Gangliosides and bacterial toxins in Guillain-Barré syndrome

Journal of Neuroimmunology, 46 (1993) 105-112 © 1993 Elsevier Science Publishers B.V. All rights reserved 0165-5728/93/$06.00

105

JNI 02410

Gangliosides and bacterial toxins in Guillain-Barr6 syndrome H.J. Willison and P.G.E. K e n n e d y University Department of Neurology, Institute of Neurological Sciences, Southern General Hospital, Glasgow, UK (Received 1 February 1993) (Revision received 18 March 1993) (Accepted 19 March 1993)

Key words: Autoantibody; Guillain-Barr6 syndrome; Miller-Fisher syndrome; Ganglioside; Bacterial toxin

Summary Autoimmune factors are strongly favoured as mediating Guillain-Barr6 syndrome (GBS); however, the precise mechanisms by which this occurs remain unknown. Microbial infections in a susceptible host resulting in an idiosyncratic immune response which cross-reacts with nerve constituents still remains the most plausible working hypothesis on which much current research is based. Considerable recent evidence indicates that this humoral immune response is at least in part directed to gangliosides. Interestingly, many bacterial toxins, including botulinum and tetanus neurotoxins, also bind to gangliosides and induce diseases with some similarities to GBS. This article discusses the evidence in favour of a pathogenic role for anti-ganglioside antibodies in GBS in the context of our knowledge of the biology of gangliosides and the factors that determine their immunogenicity.

Introduction Guillain-Barr6 syndrome (GBS) is believed to be mediated by an autoimmune reaction to peripheral nerve antigens, as has been cogently argued by McFarlin (1990) and others in recent reviews (Toyka and Heininger, 1977; Hartung et al., 1988). The relative contributions of humoral and cellular immunity to this reaction are unknown although it is likely that both are necessary for the full expression of the disease. Evidence accummulated throughout the 1980s to indicate that humoral immunity to gangliosides and related glycolipids is frequently observed in patients with chronic demyelinating peripheral neuropathies associated with IgM paraproteinaemia (Quarles et al., 1986; Latov, 1990). Considerable debate still surrounds their relevance to this disease process. However, in the light of the paraproteinaemic neuropathy data, fruitful searches were made for antibodies of a similar specificity for gangliosides in other acute and chronic demyelinating neuropathies, particularly the GuillainBarr6 syndrome. A large amount of recent evidence is

Correspondence to: H.J. Willison, University Department of Neurology, Southern General Hospital, Glasgow, G514TF, Scotland, UK.

now available to indicate that anti-ganglioside immune responses play a significant role in the pathogenesis of GBS. This article discusses these findings in relation to our current understanding of ganglioside structure, function and immunology.

Clinical and serological correlations The first report on the presence of anti-ganglioside antibodies in GBS demonstrated reactivities of various specificities in 20% of patients (Ilyas et al., 1988). Many subsequent reports have observed broadly similar findings (summarised and referenced in Table 1). Although the data remain incomplete, several patterns are emerging. Firstly, anti-GM1 ganglioside antibodies have been observed in up to 30% of cases in some series (Oomes et al., 1991; Gregson et al., 1993). In contrast to the IgM isotype of anti-GM1 antibodies observed in chronic neuropathies (Pestronk, 1991), t h e GBS-associated anti-GM1 antibodies are often IgG or IgA (Ilyas et al., 1992b). The antibodies frequently but not invariably cross-react with other terminal GaI(/313)GalNAc containing glycolipids such as GDlb and asialo-GM1 (Walsh et al., 1991). The antibody titres are often high and disappear with clinical recovery.

106 TABLE 1 Frequency and specificity of anti-acidic glycolipid antibodies in GBS and Miller-Fisher syndrome (MFS) Reference

Syndrome

Frequency

Isotype

Glycolipid antigens

Ilyas et al., 1988

GBS

Svennerholm and Fredman, 1990

GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS GBS MFS MFS GBS GBS

1/26 2/26 2/26 29/50 1/50 1/50 2/2 1/1 14/95 34/100 10/23 15/23 4/22 5/16 36/53 11/53 12/53 16/53 3/10 18/53 6/6 4/4 2/20 12/42

IgG IgG IgM IgG/IgM IgG/IgM IgG/IgM IgG IgG/IgM IgG/IgM IgG/IgM IgG > IgM IgG IgG/IgM/IgA IgG/IgM IgG/IgM IgG IgG/IgM IgG/IgM IgG IgA IgG IgG IgG IgG/IgM

LM1, hexaose GDlb GDla, GTlb LM1 GM1 GDlb, GTlb GM1 GM1 GM1 + GDlb GM1 LM1 Sulphatide GM1, GDlb, GA1 GM1 + GDlb Acidic glycolipids LM1 GDla and/or GTlb GM1 and/or GDlb GDla 5: GTlb GM1 GQlb GQlb GM1 GM1, GDlb, GA1

Yuki et al., 1990 Gregson et al., 1991 Walsh et al., 1991 Oomes et al., 1991 Fredman et al., 1991 Van den Berg et al., 1992, Nobile-Orazio et al., 1992 Ilyas et al., 1992a,

Yuki et al., 1992 Ilyas et al., 1992b Chiba et al., 1992 Willison et al., 1993 Gregson et al., 1993

Patients with GBS who are positive for anti-GM1 antibodies have a more severe illness with prominent motor axonal involvement and incomplete recovery when compared with anti-GM1 antibody negative cases (Yuki et al., 1990; Gregson et al., 1993). The prominent motor involvement is consistent with the clinical pattern observed in chronic motor syndromes associated with anti-GM1 IgM antibodies. There is also a significant association with a prodromal infection by certain strains of Campylobacter jejuni (Yuki et al., 1990; Koblar et al., 1991; Oomes et al., 1991; Saida et al., 1991), the relevance of which is discussed below. Secondly, anti-GQlb IgG ganglioside antibodies appear with a very high frequency, currently 100% of ten reported cases in the clinical variant of GBS termed the Miller-Fisher syndrome (Chiba et al., 1992; Willison et al., 1993). Antibodies to LM1, a major ganglioside of peripheral nerve, have also been noted in 60% of cases in one series (Svennerholm and Fredman, 1990) and 20% in another (Ilyas et al., 1992a). Other specificities occur with lower frequency although may be of pathogenic importance in those cases in which they are found. For example two cases of fulminant GBS have recently been reported in association with anti-GDla IgG antibodies (Yuki et al., 1992). Considerable overlap exists in the clinical features of the regional syndromes seen in GBS. How these correlate with anti-ganglioside antibody specificity is at present unknown. It will be particularly interesting, for example, to examine the anti-GQlb antibody profile of patients with ophthalmoplegia at the onset of GBS

who rapidly evolve widespread limb weakness and, conversely, patients who present with limb weakness and then later develop ocular involvement. One might predict that a severe, global, peripheral nerve root or trunk demyelination affecting all myelinated nerves including ocular motor nerves would have a different immunopathological basis from the selective ocular motor nerve failure seen in Miller-Fisher syndrome (MFS); the relationship to varying regional concentrations of target antigens is clearly of great interest. Until the methodology for detecting and reporting anti-ganglioside antibodies is standardised, it will remain impossible to derive demographic data about the frequency of particular antibodies in different patient groups, which are likely to exist. For example, the frequency of anti-GM1 antibodies in different series of GBS patients varies from 2% or less (Ilyas et al., 1988; Svennerholm and Fredman, 1990) to around 30% (Oomes et al., 1991; Ilyas et al., 1992; Gregson et al., 1993). We have observed anti-GM1 IgG antibodies in 10% of our last 20 consecutive cases (Willison et al., 1993). Regardless, it is clear to many researchers that the clinical observations on anti-ganglioside antibodies in GBS are highly significant. Establishing their origin and pathogenic potential is now a major goal.

Generating an i m m u n e response to gangliosides

Anti-ganglioside antibodies may arise through a number of different mechanisms, resulting in differ-

107 ences in antibody isotype, affinity, quantity and duration which appear to have pathogenic relevance, reflected in differences in the disease pattern induced by such antibodies. For example, the transient presence of high affinity IgG anti-ganglioside antibodies seen in GBS is in clear distinction to the persistent low affinity monoclonal or polyclonal IgM antibodies of similar specificity found in chronic motor neuropathies (Latov, 1990; Pestronk, 1991). Anti-GM1 and anti-asialo-GM1 antibodies are frequently found in normal sera (Latov, 1990). In addition, Epstein-Barr virus (EBV) transformed umbilical cord B-cells which are naive to foreign antigen (Lee et al., 1990) and adult B-cells (Willison, unpublished resuits) can secrete anti-GM1 IgM antibodies from donors with low or absent serum antibody titres. Pokeweed mitogen stimulated peripheral blood B-cells (Heidenreich et al., 1992) and normal tonsillar lymphocyte preparations stimulated with bacterial superantigens (Willison, unpublished results) can also be readily induced to secrete anti-GM1 and anti-asialo-GM1 IgM antibodies. Normal sera more rarely contain anti-ganglioside antibodies of other specificities such as GM3, GDla or the polysialylated gangliosides, GTlb and GQlb but frequently contain IgM antibodies against neutral or acidic glycolipids such as globoside, sulphatide and sulphate-3-glucuronyl paragloboside (SGPG) (Latov, 1990; Ilyas et al., 1991; Willison et al., 1993). In our experience, it is uncommon to find any anti-ganglioside IgG antibodies in normal serum. Taken together, these data suggest that some anticarbohydrate antibodies form part of the naturally occurring autoantibody repertoire that comprises lowaffinity, polyreactive IgM antibodies encoded by immunoglobulin variable region genes in germline configuration. These antibodies may have an immunoregulatory role in shaping immune maturation and may act as a first-line defence against invading micro-organisms. This is supported by the observation that anti-asialoGM1 (Graves and Ravindranath, 1992) and anti-MAG (Lee et al., 1991) antibodies can arise from the CD5 ÷ B-cell subset and it may be that other anti-ganglioside antibodies also arise from this subset. CD5 ÷ B-cells have recently received considerable attention in relation to their role in autoimmunity (Casali and Notkins, 1989; Talal et al., 1992). Whether disease-associated anti-ganglioside IgM a n d / o r IgG antibody producing B-cells arise from this or a distinct B-cell population is not yet known. The factors that regulate the immune response to carbohydrate antigens are entirely different from those that govern antibody responses to proteins. Carbohydrates containing repeating sugar sequences can activate B-cells by cross-linking their surface immunoglobulin receptors in a T-cell-independent (TI) but antigen-specific manner or alternatively can polyclon-

ally activate B-cells. This results in the predominant production of long-lived IgM responses and any IgG which is produced is transient and usually of the immediately downstream IgG3 isotype. Activation via this route does not induce classical memory and thus on re-exposure to antigen there is no secondary immune response. Synaptosomal or myelin membranes in the form of liposomes or microbial cell wall and membrane fragments containing repeating arrays of gangliosides or cross-reactive glycoproteins may be able to activate B-cells in this way. It is possible that the polyclonal IgM anti-GM1 responses seen, for example, in patients with multifocal motor neuropathies (Pestronk, 1991) arise through such TI mechanisms. T-cell-derived cytokines present in the vicinity of activated B-cells in peripheral lymphoid organs may be capable of modulating this type of TI response by providing non-cognate help which could lead, for example, to class switching from IgM to IgG isotypes or to affinity maturation; these features, however, are more typical of the cognate T-cell help that occurs with T-dependent antibody responses. T-helper cells help B-cells to generate a humoral immune response by binding processed peptide antigen presented to the T-cell receptor (TCR) by major histocompatibility complex (MHC) class II molecules expressed on the B-cell surface. This trimolecular complex comprising TCR, antigen and MHC class II forms the basis for the cognate, T-cell-dependent interactions seen with peptide antigens. Affinity maturation, class switching from lgM to IgG and recruitment of B-cells into the memory compartment are typical features of this type of response. There is strong evidence that complex carbohydrate molecules, such as gangliosides, cannot be intracellularly processed and presented by MHC class II molecules in this way (Ishioka et al., 1992). Thus, in order to induce the high affinity IgG anti-ganglioside antibody responses that are seen in GBS, gangliosides cannot stand alone as immunogens but must exist as a hapten-carrier complex (i.e., bound to a protein) or as a cross-reactive glycoprotein. In this situation, a pre-existing anti-carbohydrate antibodypositive B-cell (possibly from the CD5 ÷ B-cell pool) would act as an antigen-presenting cell (APC) by (i) endocytosing the hapten-carrier complex via surface Ig specific for the carbohydrate moiety, and (ii) processing and presenting a peptide fragment from the carrier protein to the T-cell. The T-cell would thus be activated to provide the contact and cytokine-mediated help necessary for B-cell class-switching and affinity maturation. IgG anti-ganglioside antibodies arising in GBS are likely to be produced through this mechanism. What is the source of the carbohydrate-protein complex that drives this response? The timing of onset of GBS, usually 2 weeks following an infection, indicates

108 that the neuritis arises as result of a primary rather than secondary immune response to that infection (McFarlin, 1990). In most cases in which anti-ganglioside IgG antibodies have been identified, the titres are already falling at the time of clinical onset, as judged by analysis of sequential samples, and do not persist indefinitely (Ilyas et al., 1988; Yuki et al., 1990). The implication of this is that degenerating nerve is unlikely to be the source of the anti-ganglioside response in GBS. This is supported by the observation that antiganglioside antibodies produced following nerve injury in the mouse peak at 40 days and may be persistent (Schwartz et al., 1981). The principle that anti-ganglioside antibodies may arise in response to nerve injury and then contribute to on-going pathology may, however, be relevant to chronic neuropathies. This has been argued as a possible explanation for the higher frequency of anti-LM1 and anti-sulphatide antibodies in chronic inflammatory neuropathy compared with GBS (Fredman et al., 1991). Alternative mechanisms that invoke an endogenous neural source for the ganglioside antigen in GBS, such as association of ganglioside with a neurotropic virus or bacterial toxin, presupposes that these initiating events are clinically silent. It is more likely that initiating immunological events occur at the site of infection and its associated secondary lymphoid organs and that peripheral nerve is an innocent bystander until the immune response to the prodromal illness is fully activated. The presence of anti-ganglioside IgA antibodies in some GBS cases is good evidence that immune reactions at mucosal surface are important (Ilyas et al., 1992b; Van den Berg et al., 1992). This is consistent with common sites of preceding infection being in the upper respiratory or gastrointestinal tract (Winer et al., 1988). Many different bacterial and viral infections precede GBS and many different IgG anti-ganglioside antibody specificities have been identified which could bind the wide array of candidate antigens in peripheral nerve. Some associations, such as that between Campylobacter jejuni infection and anti-GM1 antibodies are emerging (Yuki et al., 1990; Oomes et al., 1991) although are clearly not absolute (Saida et al., 1991; Gregson et al., 1993). The search for a single antigen in GBS thus seems naive since it is likely that there are many routes to the same end which share a common overall mechanism. By analogy, the fine specificity of anti-carbohydrate antibodies found in cold agglutinin disease varies considerably although there is a common clinico-pathological outcome (Pruzanski and Shumak, 1977). It is, however, becoming clear that some of the regional variations in GBS may be due to particular anti-ganglioside antibody specificities, such as the anti-GQlb antibodies in the Miller-Fisher syndrome (Chiba et al., 1992; Willison et al., 1993) and the prominent motor involvement occurring with anti-GM1 antibodies (Yuki

et al., 1990). When more monoclonal anti-ganglioside antibodies become available, ideally derived from GBS patients, it will be possible to systematically screen relevant microbes for cross-reactive glycoprotein determinants and then to undertake the search for T-cell epitopes carried by those glycoproteins which may drive the B-cell responses. The role of cytokines and cytotoxic T-cells in GBS has not been discussed here but is clearly important. The time course of GBS in relation to the prodromal illness is consistent with the minimum time required to develop a primary cytotoxic T-cell response. There is strong evidence of increased T-cell proliferation in GBS, possibly related to increased interleukin-2 (IL-2) production by T-helper cells (Hartung et al., 1992). This may be necessary for the (full) expression of the disease, either as a primary cytotoxic response to nerve or by perturbing other factors such as the vascular endothelial cells and blood-nerve barrier, thereby allowing ingress of pro-inflammatory agents, including complement components and anti-ganglioside immunoglobulin.

Lessons from bacterial toxins

Many bacteria are more readily able to colonise and attack their hosts through production of toxins which are proteins comprising functionally different polypeptide domains. The binding domains of these toxins frequently use gangliosides or cross-reactive glycoproteins as their cell surface receptors, which on binding produces conformational changes in the cell membrane that allows the ingress of the toxicity-mediating domain. The toxins produced by strains of Campylobacter jejuni, Escherichia coli and Vibrio cholerae can all bind to GM1 ganglioside and variably to other gangliosides and glycoproteins (Cautrecasas, 1973; Hansson et al., 1977). For example, the heat-labile E. coli enterotoxin, LT-1, also interacts with asialo-GM1 and GM2 in addition to a series of brush border galactoproteins (Griffiths and Critchley, 1992). Since toxins from these organisms are rarely absorbed into the circulation, they do not have any systemic toxic effects. In situations where they have been artificially introduced into the nervous system they have produced neuropathological lesions very similar to those induced by anti-GM1 antibodies (Schwerer et al., 1986). It is, however, possible that they are involved locally in the induction of cross-reactive immunity to neural gangliosides. A possible mechanism that has been suggested is that the GMl-binding bacterial toxin may act as the carrier in a hapten-carrier complex, thereby providing the protein component required for T-cell help. Since a GM1 binding toxin would block the ganglioside domain recognised by the B-cell surface immunoglobulin, this

109 would have to occur in the context of a polyvalent carbohydrate antigen incompletely saturated by toxin. Although Campylobacter jejuni is a frequent preceding infection in GBS associated with anti-GM1 IgG antibodies, there are complicating factors to this argument: (i) not all Campylobacter strains produce GM1 binding toxins, (ii) Campylobacter strains may contain cell wall glycoproteins cross-reactive with GM1 which could in themselves act as immunogens, (iii) anti-GM1 antibody associated GBS occurs without evidence of Campylobacter infection, (iv) GBS preceded by Campylobacter infection occurs without anti-GM1 antibodies, and (v) Vibrio cholerae infection, which invariably produces GMl-binding cholera toxin, is not reported in association with GBS. Bacterial toxins produced by Clostridium botulinum and tetani are systemically absorbed from the site of infection and induce profound neurological disease following uptake at presynaptic membranes in cholinergic neurons. Curiously, botulism causes a clinical syndrome very similar to Miller-Fisher syndrome, comprising prominent ophthalmoplegia but can affect all neuromuscular junctions. Tetanus toxin blocks inhibitory spinal cord neurons following retrograde transport from the periphery. The seven subtypes of botulinum toxin and tetanus toxin are biologically similar. Tetanus toxin binds with highest affinity to GTlb and GQlb, less well to GDlb and with low affinity to GDla and monosialo-gangliosides (Walton et al., 1988). Botulinum toxins A - G have slightly variable binding profiles but in general bind with highest affinity to GTlb and GQlb (Kitamura et al., 1980; Kamata et al., 1986). In Miller-Fisher syndrome, anti-GQlb antibodies are strongly linked to the disease in that they have been observed in all patients so far reported (Chiba et al., 1992; Willison et al., 1993). Since Miller-Fisher syndrome and botulism share common clinical features, it is likely, at least in the motor system, that the neurotoxin and the anti-GQlb antibodies bind to the same GQlb (or GQlb cross-reactive) receptor which has regional variations in concentration, being particularly high in the extra-ocular muscles. This may in turn be a reflection of the high density of neuromuscular junctions at that site. Interestingly, a reference laboratory for human butulism (Notermans et al., 1992) has been able to passively transfer a botulism-like illness to recipient mice from patients suspected of having botulism who were later diagnosed as having GBS. They localised the pathogenic activity to the serum immunoglobulin fraction, concluding it was antibody: it is possible that it contained anti-GQlb antibodies although this was not tested. The similarities between botulism (mediated by a toxin known to bind GQlb) and Miller-Fisher syndrome are very strong evidence that the anti-GQlb antibodies in Miller-Fisher syn-

drome account for a central element of the disease. This can readily be tested in experimental models, particularly since the proposed site of antibody binding at the presynaptic region of the neuromuscular junction is outwith the blood-nerve barrier. Tetanus has a broader regional distribution in that it frequently involves limb, paraspinal, respiratory and facial muscles; ophthalmoplegia is not a usual feature. It is likely that the differences in regionality of botulism and tetanus can be accounted for by differences in ganglioside or cross-reactive glycoprotein antigen distribution. Although this evidence strongly reinforces the role of anti-GQlb antibodies in mediating Miller-Fisher syndrome, many paradoxes persist. For example, in chronic sensory neuropathies associated with monoclonal IgM cold agglutinins, binding to GQlb and cross-reactive gangliosides is frequently observed (Arai et al., 1992; Duane et al., 1992). Although there is some clinical overlap with these cases and the MillerFisher syndrome, such as the prominence of ataxia (Willison et al., 1993), it is marginal and botulism is clearly a very different illness since sensory features are absent. Either the neural ligands for botulinum toxin and cold agglutinin associated anti-GQlb antibodies have some structural and regional differences or the toxin and antibodies have differences in fine specificity to account for differences in clinical manifestations despite apparently similar reactivity.

Ganglioside function and localisation Following induction of high circulating levels of anti-ganglioside antibodies as a result of the prodromal illness, there are two major ways in which the antibodies may induce tissue damage. On binding to antigen they may either activate pro-inflammatory pathways or disrupt ganglioside-mediated metabolic functions. Although gangliosides are enriched i n the peripheral nervous system (Fong et al., 1976) making them potential targets for autoimmunity in this site, they are widely distributed plasma membrane components of all cell types ranging from epithelial cells to lymphocytes (Hakamori, 1990). They have an amphipathic structure comprising a ceramide portion inserted in the lipid bilayer and a sialic acid containing carbohydrate structure of variable composition exposed on the extracellular cell surface which may readily act as a ligand for antibody. They may be secreted into the extracellular fluid, particularly when shed by tumours, and can reach high levels in the circulation where they bind to plasma proteins and lipoproteins (Ladisch et al., 1992). Gangliosides exhibit enormous functional diversity. They are frequently recognised by monoclonal antibodies as stage-specific differentiation antigens and blood group antigens; they are involved in cell-cell recogni-

110 tion and act as receptors for bacterial lectins and toxins; they are potent immunomodulators and they influence cell growth and differentiation in a wide range of systems (Hakamori, 1990). Most of the data on ganglioside function have been derived by observing the influence of adding gangliosides to the experimental system; however, it is equally possible that antiganglioside antibodies might exert a negative effect on the same ganglioside-mediated processes, although this approach has not been widely explored experimentally. In the immune system, for example, gangliosides are potent inhibitors of T-cell proliferation and it is possible that the presence of anti-ganglioside antibodies might reverse this effect, thereby enhancing T-cell activation (Ladisch et al., 1992). Several studies on experimental allergic neuritis (EAN) have shown that the administration of exogenous gangliosides does not enhance or attenuate the disease through an immunomodulatory mechanism (Ponzin et al., 1991; Zielasek et al., 1993). Since the experimental protocols in these EAN studies have not resulted in the induction of anti-ganglioside antibodies, it remains unclear whether such antibodies, if present, would influence the course of the disease. In an interesting series of experiments, GM1 ganglioside has been shown to inhibit complement fixation by asialo-GM1 antibodies bound to asialo-GM1 in lymphocyte membranes which readily fix complement in the absence of GM1 (Ishii and Watanabe, 1992). Thus the function of gangliosides and naturally occurring anti-ganglioside antibodies may in part be immunomodulatory and a similar but aberrant role for GBS-associated anti-ganglioside antibodies has to be considered, in addition to a direct immunopathogenic effect on nerve. In view of the ubiquitous distribution of gangliosides, the restriction of pathology to the peripheral nervous system in GBS associated with anti-ganglioside antibodies seems surprising. In particular, red blood cell antigens frequently cross-react with gangliosides: cold agglutinin disease occurs in association with peripheral neuropathy and IgM anti-ganglioside antibodies reactive with disialosyl groups, found for example on GDlb, GTlb and GQlb (Arai et al., 1992). The incidence of anaemia in GBS is around 5% and closer scrutiny may reveal this to be due to increased destruction of red cells in such cases. The blood-nerve/brain barrier may play an important role in limiting the neuropathology to the peripheral nervous system (PNS) rather than the central nervous system (CNS), particularly to areas where the barrier is absent or relatively deficient, adjacent to peripheral nerve terminals and nerve roots. However, the absence of pathology in other tissues expressing surface ganglioside or cross-reactive glycoprotein antigens has no explanation. Apart from possibly inducing alterations in immune function, it is most likely that anti-ganglioside antibod-

ies in GBS exert their pathological effect by inducing complement fixation and inflammation in neural tissue. Any metabolic effects of anti-ganglioside antibodies, such as growth factor receptor modulation, are unlikely to create the acute peripheral nerve failure seen in GBS. This type of mechanistic model may, however, apply to chronic neuropathies. GM1 ganglioside, for example, promotes neurite outgrowth in many experimental systems (Barletta et al., 1991; Juurlink et al., 1991) and anti-GM1 antibodies in lower motor neuron diseases may block the restorative or trophic effects of natural GM1 in supporting the motor unit (HeimanPaterson et al., 1991), rather than inducing local inflammation. In the paraproteinaemic neuropathies, tissue deposition of immunoglobulin is not necessarily associated with inflammatory changes (Mendel et al., 1985). Finally, the conformation of a cell surface antigenic determinant, and hence its ability to bind antibody, may be profoundly influenced by its density and its positional relationship to adjacent molecules in the lipid bilayer: this phenomenon has been widely described. For example, anti-asialo-GM1 antibodies which bind asialo-GM1 when presented in a liposomal membrane may not bind asialo-GM1 when it is presented as a purified lipid in a solid-phase assay system (Marcus et al., 1989). We have recently produced a panel of human monoclonal anti-GM1 IgM antibodies. Two of these antibodies have a very similar specificity by immunoassay and thin-layer chromatography overlay for the epitope shared by GM1, asialo-GM1 and GDlb. However, only one of the antibodies reacts with unfixed antigens in mixed spinal cord cultures (Willison, unpublished results). These subtle differences in the characteristics of antibody specificity and antibody/antigen interaction may be of central importance to disease pathogenesis.

Conclusions

An apparently simple relationship exists between anti-ganglioside antibodies and GBS: during the course of a primary immune response to infection, anticarbohydrate antibodies arise which cross-react with neural ganglioside antigens, leading to peripheral nerve inflammation which resolves with disappearance of the antibody. Modulating factors include the nature of the preceding infection, the genetic control of the host immune response to that infection, the avidity, duration and isotype of the humoral response as influenced by T-helper cells, the co-existence of T-cell cytotoxicity to neural antigens and the structure of the blood-nerve barrier and nerve biochemical anatomy which determine the ingress and binding of antibody. Anti-ganglioside antibody-negative cases may have humoral im-

111

munity to other nerve components such as neutral glycolipids or myelin proteins, areas which continue to be widely explored. Despite suggestive evidence for autoimmunity to gangliosides as mediating some features of GBS, it is clear that many of Witebsky's postulates have yet to be fulfilled (Witebsky, 1959); in his 1990 article on the pathogenesis of GBS, McFarlin outlined this and many other factors with great clarity. Although it is encouraging to note that some progress has been made since he wrote his article, each of these factors contains levels of complexity about which we know very little, ensuring that GBS researchers will be kept busy for many years to come.

Acknowledgements We are grateful to Professor Hans-Peter Hartung for his helpful comments in a critical preview of the manuscript. Financial support was kindly provided by the Brims and Halliday bequests and the Scottish Motor Neurone Disease Association.

References Arai, M., Yoshino, H., Kusano, Y., Yasaki, Y., Ohnishi, Y. and Miyatake, T. (1992) Ataxic polyneuropathy and anti-Pr 2 IgMK M proteinaemia. J. Neurol. 239, 147-151. Barletta, E., Bremer, E.G. and Culp, L.A. (1991) Neurite outgrowth in dorsal root neuronal hybrid clones modulated by ganglioside GM1 and disintegrins. Exp. Cell Res. 193, 101-111. Casali, P. and Notkins, A.L. (1989) CD5 + B lymphocytes, polyreactire antibodies and the human B cell repertoire. Immunol. Today. 9, 364-368. Chiba, A., Kusonoki, S., Shimizu, T. and Kanazawa, I. (1992) Serum IgG antibody to ganglioside GQlb is a possible marker of Miller-Fisher syndrome. Ann. Neurol. 31, 677-679. Cuatrecasas, P. (1973) Interaction of Vibrio cholerae enterotoxin with cell membranes. Biochemistry 12, 3547-3565. Duane, G.C., Farrer, R.G., Dalakas, M.C. and Quarles, R.H. (1992) Sensory neuropathy with monoclonal immunoglobulin M to GDlb ganglioside. Ann. Neurol. 31, 683-685. Fong, J.W., Ledeen, R.W., Kundu, S.K. and Brostoff, S.W. (1976) Gangliosides of peripheral nerve myelin. J. Neurochem. 26, 157162. Fredman, P., Vedeler, C.A., Nyland, H., Aarli, J.A. and Svennerholm, L. (1991) Antibodies in sera from patients with inflammatory demyelinating polyradiculoneuropathy react with ganglioside LM1 and sulphatide of peripheral nerve myelin. J. Neurol. 238, 75-79. Graves, M.C. and Ravindranath, R.M.H. (1992) Do CD5 + B cells secrete anti-asialoGM1 antibodies in motor neuron disease? Ann. N.Y. Acad. Sci. 651,570-571. Grayson, G. and Ladisch, S. (1992) Immunosuppression by human gangliosides. II. Carbohydrate structure and inhibition of human NK activity. Cell. Immunol. 139, 18-29. Gregson, N.A., Jones, D., Thomas, P.K. and Willison, H.J. (1991) Acute motor neuropathy with antibodies to GM1 ganglioside. Neurology 238, 447-451. Gregson, N.A., Koblar, S. and Hughes, R.A.C. (1993) Antibodies to gangliosides in Guillain-Barr6 syndrome: specificity and relationship to clinical features. Quart. J. Med. (in press).

Griffiths, S.L. and Critchley, D.R. (1992) Characterisation of the binding sites for Escherishia coli heat labile toxin type I in intestinal brush borders. Biochim. Biophys. Acta. 1075, 154-161. Hakamori, S. (1990) Bifunctional role of glycosphingolipids. J. Biol. Chem. 265, 18713-18716. Hansson, H.-A., Holmgren, J. and Svennerholm, L. (1977) Ultrastructural localisation of cell membrane GM1 ganglioside by cholera toxin. Proc. Natl. Acad. Sci. USA 74, 3782-3786. Hartung, H.P., Heininger, K., Schafer, B., Fierz, W. and Toyka, K.V. (1988) Immune mechanisms in inflammatory polyneuropathy. Ann. N.Y. Acad. Sci. 540, 122-161. Hartung, H.P., Reiners, K., Schmidt, B., Stoll, G. and Toyka, K. (1992) Serum interleukin-2 concentrations in Guillain-Barr6 syndrome and chronic idiopathic demyelinating polyneuropathy: comparison with other neurological diseases of presumed immunopathogenesis. Ann. Neurol. 30, 48-53. Heidenreich, F., Leifeld, L. and Reiners, K. (1992) The immune response to the ganglioside GM1 in motor nerve disorders: specific antibody production by cultured lymphocytes. J. Neurol. 239, S100-S101. Heiman-Patterson, T., Tahmoush, A.J., Thompson, P., Hoffman, R., Krupa, T. and Shy, M.E. (1991) Effects of anti-GM1 M-proteins on primate nerve-muscle co-culture. Neurology 41,340. Ilyas, A.A., Willison, H.J., Quarles, R.H., Jungalwala, F.B., Cornblath, D.R., Trapp, B.D., Griffin, D.E., Griffin, J.W. and McKhann, G.M. (1988) Serum antibodies to gangliosides in Guillain-Barr6 syndrome. Ann. Neurol. 23, 440-447. Ilyas, A.A., Mithen, F.A., Chen, Z.-W. and Cook, S.D. (1991) Search for antibodies to neutral glycolipids in sera of patients with Guillain-Barr6 syndrome. J. Neurol. Sci. 102, 67-75. Ilyas, A.A., Mithen, F.A., Chen, Z.-W. and Cook, S.D. (1992a) Anti-GM1 IgA antibodies in Guillain-Barr6 syndrome. J. Neuroimmunol. 36, 69-76. Ilyas, A.A., Mithen, F.A., Dalakas, M.C., Chen, Z.-W. and Cook, S.D. (1992b) Antibodies to acidic glycolipids in Guillain-Barr6 syndrome and chronic inflammatory demyelinating polyneuropathy. J. Neurol. Sci. 107, 111-121. Ishii, N. and Watanabe, K. (1992) Aberrant expresion of GM1 on lymph node cells of MLR/Mp-lpr/lpr mice: influences on the autoreactivities of anti-asialo-GM1 antibodies. Autoimmunity 13, 107-116. Ishioka, G.Y., Lamont, A.G., Thomson, D., Bulbow, N., Gaeta, F.C.A., Sette, A. and Grey, H.M. (1992) MHC interaction and T cell recognition of carbohydrates and glycopeptides. J. Immunol. 148, 2446-2451. Juurlink, B.H.J., Munoz, D.G. and Ang, L.C. (1991) Motoneuron survival in vitro: effects of pyruvate, a-ketoglutarate, gangliosides and potassium. Neurosci. Lett. 133, 25-28. Kamata, Y., Kozaki, S., Sakaguchi, G., Iwamori, M. and Nagai, Y. (1986) Evidence for direct binding of Clostridium botulinum type E derivative toxin and its fragments to gangliosides and free fatty acids. Biochem. Biophys. Res. Commun. 140, 1015-1019. Kitamura, M., Iwamori, M. and Nagai, Y. (1980) Interaction between Clostridium botulinum neurotoxin and gangliosides. Biochim. Biophys. Acta 628, 328-335. Koblar, S.A., Gregson, N.A., Hughes, R.A.C., Doherty, P. and Walsh, F.S. (1991) Campylobacter neuropathy. Neurology 41, 1327-1328. Ladisch, S., Becker, H. and Ulsh, L. (1992) Immunosuppression by human gangliosides. I. Relationship of carbohydrate structure to the inhibition of T cell responses. Biochim. Biophys. Acta 1125, 180-188. Latov, N. (1990) Antibodies to glycoconjugates in neurological disease. Clin. Aspects Autoimmun. 4, 18-29. Lee, K.-W., Inghrami, G., Sadiq, S., Thomas, F.P., Spatz, L.A., Knowles, D.M. and Latov, N. (1990) B-cells that secrete antiMAG or anti-GM1 antibodies are present at birth, and anti-MAG antibody-secreting B-cells are CD5 +. Neurology 40, 367 (abstract).

112 Lee, K.-W., Inghrami, G., Spatz, L.A., Knowles, D.M. and Latov, N. (1991) The B-cells that express anti-MAG antibodies in neuropathy and non-malignant monoclonal gammopathy belong to the CD5 subpopulation. J. Neuroimmunol. 31, 83-88. Marcus, D.M., Perry, L., Gilbert, S. et al. (1989) Human monoclonal antibodies that bind 3-fucosyllactosamine, asialo-GM1 and GM1. J. Immunol. 143, 2929-2932. McFarlin, D.E. (1990) Immunological parameters in Guillain-Barr6 syndrome. Ann. Neurol. 29, $25-$29. Mendel, J.R., Sahenk, Z., Whitaker, J.H., Trapp, B.D., Yates, A.J., Griggs, R.C. and Quarles, R.H. (1985) Polyneuropathy and IgM gammopathy: studies on the pathogenic role of anti-myelin-associated glycoprotein antibody. Ann. Neurol. 17, 243-254. Nobile-Orazio, E., Carpo, M., Meucci, N., Grassi, M.P., Capitani, E., Sciaccc, M., Mangoni, A. and Scarlato, G. (1992) Guillain-Barr6 syndrome associated with high titers of anti-GM1 antibodies. J. Neurol. Sci. 109, 200-206. Notermans, S.H.W., Wocke, J.H.J. and van den Berg, L.H. (1992) Botulism and Guillain-Barr6 syndrome. Lancet 340, 303. Oomes, P.G., van der Meche, F.G.A., Jacobs, B.C., Hazenberg, M.P. and Banffer, J.R.J. (1991) Antibodies to the ganglioside GM1 in sera of Guillain-Barr6 patients recognise epitopes on Campylobacter bacteria. Peripheral Nerve Study Group, Arden House, New York. (abstract). Pestronk, A. (1991) Invited review: Motor neuropathies, motor neuron disorders and anti-glycolipid antibodies. Muscle Nerve 14, 927-936. Ponzin, D., Menegus, A.M., Kirschner, G., Nunzi, G., Fiori, M. and Raine, C.S. (1991) Effects of gangliosides on the expression of autoimmune demyelination in the peripheral nervous system. Ann. Neurol. 30, 678-685. Pruzanski, W. and Shumak, K.H. (1977) Biological activity of cold-reacting autoantibodies. New Engl. J. Med. 297, 538-542. Quarles, R.H., Ilyas, A.A. and Willison, H.J. (1986) Antibodies to glycolipids in demyelinating diseases of the human peripheral nervous system. Chem. Phys. Lipids 42, 235-248. Saida, T., Kuroki, S. and Saida, K. (1991) Guillain-Barr6 syndrome associated with Campylobacter jejuni infection: clinical, bacteriological and immunisation studies. Peripheral Nerve Study Group, Arden House, New York. (abstract). Schwartz, M., Seal, B.A. and Eshhar, N. (1981) Antibodies to gangliosides and myelin autoantigens are produced in mice after sciatic nerve injury. J. Neurochem. 38, 1192-1195. Schwerer, B., Lassmann, H., Kitz, K. and Bernheimer, H. (1986) Ganglioside GM1, a molecular target for immunological and toxic attacks: similarity of neuropathological lesions induced by ganglioside antiserum and cholera toxin. Acta Neuropathol. 72, 5561.

Svennerholm, L. and Fredman, P. (1990) Antibody detection in Guillain-Barr6 syndrome. Ann. Neurol. 27, $36-$40. Talal, N., Dauphinee, M. and Ahmed, A. (1992) CD5 B cells in autoimmunity. Ann. N.Y. Acad. Sci. 651,551-556. Toyka, K.V. and Heininger, K. (1977) Humoral factors in peripheral nerve disease. Muscle Nerve 10, 222-232. Van den Berg, L.H., Marrink, J., de Jager, A.E.J., de Jong, H.J., van Imhoff, G.W., Latov, N. and Sadiq, S.A. (1992) Anti-GM1 antibodies in patients with Guillain-Barr6 syndrome. J. Neurol. Neurosurg. Psychiatry 55, 8-11. Walsh, F.S., Cronin, M., Koblar, S., Doherty, P., Winer, J., Leon, A. and Hughes, R.A.C. (1991) Association between glycoconjugate antibodies and Campylobacter infection in patients with Guillain-Barr6 syndrome. J. Neuroimmunol. 34, 43-51. Walton, K.M., Sandberg, K., Rogers, T.B. and Schnaar, R.L. (1988) Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells, J. Biol. Chem. 263, 2055-2063. WiUison, H.J., Veitch, J., Paterson, G. and Kennedy, P.G.E. (1993a) Miller-Fisher syndrome is associated with serum antibodies to GQlb ganglioside. J. Neurol. Neurosurg. Psychiatry 56, 204-206. Willison, H.J., Chancellor, A.M., Paterson, G., Veitch, J., Singh, S., Whitelaw, J., Kennedy, P.G.E. and Warlow, C.P. (1993b) Antiglycolipid antibodies, immunoglobulins and paraproteins in motor neuron disease: a population based case control study. J. Neurol. Sci. 114, 209-215. Willison, H.J., Paterson, G., Veitch, J., Inglis, G. and Barnett, S.C. (1993c) Peripheral neuropathy associated with monoclonal IgM anti-Pr2 cold agglutinins. J. Neurol. Neurosurg. Psychiatry (in press). Winer, J.B., Hughes, R.A.C., Anderson, M.J., Jones, D.M., Kangro, H. and Watkins, R.F.P. (1988) A prospective study of acute idiopathic neuropathy. II. Antecedent events. J. Neurol. Neurosurg. Psychiatry 51, 613-618. Witebsky, E. (1959) Historical roots of present concepts of immunopathology. In: P. Graber and P. Meischer (Eds.), Immunopathology (Ist International Symposium), Benno Schwabe, Basel, p. 1. Yuki, N., Yoshino, H., Sato, S. and Miyatake, T. (1990) Acute axonal polyneuropathy associated with anti-GM1 antibodies following Campylobacter enteritis. Neurology 40, 1900-1902. Yuki, N., Yoshino, H., Sato, S., Shinozawa, K. and Miyatake, T. (1992) Severe acute axonal form of Guillain-Barr6 syndrome associated with IgG anti-GDla antibodies. Muscle Nerve 15, 899-903. Zielasek, J., Jung, S., Schmidt, B., Ritter, G., Hartung, H.-P. and Toyka, K. (1993) Effects of gangliosides on experimental autoimmune neuritis induced by peripheral nerve myelin or P2 specific T cell lines. J. Neuroimmunol. 43, 103-112.