP, 0.5-3.1 pg/ml Con A, 25 pg/ml PHA, or 100 pg/ml PPD, ± exogenous gangliosides (3, 7, 13, or 26 pM sialic acid). Proliferation was assessed by PH)-thymidine incorporation. Results are expressed as the mean percentage inhibition t S.D., caused by the addition of gangliosides, as compared with antigen or mitogen-stimulated cultures that contained no exogenous gangliosides. Determinations were done in quadruplicate. Data points represent averages from 12 rats for MBP, 9 rats for Con A, 6 for PHA, and 3 rats for PPD stimulation groups. 56
Table 2. Effects of gangliosides on the Con A-induced proliferation of normal unsensitized Rat LNC*
Exp. Ganglioside Concentration, pM sialic actd No. 0 3.2 6.5 13 26
Mean CPMx 10'3 i S. D.
1 175+9.97 113i17.8b 104120.5 72.8130.9 65.8i4.70
2 92.6+7.39 54.8i4.54 35.6i4.69 20.71439 6.5810.67
3 199110.7 108115.9 107115.0 41.612.81 22.0i4.24
^ach experiment represents data from one normal Lewis rat. LNC were obtained from periaortic, popliteal, inguinal, cervical, peritracheal, and axillary lymph nodes. LNC were cultured at 2 x 105 cells/well with 3.1 pg/ml Con A and rat brain gangliosides at the indicated concentrations. Cultures were incubated for a total of 66 hours including a final 18 hour pulse withl pCi/well [3H]-thymidine. Mean cpm for LNC alone were 1417 i 98, 9 4 91 120, and 3952 i 841 for experiments 1, 2, and 3 respectively. b Mean cpm differed significantly (0.05 level) from control for all ganglioside concentrations tested. 57
Table 3. Effects of gangliosides, asialo GMI, and sulfatide onMBP-induced LNC proliferation
% Inhibition of [3H]-thymidine Incorporation
Ganglioside Asialo Gm 1 Sulfatide LNC (Glycolipid concentration in pg/ml) treatment3 6.3 12.5 25 25 50 100 25 50 100
MBP -22 0 44b -25 -22 -23 -17 -8 -12
MBP -18 11 52 -32 -12 2 -19 -2 -16
MBP -22 14 53 -28 -15 -13 -36 -12 -8
Con A 2 1 17 -3 -4 -1 7 8 6
^hree rats were sacrificed 12 days after MBP-CFA sensitization. LNC were cultured with either 3.1 ug/ml MBP or 2 ug/ml Con A. To these cultures were added the glycollpids gangliosides, asialo GMi, or sulfatldes. In the concentrations listed above (ug/ml). The data are expressed as the percentage inhibition In the presence of these glycollpids, compared with Identical cultures containing no exogenous glycollpid.
^Statistical analysis of each glycol ipid at 25 ug/ml, revealed that all three exerted a significant effect on LNC proliferation. The positive inhibitory effect of gangliosides was significantly different from the effects of either asialo GMi or sulfatides (0.05 level). The effects of the two control glycollpids were not significantly different from each other. 58
Instance, only gangliosides showed a significant suppressive effect, at the
highest ganglioside concentration tested (25 pg ganglioside/ml is
approximately equivalent to 26 pM ganglioside sialic acid). Asialo GMi and
sulfatides were not suppressive even at 100 pg/ml, which is 4-fold
greater by weight than the highest ganglioside level (approximately 5 and 8
times greater respectively, on a molar basis) All of the glycollpids
produced enhancement of the proliferative response (negative Inhibition),
particularly at the lower concentrations tested. These data demonstrate
that glycollpids with chemical structures similar to gangliosides do not
exert the same type of suppressive effect on proliferative responses.
Kinetics of Ganglioside inhibition
To examine the kinetics of this inhibition, gangliosides were added at 0,
6, 12, 18, 24, 48, and 72 hours after the initiation of the three day culture period. As shown in Figure 4, gangliosides added at the beginning of the in
vitro culture (time 0) showed maximum suppression of proliferation to
MBP, with mean inhibition of 68%. A significant degree of suppression
(43%) was observed when gangliosides were added as late as 24 hours.
However, gangliosides added at 48 hours were less effective and were not suppressive at all when added immediately prior to the termination of the proliferative assay. Thus, gangliosides appear to require a finite time period in culture in order to exert their suppressive effect(s) fully.
Effect of Supraootlmal MBP on Ganqlioslde-Medlated Suppression
The negative charge of gangliosides and the positive charge of MBP could
lead to a direct interaction, as reported by Ong and Yu (1984), resulting in
the inability of MBP to combine with its T-cell antigen receptor. 59
8 0 -
6 0 -
4 0 -
46 72 TIME (HOURS)
Figure A. Kinetics of ganglioside-mediated suppression of MBP-induced LNC proliferative responses
LNC from three MBP-sensitized rats were cultured separately with 3.1 pg/ml MBP ± 26 pM ganglioside sialic acid. The gangliosides were added to the 96-w ell plate at the indicated times (hours) after culture was initiated. Each data point represents the mean percentage Inhibition values comparing cultures containing MBP-activated LNC ♦ gangliosides, with cultures of LNC and MBP only. Each test was done in quadruplicate. These data are from one of three similar experiments. Comparison of the mean cpm showed significant ganglioside-mediated inhibition at 0, 6, 12, and 18 hours (*p< 0.005). Therefore, I tested the effect of adding supraoptlmal concentrations of MBP
to LNC cultures, In the presence of gangllosldes, to see whether excess MBP
could abrogate the observed suppression. LNC were cultured with MBP at
the optimal stimulatory concentration of 3.1 pg/ml or up to 20 times
higher MBP concentrations, along with a suppressive level of ganglioside.
Table 4 shows that, in all cases, gangllosldes significantly inhibited
MBP-induced proliferation, and the degree of ganglioside inhibition was virtually unchanged, regardless of the MBP concentration. At the highest concentration of MBP tested in this experiment (62 pg/ml) there is still a ganglioside-to-MBP molar ratio of approximately 5. There was a statistical difference between the percent inhibition at 62 pg/ml, when compared with either 12.4 or 3 1 pg/ml. Higher levels of MBP could not be used, as shown in Figure 5, since 124 and 310 ug/ml MBP did not stimulate
LNC proliferation (S.I. of 1.6 and 0.5 respectively) in the absence of gangllosldes. LNC viability was decreased to 37% and 568 of control values respectively in the presence of these known toxic levels of MBP.
Effect of Individual Gangllosldes on LNC Proliferation
Previous studies have shown that individual gangllosldes can produce differing effects on lymphocyte proliferative responses with some gangllosldes being more suppressive than others. Lengle et al. (1979) found that GTi ganglioside was a potent inhibitor of thymocyte proliferation, whereas GMi produced much less of an inhibitory effect. Similarly, Yates et al. (1980) found differential suppressive effects exerted by gangllosldes with GDib, GTib, and GM 2 showing the greatest effects. Therefore, I tested four of the major individual gangllosldes found in mammalian brain tissue 61
Table 4. Assessment of ganglloside-mediated suppression of MBP-Induced LNC proliferation, in the presence of supraoptimal MBP concentrations3
Experiment MBP Concentration Percentage Number (pg/ml) Inhibition6
1 3.1 60.8 6.2 64.0 12.4 72.3 31.0 73.7 62.0 70.3
2 3.1 71.6 6.2 83.5 12.4 69.1 31.0 75.0 62.0 72.8
3 3.1 61.6 6.2 70.3 12.4 74.5 31.0 71.7 62.0 53.3
3 LNC from 3 MBP-CFA sensitized rats were stimulated with MBP at 3.1 pg/ml (optimal concentration), 6.2, 12.4,31.0, or 62.0 pg/ml which represent values of 1 ,2 ,4 , 10 and 20 times the optimal MBP concentration. Gangllosldes (26 pM) were added to each culture. b At all MBP concentrations, gangllosldes exerted significant inhibition (0.05 level). In comparing the effects of the different MBP concentrations, the mean percent inhibition at 62 pg/ml was found to be significantly different from 12.4and31 pg/ml. 0 LNC+MBP+6angl
3.1 12.4 31 62 124 310
MBP Concentration (ug/ml)
Figure 5. Effects of supraoptimal MBP concentrations on ganglloside- mediated suppression of LNC proliferative responses
LNC were obtained 13 days after MBP-CFA sensitization and cultured at 2 x 105 cells/w ell with MBP concentrations of 3.1, 12.4, 31, 62, 124, and 310 pg/ml, ± 12 pM ganglioside. Cultures were incubated for 3 days including a final 18 hour pulse with [3H]-thymidine. The data are represented in terms of the resulting stimulation index (See formula 2 in Materials and Methods). 63 for differentia) Inhibitory effects on LNC proliferative responses to MBP.
Figure 6 shows representative data from one of four experiments testing the effects of Individual gangllosldes obtained from bovine brain. In each test, the rat brain ganglioside mixture was significantly more suppressive than any of the Individual bovine gangllosldes tested (GMi, GDu, GDib, and
GTit), at all three concentrations. Of the Individual gangllosldes tested, the more highly slalylated molecule, GT ib. was significantly more
Inhibitory than GMi and GDi* (p < 0.05) at 2.5 pM and 5 pM.
Effect of Gangllosldes on the Proliferation of an MBP-Reactlve Cell Line
All studies to this point have examined the effects of gangllosldes on a heterogeneous cell population (viz, freshly Isolated LNC), a mixture of cells which has only a small percentage of CNS antigen-reactive cells. In order to examine the effects of gangllosldes on a more homogeneous encephalltogenlc cell population, I tested the effects of gangllosldes on the proliferative responses of an encephalltogenlc Lewis rat T-cell line,
Tmbp- i ,which was produced and maintained by Dr. Caroline Whltacre and Mrs.
Ingrid Glenapp, following published procedures (Ben-nun et al., 1981 and
Vandenbark et al., 1985). This cell line, which could mediate EAE when
Injected Into naive rats, responded specifically to stimulation with MBP in vitro, In the presence of syngeneic accessory thymocytes, as shown in
Table 5. Tmbp- i cells proliferated vigorously, in the presence of accessory thymocytes, to the selecting antigen, MBP, and also to Con A, but not to
PPD. Tmbp- i cells were cultured at I04 cel Is/well with 106 irradiated syngeneic thymocytes as accessory cells and 3 .1 pg/ml MBP. Various concentrations of rat brain gangllosldes were added to these cultures 64
100
■ LNC+MBP+ IOmM c ganglioside •Ho 4J 0 LNC+MBP+ 5 mM X) ganglioside
M ED LNC+MBP+ 2.5pM 6M 6 0 1 « h 601b 6T1b Rat Mix ganglioside -20
-4 0
Figure 6. Differential suppressive effects of individual gangliosides on the MBP-induced proliferative responses of LNC
LNC were cultured with MBP as described previously, + bovine gangliosides GMi, GDia, GDib, GTib, or a mixture of rat brain gangliosides. Ganglioside concentrations are expressed as pM ganglioside rather than pM sialic acid to test equimolar concentrations of each ganglioside species. Each bar represents the mean percentage inhibition + S.D. of [3H)-thymidine incorporation for three rats, when compared with controls (no added gangliosides). Statistically significant effects of each of the gangliosides was observed at 10 pM. GMi showed significant inhibition at 2.5 pM, while GDib, GT ib, and the rat brain mixture showed significant effects at the 5 pM level. The rat brain ganglioside mixture produced effects that were significantly different from each of the other individual gangliosides tested, at all three levels (2.5, 5, and 10 pM). GT Ib was significantly different from GMI, GDI a, and GDib at 2.5 pM and 5 pM, but differed significantly from only the rat mixture at 10 pM. 65
Table 5. Proliferative responses of an MBP-reactive rat T cell line3
Treatment of T cells Etean CPM i-SJ). in culture - thymocytes ♦ thymocytes
None 3531 102 T cells 4 3 9 ± 183 422± 149 T cells ♦ 1 ug/ml MBP 171 ± 54 18,752 ± 2674 T cells + 10 ug/ml liBP 164 1 62 31,1341 1895 T cells + 60 ug/ml MBP 19 4 ± 19 36,207 1 2594
T cells + 1 ug/ml PPD 4 5 0 ± 126 2861 119 T cells ♦ 10 ug/ml PPD 296 ± 7 8 465 l 379 T cells + 50 ug/ml PPD 374 ± 124 3931315
T cells ♦ 1 ug/ml Con A 436 i 235 54,848 i 8425 T cells ♦ 2.5 ug/ml Con A 1 9 8 7 i 1600 60,225 i 7505 T cells ♦ 5 ug/ml Con a 8 1 2 5 1 6 3 6 41,569 1 4690
3 The T-cell line (Tmbp- i) was cultured ( 1 O'4 cells/w ell) with the antigen concentrations listed ± accessory irradiated thymocytes (106), in 96-w ell plates, for 72 hours, including a final 18 hour pulse with l3H]-thymidine. 66 which resulted in significant inhibition of proliferation, as shown in Table
6. As much as 50% inhibition or more was seen in two of the four experiments, at the highest ganglioside concentration tested (20 pM sialic acid).
Etfecl of Gangllosldes on EAE Transfer
The data presented thus far have demonstrated a ganglioside-induced suppression of the proliferative response of encephalltogenlc cells, tested
in vitro. The next goal was to investigate whether gangliosides could suppress EAE in the Lewis rat. I utilized the adoptive cellular transfer system, modified by thein vitro activation of the donor cells with MBP and/or Con A (Panitch and McFarlin, 1977; Richert et al., 1979; Ovadiaand
Paterson, 1981). This activation step has been shown to enhance the cellular transfer of EAE, thus reducing the number of cells necessary to
Induce disease.
Donor Lewis rats were sensitized with either guinea pig spinal cord homogenate and CFA, or MBP-CFA, then sacrificed 9 or 12 days later.
Draining LNC were obtained from these donors and cultured for 3 days with either 50 pg/ml MBP or a combination of 5 pg/ml MBP plus 1 pg/ml Con A in accord with publIshed procedures (Ovadia and Paterson, 1981). Various concentrations of gangliosides (8-155 pM gangliosides) were added to the culture flasks during the 72 hour culture period. As shown in Table 7, the addition of gangliosides had no effect on the ability of the cells to transfer
EAE, even though exogenous gangliosides reduced cellular proliferation in those cultures. In one experiment, LNC cultures containing gangliosides exhibited a 45% reduction in the cell number recovered at the end of the 3 67
Table 6. Proliferative responses of an MBP-reactive encephalltogenlc cell line: Effects of gangliosides on MBP-induced proliferation8
Exp. Ganalioside Concentration (uM sialic acid) No. 0 5 pM 10 pM 20 pM
Mean CPM x 10"3 ± S. D. (percentage inhibition)1*
1 254 ± 26 204 ± 14(20) 189 ± 15(25)° 128 ± 40 (50)°
2 263 ± 21 211 i 13(20) 175± 4.8 (34)° 125 ±7.2 (52 f
3 184 ± 14 160 ±3.6 (13) 163 ±7.5 (11) 159 ±9.8 (14)
4 76.0 ± 3.7 82.8 ± 6.4 (-9) 53.3 ± 8.9 (30)° 39.6 ±6.1 (48)°
a The MBP-reactive encephalltogenlc cell line, Tmbp-i, was cultured at 104 cells/well with 106 irradiated syngeneic thymocytes (3300 R, 137Cesium source) plus 3.1 pg/ml MBP, in the presence or absence of gangliosides (5, 10, and 20 pM sialic acid). All cultures were then incubated 72 hours including a final 6 -8 hour pulse with [3H]-thymidine. b The data is expressed as the mean cpm x 10"3 ± S.D. from quadruplicate wells. For comparison, control trltiated thymidine incorporations for one of the experiments shown (experiment * 4 ) were: Tmbp- i alone, 221 cpm; Tmbp- i ♦ thymocytes, 106 cpm; Tmbp- i ♦ MBP (no thymocytes),307 cpm; thymocytes ♦ MBP (noT mbp- i ), 133cpm. c Statistically significant, p< 0.005, by Student's t-test analysis. 68
Table 7. Effects of gangliosides on the in vitro activation of LNC to transfer EAE®
No. of LNC Culture Ganglioside Incidence of Group3 Transferred Stimulant Concentration EAE (score)b
1 1.1 -4 x 107 Con A ♦ MBP 8/8 (2.1)
2 1.1 -4 x 107 Con A ♦ MBP 8-155 pM 15/18(2.2)
3 1 -3 x 107 MBP 8/8 (2.6 )
4 1 -3 x 107 MBP 26 pM 7 /7 (2.9)
a LNC were cultured In the presence of 50 ug/ml MBP, or 5ug/ml MBP plus 1 U9/m l Con A for 3 days In the presence or absence of various concentrations of either bovine or rat brain gangllosldes. LNC were then Injected i.v. Into syngeneic Lewis recipients. b Numbers in parenthesis represent the mean clinical score on the scale: 0 - no disease, 1 - distal limp tail, 2 - total limp tail, 3 - hind limb paresis or ataxia, 4 - total hind limb paralysis or death. 69 day incubation period, as compared to cultures containing no gangliosides.
Gangliosides were further tested for their effects on the cellular transfer of in vitro MBP-activated LNC using three approaches: J) ganglio sides were mixed with the activated LNC and injected together I.v., 2) gangliosides were mixed with the activated LNC and incubated for 1 hour at
37*C, then injected together i.v. (Offner and Vandenbark, 1985), 3) ganglfo- sides were injected i.v. 1 day, 3 days, or both 1 and 3 days after the i.v. cellular transfer. The latter approach was designed to produce inhibitory but not cytotoxic levels of gangliosides in the peripheral blood, and to test whether hematogenous gangliosides could affect the passive transfer of
EAE. For this experiment, 400 nanomoles of rat brain ganglioside was injected i.v. at each time point. Based upon a blood volume estimate of 20 ml (Petty, 1982), this would result in a level of 20 nanomoles ganglioside/ml (20 pM). The data from this experiment are shown in Table
8. Two of three rats that received 40 x 106 MBP-activated LNC developed
EAE with moderate clinical signs (group A). As a control for the ganglioside injections, one group of rats (D) received i.v. injections of HBSS on days 1 and 3 post-transfer, and these rats also developed EAE. Rats that received i.v. gangliosides 1 day, 3 days, or both I and 3 days after transfer
(groups E, F, and G) all developed EAE indistinguishable from controls.
Likewise, the recipients of a mixture of cells and gangliosides (20 pM) showed EAE clinical signs, even when the mixture was first incubated for 1 hour prior to transfer (groups B and C), although the latter group did show some delay in disease onset (not significantly different from control), from
6 days to 7.7 days. Therefore, under the conditions tested here, 70
Table 8. Assessment of LNC transfer of EAE in recipients injected with gangliosides8
Mean Day Mean Treatment of Treatment of Incidence of Onset Severity Donor cells Recipient of EAE t S. D. ± S. D.
A LNC 2/3 6.0 ±0 2.0 i 0
B LNC + 20 uM 3/3 6 .3 1 0.6 2.0 ♦ 0 ganglioside
C LNC ♦ 20 pM 3 /3 7.7 ±1.2 2.7 i 1.2 ganglioside, 1 hr
D LNC HBSS day ♦ I, *3 3 /3 6.3 ♦ 0.6 2 .0 1 0
E LNC 400 nmoles 3 /3 6.7 ± 0.6 2.3 i 1.5 ganglioside, day ♦ I
F LNC 400 nmoles 3/3 6.7 ± 0.6 2.0 i 0 ganglioside, day +3
G LNC 400 nmoles 3 /3 6.3 ♦ 0.6 2.0 t 0 ganglioside, day ♦!, *3
a Recipient rats received 40 x ^MBP-activated LNC, i.v., on day 0. Recipient rats received 400 nmoles of rat brain gangliosides i.v. at the times indicated (concentration of gangliosides in the blood, based on a blood volume of 20 mis is 20 pM). Groups B and C had MBP-activated LNC mixed with 20 pM gangliosides then injected i.v., and in group C, this mixture was first incubated for 1 hour at 37’ C before injection. b Severity of clinical signs were graded on a scale described in Table 6. 71 gangliosides, whether Introduced in vitro or in vivo, had no effect on the development of EAE clinical signs following LNC passive transfer.
The working hypothesis which Initiated this study was that In EAE, there Is marked damage to the ganglioside-rich brain tissue, which might cause the release of gangliosides into the bloodstream. Such hematogenous gangliosides could then act to suppress further disease by inhibiting the expansion of the CNS antigen-reactive cells which cause EAE, and thus aid in recovery. Therefore, I examined serum ganglioside levels in naive rats and rats 15-18 days after MBP-CFA sensitization, a time during which
Lewis rats show peak clinical signs and begin to recover from EAE.
Figure 7 shows tests of eight normal rat sera (four of the sera were pooled prebleeds from animals later sensitized and used as donors of EAE sera), which showed a mean sialic acid level of 2.03 ± 0.92 pg/ml sialic acid (range of 0.8 - 3.3 pg/ml). A total of 17 sera taken on day 15 or 18 postsensitization from rats with EAE was also tested. In this group, the mean serum sialic acid was 2.08 ♦ 0.42 pg/ml (range of 1.3 - 3.0 pg/ml).
Therefore, there was no difference in the detectable levels of serum gangliosides in rats with EAE as compared with normal rats.
I also examined the TLC profile of normal and EAE serum gangliosides to see whether EAE sera contained more of the gangliosides more commonly found in the brain. As shown in Plate II, the ganglioside pattern of normal
(lanes 3 and 4) and EAE sera (lanes 6 and 7) were quite similar qualitatively, with the predominance of GMa, GDia. and GT ib. In the sera of rats with EAE, there was no detectable Increase in gangliosides GMi, GD 2, or GDib, which are found in abundance in the rat brain. The ganglioside 72
3 - E ■g o
* * 2 - O "o CO O*
Normal Sera E A E S e r a
Figure 7. Sialic acid concentrations in sera from unsensitized rats and in the sera from rats with EAE
Gangliosides were extracted from the sera of normal, unsensitized rats, and rats 15-18 days post MBP-CFA sensitization. Sialic acid was quantitated by the resorcinol-HCl method. Each data point is from one individual rat. The bars represent the mean concentration of sialic acid i S.D. The means of the two sera groups are not statistically different. 73
Plate II. TLC Pattern of Gangliosides Extracted from Rat Sera
GM3
0M2 GMI m 6D3 GDIo M fc GD2 GDib ^
GT 1 b
1 6 7
1. Standards, bovine brain gangliosides GMI, GDI a, GDib, GT I b
2. Rat brain ganglioside mixture
3. Gangliosides extracted from the serum of one naive rat
4 Gangliosides from the pooled serum extractions of 3 naive rats
6. Gangliosides extracted from the serum of MBP-CFA-sensitized rat
7. Gangliosides extracted from the serum of an MBP-CFA- sensitized rat
Arrows indicate non-ganglioside contaminants in lanes 3, 4 ,6 , and 7. 74
patterns In lanes 6 and 7 were obtained from rats that had progressed
totally to paralysis, and one of the two rats had begun to recover by the
time of sacrifice.
In light of the Inability of rat gangllosldes to inhibit cellular transfer
of EAE, and the lack of any detectable release of brain gangliosides into the
peripheral circulation during disease, I conclude that circulating
CNS-derived gangliosides play no major role in the recovery from EAE. This does not, however, exclude the possible effects of gangliosides within the
CNS compartment itself.
II. Gangliosides and Interleukin 2
Having established that gangliosides suppress the proliferative responses of encephalitogenic cells, I investigated one possible mechanism for this inhibition, namely, the effect of gangliosides on the activity of interleukin 2 (IL 2). IL 2 Is known to be necessary for antigen or mitogen-induced in vitro proliferation of lymphocytes (Smith, 1980).
Recent studies have reported that gangliosides can bind to IL 2 and remove
IL 2 activity from solution (Parker et al., 1984). Gangliosides have also been reported to suppress the IL 2-induced proliferation of a murine IL 2- dependent cell line (Merritt et al., 1984). EnficLQLGanQllosldes on IL 2 Production The first question addressed was whether gangliosides inhibited the antigen or mitogen-stimulated production of IL 2. IL 2 levels were 75 measured in supernatants taken from cultures containing 2 x 105 LNC plus
3.1 pg/ml Con A in the presence or absence of gangliosides (9 and 17 pM).
These culture supernatants were diluted two-fold, and I04 CTLL-20 cells
(murine IL 2-dependent cell line) were added and incubated for 24 hours.
CTLL-20 cell proliferation in response to IL 2 was measured by l3H]-thymidine incorporation. Figure 8 (A, B, and C) shows representative data from LNC of three individual rats (one of three experiments) in which the IL 2 levels detected were not decreased in cultures containing gangliosides, even though LNC proliferative responses were suppressed. In all three cases, ganglloside-mediated suppression of LNC (3HJ-thymidine
incorporation was not accompanied by a corresponding decrease in IL 2 activity in the culture supernatants removed 24 hours after culture
initiation. Similar data were obtained when supernatants were removed
48 and 72 hours after the start of incubation. Supernatants were also assayed from cultures containing Tmbp-i cells stimulated with antigen and accessory cells, in the presence or absence of gangliosides (10 and 20 pM).
As was seen with Con A-activated LNC, gangliosides did not decrease IL 2 production from MBP-stimulated Tmbp-1 cells (Figure 8D). The corresponding LNC and Tmbp-i cell proliferations, as measured by
[3H]-thymidine incorporation, were decreased 64,60, 74, and 50% in the presence of the highest ganglioside concentrations used, yet IL 2 levels remained virtually unchanged (Figure 8A, B, C, and D respectively).
Freshly isolated LNC stimulated with MBP produced very low amounts of
IL 2 (compared with Con A stimulation), as assessed by the supernatant assay. A more sensitive detection method reported by Clouse et al. (1984) 76
Figure 8. Detection of IL 2 activity in supernatants derived from Con A or MBP-activated cells: Effects of gangliosides on IL 2 activity
In panels A, B, and C, LNC were obtained from three individual MBP-CFA sensitized Lewis rats and cultured with 3.1 pg/ml Con A i 9 or 17 pM ganglioside sialic acid for 2 A hours. Supernatants were then removed and assayed for IL 2 activity by assessing the proliferation of the IL 2- dependent cell line, CTLL-20, as described in Materials and Methods. Values represent mean cpm of [3H]-thymidine incorporation for triplicate determinations. The standard deviations for each rat, at the 1:2 dilution was less than 10% of the mean. Panel D shows data from one of three experiments testing supernatants derived from cultures of 104 Tmbp- i cells ♦ 106 syngeneic irradiated thymocytes ♦ 3 .1 pg/ml MBP ♦ 10 or 20 pM gangliosides. Data represents the mean cpm of [3H]-thymidine Incorporation by the CTLL-20 cells, with each data point determined in quadruplicate. CPM X 10" 0 6 iue . eeto o I 2 ciiy n uentns derived supernatants in activity 2 IL of Detection 8. Figure 2 0 Supernotonts from cultures of of cultures from Supernotonts agisds n L activity 2 IL on gangliosides rmCnAo B-ciae cls Efcs of Effects cells: MBP-activated or A Con from -O” LNC + ConA + 9pm 9pm +ConA + LNC -O” ConA LNC+ - -o LNC +ConLNC +A 17 LNC LNC Gonglioside Gonglioside pm NES DILUTION INVERSE 2 2 0 32
el + B ♦Tyoye ♦ Thymocytes ♦ MBP + cells T - o el + B + hmcts ♦ Thymocytes + MBP + cells T - • Thymocytes ♦ MBP + cells T ■o- uentns rm clue of' cultures from Supernotonts T cells + MBP MBP + cells T 10 20 20 pm pm Gonglioside Gonglioside 77
was attempted. Using this approach, MBP-stimulated LNC cultures were
incubated for 24 hours, irradiated (2000 R) to inhibit further LNC
proliferation, then CTLL-20 indicator cells were added directly to the
wells and their proliferation measured as before after an additional 24
hours. Table 9 shows data from two direct indicator cell addition
experiments in which IL 2 levels were found to be on the threshold of
detection. Therefore, even with a more sensitive assay, MBP-stimulation
of LNC did not produce enough detectable IL 2 to be able to discern differences in IL 2 levels reliably in the presence or absence of gangliosides.
Effect of Gangliosides on IL 2-Dependent Proliferation of CTLL-2Q Cells
In light of the reports of Parker et al. (1984) and Merritt et al. (1984) which Showed that exogenous gangliosides could inhibit the IL 2-lnduced proliferation of other IL 2-dependent cell lines, I examined more closely the possibility of such direct suppressive effects of gangliosides on
CTLL-20 cells. Additionally, because CTLL-20 cells continuously express
IL 2 receptors, the addition of exogenous gangliosides to cultures containing CTLL-20 cells and IL 2, would determine whether the gangliosides could interfere directly with receptor-ligand interaction. In three experiments, 104 CTLL-20 cells were incubated with serial two-fold dilutions of IL 2-containing supernatants in the presence or absence of 20 pM gangliosides (a ganglioside concentration which suppressed proliferation by as much as 79% in cultures containing 2 x 105 LNC). As shown in Figure 9A, gangliosides had no effect on CTLL-20 cell proliferation when the IL 2-containing supernatant, gangliosides, and 79
Table 9. Direct CTLL-20 Indicator cell addition assay for the detection of IL 2 in MBP-activated LNC cultures*
Mean CPM ± 5. D. CTLL-2013H]-thymidine incorporation
Exp. No. LNC LNC + MBP ( IL 2 units/mlb)
1 871 ± 250 2 2 8 9 ± 1140 (0.16) 2 3404 i 850 16,446 ± 3120 (1.3) 3 974 ± 280 2290 ± 940 (0.16)
4 448 ±80 516 ±80 (<0.1) 5 861 ± 137 2251 ± 338 (0.16) 6 530 ± 168 2489 ± 458 (0.17)
a LNC (2 x lO Vw ell) were incubated for 24 hours with 3.1 pg/ml MBP in 96-well plates. The cells were then irradiated (2000 R, 13'Ces1um source), and then 104 CTLL-20 cells were added directly to each well. The plate was then incubated a further 24 hours, the final 6 to 8 hours including 1 pCI/well [3H]-thymidine. b IL 2 units/ml were estimated by comparing the mean cpm with a standard titration curve of CTLL-20 cell proliferation induced by an IL 2- containing supemate, which contained 315 units IL 2/ml. 80
Figure 9. Effects of gangllosldes on IL 2-lnduced CTLL-20 cell proliferation
In panel A, 104 CTLL-20 cells were incubated with two-fold dilutions of an IL 2-containing supernatant ♦ 20 pM ganglioside sialic acid. In panel B, CTLL-20 cells were first preincubated for 3 hours with 20 pM gangliosides, then the cells were added to serial two-fold dilutions of IL 2. All cultures were then incubated for 24 hours, the final 6-12 hours of which included a pulse with 1 pCi/well (3H]-thymidine. Determinations were performed in triplicate. CTLL-20 cells alone showed a mean of 434 cpm and CTLL-20 cells ♦ 20 pM ganglioside (no IL 2) showed a mean of 346 cpm. K> CPM X 10" 0 0 1 140-1 120 - 0 4 - 0 6 - 0 8 0 2 0 2 - 0 6 iue . fet o gnloie o I 2idcd CTLL-20 2-induced IL on gangliosides of Effects 9. Figure - - - - B L + 0 2 + IL2 2 2 CTLL-20 el proliferation cell CTLL-20 + IL2 + CTLL-20 pm gongliosides 8 8 CTLL-20 1L2 TL2 + 0/ gangliosides 20//m + 3hrs CTLL-20 NES DILUTION INVERSE 32 32 I 2 IL + 128 2 512 128 512
2048
81 82
CTLL-20 cells were added simultaneously.
To allow more time for ganglioside-cell interaction, CTLL-20 cells were preincubated with gangliosides for up to 3 hours before the addition of IL 2. Figure 9B shows that this pretreatment of CTLL-20 cells with 20
pM gangliosides also produced no change in the CTLL-20 proliferative response. These findings indicate that, under these conditions, gangliosides did not interfere with the interaction between IL 2 and the cell-surface IL 2 receptors continuously displayed on the CTLL-20 cells.
The possibility existed that the murine CTLL-20 cells were susceptible to suppression at higher ganglioside concentrations than were used to suppress rat LNC proliferation. Therefore, I tested the effects of 20, 100,
200, 300, and 400 pM gangliosides on CTLL-20 proliferation and viability.
As shown in Figure 10, 20 and 100 pM gangliosides did not inhibit CTLL-20 proliferation and did not adversely affect cell viability. However, at the higher ganglioside concentrations (200, 300, 400 pM), decreased proliferation was observed, which correlated with decreased viability.
Therefore, higher ganglioside levels did not affect CTLL-20 cell proliferation until there were direct effects on cell viability.
In order to allow time for direct ganglioside-IL 2 interaction, IL 2- contalnlng supernatants were preincubated with gangliosides (20, 40 and
80 pM sialic acid) for 4, 7, or 24 hours prior to the addition of CTLL-20 indicator cells. Figure 11 shows that when IL 2-containing supernatants were first preincubated with gangliosides for 4, 7, or 24 hours before addition of CTLL-20 cells, there was a ganglioside dose-related Inhibition of CTLL-20 proliferation. As preincubation times increased, ganglioside- 83
70
60 ♦- CTLL-20
^ CTLL-20* 20pM ganglioside
c/ 2 ■- CTLL-20* 100 jjfl + 1 ganglioside co 4 0 o rH CTLL-20* 200 pfl x 30 ganglioside s CL, CTLL-20* 300 pM ganglioside
CTLL-20* 400 pM 10 ganglioside
0 at G__ 2 8 32 128 512 2048
Inverse Dilution of IL 2
Figure 10. Effects of various concentrations of gangliosides on CTLL-20 cell proliferation and viability
CTLL-20 cells ( 104)were incubated with IL 2-containing supernatants for 24 hours as described in Figure 9, ♦ gangliosides (20, 100, 200, 300, and 400 pM sialic acid). The data are expressed as the mean cpm of CTLL-20 cell proliferation from triplicate wells. Concurrently, viabilities of the CTLL-20 cells were assessed by trypan blue dye exclusion, with resulting viabilities of 83, 97, 96, 1.3, 0, and 0 for ganglioside concentrations of 0, 20, 100, 200, 300, and 400 pM respectively. 84
100-1 Z o o p CM < 8 0 - Conlrol - HBSS Only ^ ° IL 2 + 2 0 /jM Gonglioside ° o IL 2 + 4 0 pM Gonglioside 6 0 - IL 2 + 8 0 pM Ganglioside 1 1 ^ L lI X 2 - 40- 5 -
O x v° I— 2 0 - O' I X ro - i ------1------"I------1------r ~ 4 7 24 NCUBATION TIME (hours)
Figure 11. Assessment of IL 2-induced CTLL-20 cell proliferation: Effects of ganglloside-IL 2 preincubation
A 1:8 dilution of an IL 2-containing supernatant (13 units/ml final concentration), was preincubated with 20, 40, or 80 pM gangliosides for 4, 7, or 24 hours prior to the addition of 104 CTLL-20 cells. CTLL-20 and the IL 2-ganglioside mixture were then incubated for 24 hours and the CTLL-20 proliferation was determined as described previously. Tests were performed in triplicate and this data is from one of two experiments giving similar results. Statistical analysis revealed significant effects of 20 pM gangliosides (at 24 hours only, p<0.05), 40 pM gangliosides (at 4 and 7 hours, p<0.05, and at 24 hours, p<0.005), and 80 pM (4, 7, and 24 hours, p<0.005). In comparing the effects of the various ganglioside concentrations, 40 and 80 pM, but not 20 pM ganglioside significantly differed from the HBSS control. 85
IL 2 interaction appeared to occur, resulting in less detectable IL 2
activity. Inhibition of CTLL-20 cell proliferation was as great as 90%
when IL 2 was preincubated for 24 hours with 80 pM gangliosides.
However, at the ganglioside concentration known to be suppressive for rat
LNC cultures (20pM), inhibition of IL 2 activity was only 38% when
gangliosides were preincubated with IL 2 for 24 hours.
Effect of gangliosides on IL 2 receptor expression
To test whether gangliosides affected expression of IL 2 receptors, LNC
from MBP-CFA-sensitized rats, were stimulated with Con A in the presence or absence of gangliosides for 48 hours. Cells were then incubated with the mouse monoclonal antibody ART 18, directed against the rat IL 2 receptor, followed by FITC-labeled anti-mouse immunoglobulin.
Fluorescence analysis revealed similar fluorescence intensity of Con A- stimulated LNC in the presence or absence of gangliosides (Figure 12). In these same cultures, LNC proliferation was suppressed by 48 and 53% at 10 pM gangliosides, for experiments 1 and 2 respectively (Table 10). As expected, IL 2 activity in the culture supernatants was not diminished by the addition of gangliosides, as indicated by the fact that there was no decrease in detectable IL 2 units. Therefore, gangliosides did not affect the binding of a monoclonal antibody to the cell-surface IL 2 receptor.
Effect of Exogenous IL 2 on Ganglioside-Mediated Suppression
Experiments were conducted to determine whether the addition of exogenous IL 2 could restore proliferative responses that had been suppressed by gangliosides. The ability of excess IL 2 to abrogate the ganglioside suppressive effect would be expected if the primary mechanism 86
100
CONTROL LNC + CON A LNC+ CON A+ 10 pm GANGLIOSIDE LNC + CON A + 5 pm GANGLIOSIDE UJ 60
UJ ce 20 -V..
—r— T T* 10 IS" 20 25 3030 35 40 45 50 55 FLUORESCENCE INTENSITY
Figure 12. Effects of gangliosides on the binding of a monoclonal anti-IL 2 receptor antibody to Con A-activated LNC.
LNC from MBP-CFA-sensitized rats were cultured at 2 x 105 cells/well with 3.1 pg/ml Con A t 5 or 10 uM rat brain gangliosides. Cultures were incubated for 48 hours, then the cells were harvested and reacted with a 1:4 dilution of mouse monoclonal antibody ART-18, directed against the rat IL 2 receptor, and incubated for 30 minutes. After washing the cells, a 1:50 dilution of FITC-labeled goat anti-mouse immunoglobulin was added, and the relative fluorescence intensity was measured, as described In Materials and Methods. 87
Table 10. Effects of gangliosides on the expression of IL 2 receptors on Con A-activated LNC*
LNC* lOpM 5pM ^Positive Mean CPM IL 2 ConA 1 *Ab 2*Ab Gang Gang Cells * S. D. Units/mlc
Experiment number 1
---- - 850 * 90 ♦ ♦ 4.4 164,309*4176 131 ♦ ♦ ♦ 64.1 164,309*4176 131 ♦ ♦ ♦ ♦ 69.8 84,882 * 2358d 126 ♦ ♦ ♦ ♦ 72.3 113,665* 607 ld 126
Experiment number 2 2546 ♦ 74 ♦ ♦ 3.0 266,199 * 4466 154 ♦ ♦ ♦ 59.0 266,199 * 4466 154 ♦ ♦ ♦ ♦ 62.5 126,437 ♦ 5274d 154 ♦ ♦ ♦ ♦ 64.7 187,070* 80 14* 185
3 LNC from MBP-CFA-sensitized rats were cultured at 2 x 105 cells/w ell with 3.1 pg/ml Con A ± 5 or 10 pM rat brain gangliosides. Cultures were incubated for 48 hours, then the cells were harvested and reacted with a 1:4 dilution of mouse monoclonal antibody ART-18 (TAb), directed against the rat IL 2 receptor, and incubated for 30 minutes. After washing the cells, a 1:50 dilution of FITC-labeled goat anti-mouse Ig (2*Ab) was added and the relative fluorescence intensity was measured. b Simultaneous cultures of Con A-stlmulated LNC ± gangliosides were pulsed for 6 hours with I pCI/well [fyj-thymidine, harvested and counted. c Supernatants were removed from the 48 hour cultures described above and tested for IL 2 activity by the CTLL-20 supernatant assay. d Statistically significant, p< 0.005 (Compared with Con A-activated cultures in the absence of gangliosides), by Student's t-test. 88 by which gangliosides inhibited proliferation was by simply adsorbing IL 2, thus preventing productive interaction between IL 2 and its cell-surface receptor. As shown in Table 11, addition of IL 2-containing supernatants at dilutions of 1:4 - 1:256 partially restored the proliferative responses, although the level of response did not approach that of control cultures containing LNC and Con A alone. It should be noted that LNC cultured with
ConA and IL 2, in the absence of gangliosides, at times showed proliferative responses greater than the response to ConA alone (Table 11, rats 5 and 6), which might contribute to the increased responses seen when excess IL 2 was added to ganglioside-suppressed cultures. For comparison, the dilutions of IL 2 used contain 1.2 units/ml (1:256 dilution) to 78.8 units/ml (1:4 dilution) of IL 2 activity, and 3 units/ml (1:115 dilution) of this particular IL 2 preparation produces one-half of the maximum proliferation of 104 CTLL-20 cells.
From the foregoing data, I conclude that exogenous gangliosides suppress the in vitro proliferative responses of autoreactive lymphocytes.
However, CNS-derived hematogenous gangliosides do not contribute significantly to the remission from EAE observed in the Lewis rat, since serum ganglioside levels in rats with EAE were not different from controls, and gangliosides did not inhibit EAE transfer. Exogenous gangliosides do not inhibit IL 2 production nor the expression of IL 2 receptors. There is some interaction between ganglioside and IL 2, but this association is probably not the primary mechanism by which gangliosides suppress 89
Table 11. Effects of excess IL 2 on ganglloslde-mediated suppression of Con A-lnduced LNC proliferative responses®
Mean CPM x 10"3 ± S. D. (X inhibition)b
Exp. LNC* LNC+ConA LNC*ConA LNC+ConA+IL2 No. ConA ♦IL 2° ♦Ganglioside ♦Ganglioside
1 319 ♦ 16 280± 14 138 ♦ 7.7(57) 162+ 14(49) 318 ± 14 290 + 12 125+11 (61) 164 + 5.8 (48) 337 + 6.0 294 + 21 112 + 5.1 (67) 161 + 1.3(52)
2 288 t 15 285 + 10 184+ 17(36) 209+ 1.6(28) 261 ± 11 288 + 11 143 + 23 (45) 201 ♦ 12(23) 247 + 5.2 295 + 9.7 148 + 5.7(40) 226 ± 11 (9)
3 381 + 15 369 + 12 186 + 21 (52) 203 + 30 (47) 400 ♦ 18 349 + 15 345 ±7.5(14) 349+ 12(12) 403 + 10 360 ± 15 171 +23(57) 207 + 11(49) aCultures were established as previously described with LNC ♦ 3.1 ug/ml Con A ♦ 1 7 -2 0 pM gangliosides ♦ exogenous IL 2. When present, all factors were added simultaneously at the initiation of culture. Exogenous IL 2 was used at 1:11 and 1:4 dilutions for experiments 1 and 2 respectively. A range of IL 2 dilutions was used for experiment 3, from 1:4-1:256, and the dilution giving the greatest change in proliferation is shown.
^Numbers In parenthesis represent the percent Inhibition, compared with control cultures, which were Con A-actlvated, in the absence of gangliosides (shown in the first column) c Statistical evaluation of all the data showed that the addition of IL 2 to cultures containing LNC ♦ Con A, produced no significant changes (0.05 level). The addition of gangliosides did cause a significant decrease in the mean cpm. Exogenous IL 2 had a significant effect upon the mean cpm, in cultures containing LNC ♦ Con A ♦ gangliosides. 90 proliferative responses, In light of the high ganglioside concentrations needed to interfere with IL 2 activity, and the fact that exogenous IL 2 only partially restored gangliosidesuppressed proliferative responses. DISCUSSION
The first portion of this study examined a possible regulatory role for
gangliosides in EAE, by three different approaches: I ) by testing the
effects of exogenously added gangliosides on the proliferative responses of encephalitogenic cells in vitro 2) by testing whether gangliosides could
Inhibit the cellular transfer of EAE 3) by comparing serum ganglioside
levels in naive unsensitized rats with serum gangliosides derived from rats with EAE. The major findings can be summarized in the following manner.
Gangliosides suppressed both the antigen and mitogen-induced proliferation of LNC obtained from rats with EAE, as well as the Con A-induced proliferation of LNC derived from unsensitized rats. Gangliosides also suppressed the MBP-stimulated proliferation of an encephalitogenic rat lymphoid cell line, Trap-i. This suppressive effect was dependent upon the dose of ganglioside, and in the case of MBP stimulation, was not abrogated by the addition of excess MBP. Ganglioside-mediated suppression of LNC proliferation was greatest when the gangliosides were added at the initiation of the in vitro culture period, but significant inhibition was also observed when the gangliosides were added as late as 24 hours after the initiation of the 72 hour culture period. Addition of the glycolipids asialo
GMi and sulfatides did not suppress LNC proliferative responses.
Ganglioside GTib was the most suppressive of the individual gangliosides tested, but was not as effective as the purified rat brain ganglioside mixture. In spite of the suppressive effect on cellular proliferative
91 92 responses, gangliosides did not inhibit the passive transfer of EAE. This finding was demonstrated whether gangliosides were added to the LNC in
vitro activation cultures prior to cell transfer, or whether the gangliosides were injected intravenously into recipient rats following transfer. The analysis of serum ganglioside levels revealed that normal unsensitized rat sera and sera derived from rats 15-18 days following sensitization with MBP-CFA, did not differ in total ganglioside sialic acid content. Moreover, their respective TLC patterns were qualitatively quite similar, showing no significant changes in the major ganglioside species.
The second part of this study focused on determining whether gangliosides affected IL 2 activity, as one possible mechanism of their inhibitory effect. Such an effect could be detected experimentally by suppressed IL 2 production, inhibition of IL 2-lnduced stimulation of responder cells, or by modulation of the cellular receptors for IL 2.
Gangliosides did not decrease antigen or mitogen-stimulated IL 2 production. Exogenous gangliosides did not inhibit the IL 2-induced proliferation of the IL 2-dependent murine cell line, CTLL-20, unless the
IL 2 and high levels of gangliosides were first preincubated, prior to the addition of CTLL-20 cells. Gangliosides cultured with Con A-activated LNC did not affect the binding of a monoclonal antibody directed against the rat
IL 2 receptor, when compared with similar cultures in the absence of gangliosides. Finally, the addition of excess IL 2 to cultures with ganglioside-suppressed proliferative responses, only partially restored the response. From these results, I conclude that ganglioslde-IL 2 interaction is not the primary mechanism by which gangliosides suppress rat 93 lymphocyte proliferation.
The data presented in this study is in accord with previous reports of
ganglioside-mediated suppression of proliferative responses (reviewed by
Marcus, 1984). The ganglioside concentrations found to suppress rat
lymphocyte proliferative responses (5 - 26 pM) were in the same range as
were reported for the suppression of human (Yates et al., 1980) and murine
cells (Ladisch et al., 1983). Yates et al. (1980) added gangliosides (5 - 158
pM) to human PBMC which were stimulated with Con A or allogeneic
lymphocytes. A concentration of 40 pM gangliosides was found to produce
56% inhibition of Con A-induced pHj-thymidine incorporation. Ladisch et
al. (1983) purified gangliosides from murine YAC-I lymphoma cells and reported that 10 - 30 pM gangliosides produced greater than 90%
inhibition of the proliferative response when added to Con A-stimulated mouse splenocytes.
Concentrations of gangliosides higher than 26 pM could not be used in the present study due to the cytotoxicity exerted by the gangliosides on rat lymphoid cells (Figures 1 and 2). The reason for this greater susceptibility of rat versus human lymphoid cells is not clear, although some possible explanations include differential incorporation of gangliosides into rat versus human lymphoid cell membranes, differential effects of gangliosides on essential cell membrane enzymes, or differential effects of gangliosides on cell membrane ion channels. Although studies comparing the interactions between rat and human cells with gangliosides have not been conducted, gangliosides have been shown to enhance the activity of
(Na*,K*)ATPase in crude rat brain synaptosomal preparations (Leon et al., 1981) and to Increase the activity of rat cerebral cortex membrane adenylate cyclase and phosphodiesterase (Daly, 1981). Gangliosides have also been shown to act with calcium, to affect the activity of membrane protein kinases in a human neuroblastoma cell line (Nagai et al., 1985), and
in rat brain membranes (Goldenrlng et al., 1985). The exchange of essential ions, such as calcium could be differentially altered by exogenous gangliosides. In one report, addition of 13 pM GDia produced a 508 reduction in the Con A-induced influx of ^C a** Into AKR/J mouse lymphocytes (Krishnaraj et al., 1983).
Maximum inhibition of LNC proliferation occurred when gangliosides were added at the initiation of the culture period, but there was also significant suppression when the gangliosides were added as late as 24 hours after culture initiation (Figure 4). Only slight inhibition was seen at
48 hours, and no inhibition of proliferation was observed when gangliosides were added just prior to culture termination. Lengle et al. (1979) similarly added exogenous gangliosides at different times during a 48 hour in vitro culture of mouse thymocytes stimulated with Con A. Gangliosides added at the initiation of culture produced a 100% inhibition of proliferation, with a decreasing effect seen when gangliosides were added at later times. Yates et al. (1960) added gangliosides at the initiation of culture as well as 4 hours and 18 hours later to four-day cultures of human PBMC, stimulated with Con A or pokeweed mitogen. Gangliosides added at time 0 produced maximum reduction of l3H]-thymidine incorporation (90% for Con A- stimulated cells). Gangliosides added at 4 hours and 18 hours reduced
Con A-induced proliferation by 66 and 21% respectively. Therefore, 95
ganglioside suppressive effects may begin relatively rapidly, as evidenced
by the 13% inhibition observed here when gangliosides were added as late
as 48 hours into a 72 hour culture (Figure 4). However, the inhibitory
effects of gangliosides are greatest when the gangliosides are in culture
for longer periods. This observation suggests that gangliosides require a
mimimum time period to exert their effects, possibly by intercalating into
the target cell membrane or by being internalized. If such incorporation
were necessary for gangliosides to have an optimal suppressive effect,
then one would expect the greatest inhibition to occur when gangliosides are present in culture for the entire period.
Several investigators have reported that gangliosides rapidly adhere to cell surfaces in vitro, yet, the majority of these glycolipids are sensitive
to trypsin treatment (Moss et al., 1976; Callies et al., 1977; Bremer et al.,
1984). Bremer et al. (1984) have reported that a 48 hour period is necessary in order for exogenous gangliosides to intercalate into the cell plasma membrane in a trypsin-insensitive manner. Therefore, although gangliosides may adhere rapidly to the target cell surface, possibly by binding to surface glycoproteins, a finite period of time may be required for gangliosides to insert into the membrane and exert their suppressive effect. In the report by Lengle et al. (1979), ganglioside-mediated suppression was shown to be reversible if cells were washed free of gangliosides after 4 hours, and recultured with Con A. Yates et al. (1980) found that preincubation of human PBMC with gangliosides for 18 hours followed by washing resulted in Con A-induced proliferative responses which were not significantly different from control cultures not pretreated 96
with gangliosides. However, if the cells were preincubated with
gangliosides for 72 hours before washing, the subsequent proliferative
response to Con A was suppressed. Again, these observations suggest that
the initial binding of gangliosides to the cell surface was easily
dissociated by washing, whereas, after 2-3 days of incubation, the
gangliosides were more stabily inserted into the membrane, and could thus
exert their suppressive effects.
The opposite charge of ganglioside and MBP could potentially result in a
ganglioside-MBP Interaction, which might partially explain the observed
inhibition of MBP-induced cell division. Ong and Yu (1984) analyzed the
nuclear magnetic resonance spectrum of MBP, and found that exogenous GMi
ganglioside bound to an estimated four saturable binding sites on MBP, and
caused conformational changes in the MBP molecule. However, my findings
argue against this ganglioside-MBP association as a principal mechanism
for the suppressive effects of gangliosides. First, gangliosides also
suppressed PPD, PHA, and Con A-induced stimulation, the latter of which
has been shown not to be caused by adsorption of Con A to the gangliosides
(Whisler and Yates, 1980). Additionally, excess MBP (2, 4, 10, and 20 times
the optimal) was unable to abolish suppression (Table 4).
The glycolipids aslalo GMi and bovine brain sulfatide had no inhibitory effects on the antigen or mitogen-induced rat lymphocyte proliferative response (Table 3). The fact that asialo GMi was not found to be
suppressive, along with the reports that neuraminidase treatment of gangliosides partially or totally abrogates ganglioside suppressive effects
(Ryan and Shinitzky, 1979; Gonwaetal., 1984), would suggest that sialic 97
acid is important to the suppressive effect. Other investigators have
shown that sialic acid alone is also not inhibitory (Lengle et al., 1979;
Offner and Vandenbark, 1985). Therefore, sialic acid moieties, together with other portions of the ganglioside molecule, are necessary for maximum ganglioside-mediated suppression of lymphocyte proliferative responses. Sulfatides share the same ceramide backbone structure with gangliosides and have sulfate groups covalently attached to galactose residues, resulting in a net negative charge. The fact that sulfatides did not inhibit MBP or Con A-induced proliferation demonstrates that the negative charge of gangliosides is not solely responsible for their inhibitory effect.
Differential suppressive effects of individual gangliosides have been reported previously ( Lengle et al., 1979; Yates et al., 1980; Offner and
Vandenbark, 1985) and is demonstrated here (Figure 6) where GDib and GTib were shown to be more effective at suppressing MBP stimulation than were the other individual gangliosides tested. The greater suppressive effect of the rat brain mixture could be largely due to other individual ganglioside(s) not tested, or to a synergistic effect of two or more gangliosides. For example, GD2 and GDs gangliosides, both of which contain disialo linkages, were found to constitute 12 and 16% of the rat brain mixture respectively.
Such disialo groups have been shown to be important for the expression of ganglioside-mediated suppression (Yates et al., 1980)
The present study and the report by Offner and Vandenbark (1985) examined the suppressive effects of gangliosides in EAE. Offner and
Vandenbark (1985) utilized an MBP-reactive T-lymphocyte cell line (BP-1), 98
which bears the W3/25 rat T-helper cell marker and is*ncephalitogenic in
Lewis rats. Gangliosides added to cultures containing BP-1 cells, MBP, and
irradiated Lewis thymocytes as a source of accessory cells, significantly
reduced the incorporation of [3Hj-thymidine. The tetrasialylated
ganglioside, GQib, showed the greatest suppressive effect. In other
experiments, BP-1 cells were activated with MBP and accessory cells,
followed by expansion in culture with IL 2. The addition of gangliosides to
these cultures increased cell division time from 26 to 49 hours, and
reduced l3H]-thymidine uptake. Gangliosides also blocked the cel 1-surface
binding of monoclonal antibody W 3/25 to BP-1 cells, as assessed by
immunofluorescence, without similarly affecting the binding of monoclonal
antibodies W3/13 (anti-T-total), OX-8 (anti-T-nonhelper), or OX-6
(anti-rat la).
Offner and Vandenbark (1985) also reported that the addition of
gangliosides at the beginning of a 3 day in vitro culture, containing BP-1
cells, MBP, and accessory cells, did not inhibit subsequent EAE
development, following injection of BP-1 cells into syngeneic Lewis rats.
However, when in vitro activated BP-1 cells were incubated with gangliosides for only one hour prior to injection Into naive rats, EAE
incidence and severity were reduced. In fact, five of the fourteen recipients of ganglioside-treated BP-1 cells showed no clinical signs. In the present study, gangliosides were tested for their effects on the in v itn activated LNC transfer of EAE. I found that the addition of gangliosides to activation cultures did not significantly alter LNC-mediated transfer
(Table 7). LNC, activated in vitro with MBP for 72 hours, then incubated 99
for one hour with gangliosides, still transferred EAE in 3 of 3 recipient
rats (Table 8, group C). There was a slight delay (not significantly
different from group A, Table 8) in the mean day of onset of clinical signs.
Therefore, a one hour incubation with gangliosides did not suppress LNC
transfer of EAE, as was observed with the suppression of BP-1
line-mediated EAE (Offner and Vandenbark, 1985).
This difference in the ability of gangliosides to affect LNC versus BP-1 cell transfer of EAE could be explained in at least two ways. First, in the present study, a heterogeneous population of MBP-activated LNC were used as donor cells and transferred into recipient rats, whereas, Offner and
Vandenbark (1985) used an MBP-reactive long term T-cell line (BP-1).
These two cell populations may differ in their susceptibility to gangliosides. Selection and long-term culture of the BP-1 cell line could result in membrane alterations, which lead to differences between the
BP-1 cells and freshly isolated LNC, in the ganglioside-membrane interactions which occur. Additionally, the mechanisms by which these cells (LNC or BP-1 cells) initiate EAE could also differ, as evidenced by differences in histopathologic changes following transfer. Con A-activated spleen cells and LNC have been shown to produce primarily perivascular mononuclear cell infiltrates in the CNS, following the passive transfer of
EAE (Panitch and McFarlin, 1977). Lesions were shown to be more prominent in the spinal cord, and less intense in the brain stem, cerebrum, and cerebellum, in contrast, EAE induced by the BP-1 encephalitogenic cell line exhibited more diffuse CNS lesions with greater involvement of the cerebrum and brainstem (Vandenbark et al., 1985). Cellular infiltrates, 100
observed in the CNS of rats following injection of BP-1 cells, were
predominantly plasma cells and neutrophils. Therefore, the differences
observed in the ability of gangliosides to alter the cellular transfer of EAE
by either LNC or BP-1 cells could result from inherent differences in the
mechanism of cell transfer.
In both the LNC and BP-1 cell-mediated transfers of EAE, the in vitro
expansion of encephalitogenic cells was not essential for disease to occur
in the recipient. As shown in Table 7, gangliosides, added at
concentrations which suppressed l3H]-thymidine incorporation, had no
effect on LNC transfer of EAE. Similarly, the addition of gangliosides to
the 3 day activation cultures containing BP-1 cells, accessory thymocytes,
and MBP, had no inhibitory effect on subsequent EAE in the cell recipients,
although BP-1 expansion was decreased by 608 (Offner and Vandenbark,
1985). Earlier transfer studies using Lewis rat spleen cells also demonstrated that cellular proliferation in culture was not a prerequisite for passive transfer (Richert et al., 1979 and Richert et al., 1981). In the
latter study, it was shown that prior treatment of the spleen cells with distilled water or ammonium chloride (to lyse erythrocytes) resulted in the
loss of (3H]-thymidine incorporation, in response to MBP stimulation.
However, these cells retained their ability to transfer EAE. These same authors suggested that the clonal expansion of the encephalitogenic cells may indeed be necessary, in vivo, following transfer into the recipient. In support of this suggestion, Richert et al. (1981) reported that the treatment of the spleen cells in culture with mitomycin C or 5-bromo-2‘- deoxyuridine plus light, treatments which inhibit some phase of the 101 replicative cycle, greatly reduced the cellular transfer of EAE.
IL 2 has been shown to be important in the in vitro activation of cells to transfer EAE (Ortiz-Ortiz and Welgle, 1982). Previous work had shown that both spleen cells and LNC, from sensitized donors, could be enhanced in their ability to transfer EAE, following in vitro culture with MBP.
However, spleen cells, but not LNC could be activated with Con A to transfer EAE (Panitch and McFarlin, 1977). Ortiz-Ortiz and Weigle (1982) found that this defect in the ability of Con A to enhance LNC transfer was due to deficient production of IL 2. By altering the Con A concentration in
LNC cultures to maximize production of IL 2, or by directly adding IL 2 to sensitized LNC cultures, these authors demonstrated that IL 2 was indeed essential for full expression of encephalitogenicity in EAE transfer. The present study, concerning the effects of gangliosides, clearly showed that gangliosides did not decrease the production of IL 2 in stimulated LNC cultures (Figure 8). Therefore, although gangliosides suppressed in vitro proliferation or expansion of autoreactive cells, gangliosides did not diminish the production of IL 2, a factor which may contribute to the expansion of the relevant encephalitogenic clones in vivo. This lack of a ganglioside-mediated inhibition of IL 2 production and the possibility that
in vitro expansion of encephalitogenic cells is not necessary for EAE transfer, would explain the inability of gangliosides to inhibit LNC transfer of EAE. Additionally, the effect of gangliosides on EAE transfer was tested in
vivo in the recipient animals. Gangliosides, injected intravenously into recipients one day, three days, or both one and three days following cellular 102
transfer, had no effect on the Incidence of EAE (1008 of the recipients exhibited clinical signs of EAE) or on its severity (Table 8). Due to the cytotoxicity of gangliosides for rat lymphocytes in vitro, I used quantities of gangliosides which would achieve a nontoxic, but still suppressive concentration in the blood (20 pM sialic acid). In the blood, gangliosides have been shown to bind to serum albumin, forming mixed miceilar complexes (Tomasi et al., 1980). In another report (Tettamanti et al.,
1981), the fate of radiolabeled GMi injected intravenously, intramuscularly, or subcutaneously into Swiss albino mice was examined
(Tettamanti et al., 1981). [3H-galactose]-GMi levels in the blood fell greater than 60% within four hours, following i.v. injection. The majority of serum GMi was found to be associated with albumin, and tissue radioactivity was localized predominantly in the liver. Therefore, in my study, possible explanations for the inability of i.v. injected gangliosides to affect EAE transfer are, the small dose of gangliosides used, the complextng of gangliosides with serum albumin, and the rapid clearance of gangliosides from the blood.
Serum ganglioside concentrations from naive rats were compared with the concentrations found in sera from MBP-CFA-sensitized rats. The data revealed no significant change in the detectable levels of ganglioside sialic acid, between the two groups of sera (Figure 7). Similar comparisons have been made between ganglioside concentrations found in normal human sera and sera from MS patients. Sela et al. (1982) reported that MS sera showed significant increases in sialic acid when compared with normal control sera. The TLC pattern of gangliosides extracted from MS sera (Sela et al., 103
1982) did not show increases in the common brain gangliosides, similar to my results with EAE sera (Plate II). This suggests that, in both MS and EAE, there is little or no detectable release of brain gangliosides into the peripheral circulation. The possibility exists that excess gangliosides may be rapidly cleared from the blood to the liver.
In the second part of this study, I examined the effect of gangliosides on IL 2 production and activity. Gangliosides have been shown to inhibit the
IL 2-dependent growth of the CT-6 murine T-cell line (Merritt et al.,
1984b). CT-6 cell division and [3H]-thymidine incorporation were observed to be inhibited by exogenous gangliosides, in a dose-dependent manner. In contrast, I found that exogenous gangliosides did not suppress the IL 2-
Induced proliferation of the murine T-cell line CTLL-20 (Figures 9 and 10), unless the IL 2 and gangliosides were first preincubated, prior to the addition of the CTLL-20 cells (Figure 11). The disparity between my results and those of Merritt et al. (1984b) concerning the ability of gangliosides to inhibit the proliferation of IL 2-dependent cell lines could result from a number of factors. First, the CT-6 cell line is reported to become relatively unresponsive to IL 2 during certain culture passages
(Merritt et al., 1984a). In fact, the addition of gangliosides was shown to restore CT-6 cell IL 2 responsiveness partially . However, CTLL-20 cells continuously express IL 2 receptors and have no IL 2 unresponsive phase
(Glllis et al., 1978). The two cell lines may also differ in their rate of uptake of rat IL 2, or in their affinity for IL 2. Secondly, Merritt et al.
(1984b) used commercially obtained bovine brain gangliosides, whereas, 104 gangliosides in the present study were purified from rat brains.
Additionally, the IL 2-containing supernatants used in the two studies were prepared differently (24 hour versus 48 hour rat ConA-activated supernatant) and thus probably vary in molar IL 2 concentration and in the levels of other secreted factors. Therefore, the various technical differences between the present study and the report by Merritt et al.
(1984b), could contribute to the disparate results obtained.
Gangliosides did not inhibit the proliferation of CTLL-20 cells when
CTLL-20 cells, IL 2, and gangliosides were added simultaneously at the initiation of culture (Figure 9A). However, when IL 2 was first preincubated with gangliosides for various times before addition of CTLL-20 cells, proliferation was decreased (Figure 11). A 50% reduction in CTLL-20 cell proliferation was observed only with 80 pM gangliosides (a concentration that is toxic for rat LNC), following a 7 - 24 hour preincubation with IL 2.
The small effect observed at 4 hours could be explained in terms of a rapid association between IL 2 and its receptor on the surface of the CTLL-20 cells, thus not allowing sufficient time for gangliosides to bind to IL 2, unless the IL 2 and gangliosides were first preincubated in the absence of cells for longer time periods. Smith (1980) demonstrated that binding of
IL 2 to Con A-activated murine splenocytes occurred rapidly, since these
IL 2 receptor bearing cells absorbed greater than 80% of the total IL 2 activity in one hour. Additionally, the IL 2-containing supernatant preincubated with gangliosides in the present study, contained only 13 units IL 2/m l, as compared with the levels detected in Con A-activated LNC cultures shown in Table 10(126- 185 units/ml). Under these 105 preincubation conditions, ganglioside-IL 2 association resulted in 508 reduction in CTLL-20 cell proliferation only when 80 pM gangliosides were incubated with 13 units IL 2/m l, for 7 or 24 hours (Figure 11). Therefore, the fact that a ganglioside-IL 2 interaction was observed, under certain conditions and, resulted in less available IL 2, may not be of prime importance to ganglioside-mediated suppression of proliferative responses, in light of the time factors and the relative ganglioside-IL 2 concentrations involved.
It has been suggested that the suppressive effect of gangliosides could be due, in part, to direct ganglioside-IL 2 association, resulting in less IL 2 available to stimulate the responder cells (Parker et al. 1984 and Robb,
1966). Parker et al. (1984) showed that gangliosides, covalently attached to poly(L-lysine)-agarose columns, were able to retain IL 2 activity when
IL 2-containing supernatants were passed through the columns. While completing my studies, a report by Robb (1986) appeared which described the suppressive effect of gangliosides on IL 2-dependent proliferation, mediated through direct IL 2-ganglioside association. In this study, gangliosides were shown to inhibit the PHA-induced proliferation of freshly isolated human lymphocytes and the IL 2-dependent growth of both murine T-cell lines and human PHA-stimulated lymphoblasts. In agreement with my findings (Table 11), Robb (1986) found that exogenous IL 2 was unable to reverse the ganglioside-mediated suppression of freshly isolated mitogen-stimulated lymphocytes. However, Robb (1986) reported that exogenous IL 2 was able to reverse up to 808 of the suppression of IL 2- dependent proliferation. This reversible inhibition caused by gangliosides 106 was shown by Robb (1986) to be due to a ganglioside-IL 2 interaction with subsequent loss of IL 2 activity. GTib inhibited the binding of [3H]-IL 2, but not (3H)-anti-Tac (anti-human IL 2 receptor) antibody to the human cell line
HUT 102B2, which expresses IL 2 receptors. Gangliosides GMi and GT ib also reduced the binding of [3H]-IL 2 to one monoclonal and one polyclonal antibody directed against the IL 2 molecule. Therefore, gangliosides are able to suppress proliferative responses by more than one mechanism, and a direct ganglioside-IL 2 association is not the primary mechanism involved in the inhibition of freshly isolated lymphocyte proliferative responses to antigen and mitogen.
Cantrell and Smith (1984) have proposed that progression of
T-lymphocytes through the cell cycle is a predictable series of events which depends upon three factors: a critical threshold of available IL 2,
IL 2 receptor density on the responding cells, and the duration of the
IL 2-receptor interaction. Therefore, a study such as the present one, which attempts to investigate the role of the IL 2 system in determining alterations in the proliferative responses of a cell population, is necessary.
While data from the present study does not argue in favor of a primary role for ganglioside-IL 2 interactions in ganglioside-mediated suppression of proliferation, there is the suggestion of some contribution. Additionally, the report by Robb (1986) using human cells suggested a primary role for ganglioside-IL 2 association in controlling cellular proliferation of IL 2- dependent lymphoid cells. The possibility of species differences in the two systems, accounting for the different level of significance placed upon this ganglioside-IL 2 interaction, cannot be overlooked. 107
If future reports point to a ganglioside-1L 2 association as being an important mechanism to explain the suppressive effects of gangliosides on lymphocyte proliferation, then further investigations may focus on ganglioside interactions with other cell growth factor systems, such as was examined by Bremer et al. (1984). These investigators examined the effects of gangliosides on the proliferative responses of mouse fibroblast
3T3 cells, incubated with platelet-derived growth factor (PD6F) or epidermal growth factor (E6F). GMi, and GM3 gangliosides inhibited PDGF and EGF-induced proliferation, but did not affect the binding of these factors to their cell surface receptors, nor did the gangliosides affect the number of receptor sites. Bremer et al. ( 1984) did find that the gangliosides increased the affinity of the PDGF receptors for their ligand, possibly through reduced phosphorylation of the receptor protein. In contrast, Robb (1986) found that gangliosides inhibited IL 2-dependent growth by directly binding to IL 2. Exogenous gangliosides did not alter the apparent dissociation constant of high affinity IL 2 receptors for their ligand.
The question of what causes the spontaneous remission from EAE, seen in the Lewis rat model, remains unanswered. A better understanding of the initiation process of EAE and effector mechanisms operative in EAE, w ill undoubtedly add to our understanding of the recovery process. I believe that a tenable model for the recovery from acute EAE, can now be proposed, based upon available data in the literature, and upon the assumption that the CNS inflammatory response in EAE, is directly involved in the 108
development of the observed clinical signs. Such a recovery model would
have three primary components. First, within the CNS itself, immune regulatory factors, which also act peripherally to lim it the duration of any
inflammatory reaction, would shut-down the destructive events (e.g. edema) within the brain and spinal cord. This would lead to clinical remission. Secondly, the encephalitogenic T-cells, which somehow initiate the inflammatory response, may have a limited effector life span. At the time of recovery, these cells would no longer be able to initiate new disease. The final piece of this model would involve suppressor cells, which act peripherally within the lymphoid organs to prevent the generation of new T-effector cells. The latter two portions of this model
(limited life span of effector T-cells and suppressor cells) were also suggested recently by Willenborg et al. (1986).
The role of macrophages in both disease pathology and recovery may be given more attention in the future, since it has been reported that macrophages are essential to the development of acute EAE in the Lewis rat
(Brosnan et al., 1981). In this report, macrophage depletion by the intraperitoneal injection of silica dust, reduced the number of peritoneal exudate macrophages, delayed EAE onset, and significantly reduced the incidence of paralysis. Macrophages have also been shown to be necessary for in vitro activation of cells for the enhanced passive transfer of EAE
(Panitch and Ciccone, 1981; Kilien and Swanborg, 1982). Panitch and
Ciccone (1981) incubated spleen cells, derived from MBP-CFA sensitized
Lewis rats, with Con A or MBP for 48 hours prior to intravenous transfer into naive recipient rats. Removal of adherent macrophages by Sephadex 109
G -10 column filtration reduced the Incidence and severity of EAE in the recipients. This response was restored with the addition of peritoneal exudate cells from either normal unsensitized or immune rats. Kiilen and
Swanborg (1982) reported that spleen cells from MBP-IFA-tolerized Lewis rats could transfer EAE after in vitro MBP or Con A activation (Killen and
Swanborg, 1982). Donor LNC transferred disease after culture with MBP, but Con A-activation was achieved only after the addition of adherent phagocytic cells or culture supernatants from Con A-stimulated adherent cells, obtained from normal Lewis rat spleens.
Macrophages have also been implicated in the pathology of MS. Cellular infiltration into the CNS in MS includes macrophages (McFarlln and
McFarland, 1982). Macrophage products, such as neutral proteases, have been suggested as contributing factors in MS demyelinatlon
(Cammer et al., 1978). Prineas and Connell (1978) reported that macrophages removed myelin directly from intact myelin sheaths. In another report, surface capped IgG was observed on macrophages, which were observed to be in close association with active lesions, In the brains of two MS patients (Prineas and Graham, I960). These authors suggested that the IgG caps were antibody directed against myelin antigens, which then attached to the surface of macrophages via Fc receptors, thus facilitating macrophage association with the myelin sheath.
Brain gangliosides may affect the course of EAE by acting upon macrophages in the brain tissue itself. Gangliosides have been shown to bind to human adherent monocytes to a greater degree than to either T or B cells (Yates et al., 1980). In this study, (14C]-gangliosides were incubated 110
with separated cel) subpopulations for 24 hours prior to harvest, and
adherent monocytes bound greater than two times more ganglioside than
the other two subpopulations. Additionally, Ladisch et al. (1984) reported
that gangliosides modulate proliferative responses of human PBMC by
preferentially inhibiting accessory cel) function. In this study, PBMC were
separated on the basis of plastic adherence (adherent cells were >95%
esterase positive), then either the adherent or nonadherent subpopulations
were treated with gangliosides. The cells were then recombined and
stimulated with the antigens, tetanus toxoid or streptokinase-
streptodomase. Pretreatment of adherent cells with gangliosides resulted
in suppressed proliferative responses, whereas, gangliosides added to nonadherent cells did not similarly affect subsequent proliferation.
Macrophage surface binding properties have been shown to be influenced by exogenous brain gangliosides (Riedl et al., 1982). Sheep red blood cells were pretreated with gangliosides, incubated with rat alveolar or peritoneal macrophages, then these cell mixtures were assayed for macrophage-erythrocyte rosette formation. Gangliosides GM 2, GDia, and a mixture of bovine brain gangliosides Induced 60-90% rosette forming cells, whereas, GMi, asialo GM 2. and no ganglioside pretreatment of sheep red blood cells, produced no rosette formation. These authors speculated on the possibility of specific receptors on the surface of rat macrophages for certain ganglioside structures. Therefore, any future examination of ganglioside regulation in CNS autoimmune disease should consider the effect of gangliosides on the inflammatory macrophage. I l l
Any study of ganglioside suppressive effects w ill necessarily have to
consider the total spectrum of ganglioside effects observed to date, which
include effects on growth factors, cell surface receptors, enzymes, and other surface proteins. Future studies must also include careful selection of the relevant gangliosides to use in a particular investigation. Earlier studies primarily used brain-derived gangliosides to examine the effects of gangliosides on lymphoid cells (Lengle et al., 1979; Whisler and Yates,
1980; Merritt et al., 1984b). However, it is known that brain gangliosides differ dramatically in composition from lymphocyte or serum gangliosides
(Ledeen, 1983). Of special importance then is the fact that individual gangliosides exert varying effects on cells, and therefore, the use of a mixture of CNS-derived gangliosides to examine immunological phenomena should be evaluated with caution. REFERENCES
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