Brain Advance Access originally published online on February 25, 2004
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Brain, Vol. 127, No. 5, 1085-1100, 2004
© 2004 Guarantors of Brain
doi: 10.1093/brain/awh127
Anti-ganglioside antibody-mediated neuronal cytotoxicity and its protection by intravenous immunoglobulin: implications for immune neuropathies
Departments of 1 Neurology and 2 Pharmacology, Johns Hopkins University, Baltimore, MD, USA and 3 Department of Neurology, The Second Teaching Hospital, Hebei Medical University, Shijiazhuang, People’s Republic of China
Correspondence to: Dr Kazim Sheikh, Department of Neurology, Johns Hopkins Hospital, 600 N. Wolfe St, 509 Pathology Building, Baltimore, MD 21287, USA E-mail: ksheik{at}jhmi.edu
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Summary |
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Antibodies against GD1a, GM1 and related gangliosides are frequently present in patients with the motor variant of Guillain–Barré syndrome (GBS), and their pathological role in this variant of GBS is now widely accepted. However, two basic issues related to anti-ganglioside antibody-mediated neural injury are not completely resolved: (i) some anti-ganglioside antibodies can cross-react with glycoproteins and therefore the nature of antigens targeted by these antibodies is not well established; and (ii) although pathological studies suggest that complement activation occurs in GBS, experimental data for the role of complement remain inconclusive. To address these issues, we developed and characterized a simple anti-ganglioside antibody-mediated cytotoxicity assay. Our results demonstrate first, that both GBS sera containing anti-ganglioside antibodies and monoclonal anti-ganglioside antibodies cause neuronal cell lysis by targeting specific cell surface gangliosides, and secondly, that this cell lysis is complement dependent. In this assay, the GD1a cell membrane pool appears to be more susceptible to anti-ganglioside antibody-mediated injury than the GM1 pool. Further, human intravenous immunoglobulin (IVIg), now a standard treatment for GBS, significantly decreased cytotoxicity in this assay. Our data indicate that the mechanisms of IVIg-mediated protection in this assay include anti-idiotypic antibodies and downregulation of complement activation. This simple cytotoxicity assay can potentially be used for screening of (i) pathogenic anti-ganglioside antibodies in patients with immune-mediated neuropathies; and (ii) new/experimental therapies to prevent anti-ganglioside antibody-mediated neural injury.
Key Words: cytotoxicity assay; Guillain–Barré syndrome; acute motor axonal neuropathy; anti-ganglioside antibodies; IVIg
Abbreviations: Ab= antibody; AMAN = acute motor axonal neuropathy; ELISA = enzyme-linked immunosorbent assay; GBS = Guillain–Barré syndrome; IgG = immunoglobulin G; IgM = immunoglobulin M; IVIg = human intravenous immunoglobulin; LDH = lactate dehydrogenase; mAb = monoclonal antibody; P4 = 1-phenyl-2-hexade-canolamino-3-morpholino-1-propanol; TLC = thin-layer chromatography
Received October 8, 2003. Revised December 18, 2003. Accepted December 22, 2003.
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Introduction |
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Anti-ganglioside antibodies (Abs) are implicated as immune effectors in several autoimmune neurological disorders including Guillain–Barré syndrome (GBS) (Hartung et al., 1996
; Hughes et al., 1999; Quarles and Weiss, 1999; Kusunoki, 2000; Ariga et al., 2001; Willison and Yuki, 2002). Recent studies show strong correlations between specific anti-ganglioside Abs and different variants of GBS. For example, anti-GQ1b Abs are found in >85% of patients with Fisher variant, and Abs to GD1a, GM1, GalNAc-GD1a and GM1b are strongly associated with motor axonal variants of GBS (Ilyas et al., 1988; Yuki et al., 1990, 1993a, 1999; Chiba et al., 1992; Willison et al., 1993; Ho et al., 1995, 1999; Lugaresi et al., 1997; Kaida et al., 2000; Ogawara et al., 2000). Anti-ganglioside Abs in patients with GBS are usually complement-fixing immunoglobulin Gs (IgGs) (Willison and Veitch, 1994; Ogino et al., 1995; Yuki et al., 1995). Different lines of investigation strongly support a pathogenic role for anti-ganglioside Abs in some forms of GBS (Roberts et al., 1994, 1995; Kusunoki et al., 1996; Goodyear et al., 1999; Paparounas et al., 1999; Plomp et al., 1999; Buchwald et al., 1998a; Yuki et al. 2001).
In autoimmune diseases it is crucial to define the nature of autoimmune responses and their target antigens. A critical question in GBS pathogenesis that has not been fully addressed is the nature of antigens targeted by anti-ganglioside Abs. Anti-ganglioside Abs not only frequently cross-react with structurally related gangliosides, but they have also been implied to cross-react with glycoproteins because of shared carbohydrate structures (Thomas et al., 1989; Apostolski et al., 1994); conversely, peptide mimotopes of carbohydrate antigens have also been described (Kieber-Emmons et al., 2000; Meloen et al., 2000; Beenhouwer et al., 2002). The nature of tissue antigens targeted by anti-ganglioside Abs is an important but difficult issue to address in complex experimental assays because of the problems inherent in manipulating expression of gangliosides in such models. Currently, lack of a robust mouse model of GBS precludes addressing this question in vivo. A short-term functional assay in which ganglioside expression of a uniform neuronal cell population can be easily and quickly manipulated should be helpful in addressing the issue of target antigens. Moreover, such a system can aid in studying the role of complement in anti-ganglioside Ab-mediated injury because pathological studies suggest that complement activation is involved in the pathogenesis of GBS (Hafer-Macko et al., 1996a, b). A cytotoxicity assay with well defined immune insult (anti-ganglioside Abs) and target antigens (ganglioside) would permit a study of mechanism(s) of action of both established and new immune therapies for Ab-mediated disorders such as GBS.
To address these issues, we developed an anti-ganglioside Ab-mediated cytotoxicity assay by using the NG108-15 cell line that expresses complex gangliosides GM1 and GD1a and has neuronal properties (Dahms and Schnaar, 1983), and NG-CR72, a mutant line derived from NG108-15, that does not express complex a-series gangliosides (Wu et al., 2001a, b). We designed the experiments to study the role of ganglioside expression and complement in this cytotoxicity assay. Whether human intravenous immunoglobulin (IVIg) can protect from anti-ganglioside Ab-mediated injury in this cytotoxicity assay was also examined. Further solid phase assays were devised to study the mechanisms involved in IVIg-mediated protection from anti-ganglioside Ab-mediated injury.
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Material and methods |
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Materials
Purified gangliosides GM1, GD1a, GT1b and GD1b were from Matreya (Pleasant Gap, PA) or Sigma Chemical Co. (St Louis, MO); Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and HAT supplements were all from Gibco (Grand Island, NY). Fluorescein isothiocyanate (FITC)- or Texas red-conjugated specific anti-mouse IgG and anti-rabbit IgG (H+L) were from Vector Laboratories (Burlingame, CA). FITC-conjugated cholera toxin subunit B was from List Biologicals (Campbell, CA), and rabbit anti-C3d and anti-C1q Abs were from Dako (Carpentaria, CA).
Monoclonal Abs and purification
Six IgG monoclonal Abs (mAbs) against gangliosides were used in this study. The generation, specificity and purification of anti-ganglioside mAbs were described previously (Lunn et al., 2000; Gong et al., 2002; Schnaar et al., 2002) and are summarized in Table 1. One ganglioside mAb GD1a/GT1b IgG2b that cross-reacts with proteins was also included (Gong et al., 2002). These mAbs are designated according to their ganglioside specificity and IgG isotype (1, 2a or 2b); for example, GM1-2b refers to an mAb with GM1 specificity and IgG2b isotype. One control IgG2b mouse mAb HB-94 (ATCC, Rockville, MD), which recognizes a combinatorial determinant of the HLA A, B, C–ß2-microglobulin complex, was used as a complement-fixing negative control. Both ganglioside-specific and control mAbs were purified according to previously described methods (Gong et al., 2002), and purified mAbs were used in all assays.
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GBS and control sera
Fourteen GBS acute phase pre-treatment sera with high titres of IgG anti-GD1a and/or anti-GM1 Abs (Table 2) were used from a repository. The clinical, electrophysiological and serological features of 13 out of 14 cases were published previously (Ho et al., 1999
) and are summarized in Table 2. Eight sera from normal healthy volunteers without reactivity against complex gangliosides were used as negative controls. All sera were kept at –70°C until use. All GBS and control sera were heat inactivated (56°C for 30 min) to deplete complement activity before use in the cell lysis assay.
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Complement
Human, rabbit, guinea pig, rat and mouse sera were used as sources of respective complements. Lyophilized rabbit and guinea pig sera were obtained from ICN Pharmaceuticals (Aurora, OH). Human, rat and mouse sera were isolated from freshly collected whole blood from healthy controls. These sera were stored at –70°C until further use. The sera were heat inactivated (56°C for 30 min) for control studies.
Cell lines
Two cell lines were used for all cytolytic assays: (i) NG108-15, a neuroblastoma x glioma hybrid neural cell line that expresses gangliosides GM2>GD1a>GM1 (Dahms and Schnaar, 1983); and (ii) its mutant derivative NG-CR72 that is deficient in GM1-synthase and hence a-series complex gangliosides, and contains significantly more GM2 than does the parent cell line (Wu et al., 2001a, b). The NG-CR72 cell line was kindly provided by Dr Robert Ledeen, UMDNJ, NJ. These cells were cultured in a 5% CO2 humidified atmosphere at 37°C in DMEM supplemented with 5% heat-inactivated FBS plus a supplement of HAT (0.1 mM hypoxanthine, 0.4 µM aminopterin and 16 µM thymidine). These cells were grown to a high density (confluence) and passaged every 2–3 days before use. Because the NG-CR72 cell line is a mutant derivative of the NG108-15 cell line, its phenotype was reconfirmed by the absence of cholera toxin subunit B staining before all experiments.
Anti-ganglioside Ab-mediated cytotoxicity assays
All cytotoxicity assays were done in triplicate wells and at least three independent experiments were performed per condition. Preliminary studies were done to determine the optimal number of cells, duration of lytic assay, and concentration and source of complement. Comparison of human, guinea pig, rabbit, rat and mouse complements (sera) indicated that the rabbit complement (1% serum) was optimal for the lysis assay; therefore, this source and concentration were used for all cytotoxicity assays in this study. Three assays were used to assess Ab-mediated cytotoxicity.
Lactate dehydrogenase (LDH) release assay
For this assay, NG108-15 cells and NG-CR72 were plated on flat-bottom 96-well plates at a density of 10 000 cells per well in serum-free medium and allowed to adhere to the bottom of the well overnight. Then these cells were incubated with the purified anti-ganglioside or control mAbs (1–20 µg/ml diluted in serum-free medium) or GBS or control sera (1 : 10–1 : 100 diluted in serum-free medium) for 30 min at 37°C before 1% rabbit serum was added and incubation was continued for a further 60 min at 37°C. Cell viability was assessed by determination of LDH in the supernatant fractions by using an LDH cytotoxicity detection kit according to the manufacturer’s instructions (Roche, Mannheim, Germany). ‘Maximal LDH activity’ was determined in 1% Triton X-100 lysates of sister control wells containing 1% rabbit serum and/or GBS or control sera in a dilution equivalent to that added to experimental wells. ‘Spontaneous LDH release’ was determined from supernatant fractions collected from cells incubated with serum-free medium alone. The cell lysis percentage (%) was calculated according to the following formula:
{(LDH released in supernatant fractions of experimental wells – spontaneous LDH release)/(maximal LDH release – spontaneous LDH release)} x 100
Reverse LDH release assay
Because GBS and control sera had high LDH activity, a reverse LDH assay (Sepp et al., 1996) was used for most experiments with sera to determine the extent of cytotoxicity by measuring LDH activity of viable cells adhering to the bottom of wells at the end of the Ab/complement incubation period. Briefly, cells were incubated with GBS and control sera and complement in 96-well plates as described above, supernatant fractions were discarded, and then wells were carefully washed, surviving adherent cells were lysed in 1% Triton X-100, and finally the LDH activity of this lysate was determined. The LDH value for total viable cells/well was obtained by determining LDH activity in 1% Triton X-100 lysates of control wells. The viability of treated cells was determined by calculating the ratio of LDH activity in treated and control wells and the lysis percentage by the formula: (1 – viability) x 100.
Live-Death vital dye
Besides LDH measurements, in a separate set of studies, cell viability and death were also determined by a Live-Death Viability/Cytotoxicity Kit (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions. After treatment with mAbs or sera as described above, cell viability was determined by counting 200–400 cells/experimental condition from six randomly selected fields from three separate wells.
Manipulation of ganglioside expression
The ganglioside expression of the NG108-15 and NG-CR72 cells was manipulated by the following techniques.
Ganglioside reconstitution
The cells were suspended in serum-free media containing 10 µM exogenous bovine brain GM1, GD1a or GT1b in Eppendorf tubes at 37°C for 2 h in a microrotator and then washed three times with serum-free medium. The resulting reconstituted cells were plated onto 96-well plates in serum-free medium at a density of 10 000 cells per well, and allowed to adhere for 4–6 h. Finally, the cytotoxicity assays were conducted on the reconstituted cells as described above. In a separate set of experiments, reconstitution with exogenous gangliosides (GM1, GD1a and GT1b) in both NG108-15 and NG-CR72 cell lines was confirmed by immunocytochemistry (described below) and analysis of extracted gangliosides according to previously described methods (Dahms and Schnaar, 1983; Schnaar, 1994; Schnaar and Needham, 1994).
Neuraminidase treatment
NG108-15 and NG-CR72 cells reconstituted with GM1 and GD1a were treated with neuraminidase to convert GD1a to GM1. The cells were plated for the lysis assay in the serum-free medium overnight, treated with 10 mU/ml of Vibrio cholerae neuraminidase for 2 h at 37°C, and washed with serum-free medium before cell lysis assays were performed as described above. The change in cellular ganglioside profile after enzyme treatment was confirmed by immunocytochemistry with cholera toxin (Wu et al., 2001b) and thin-layer chromatography (TLC) analysis of extracted gangliosides.
Metabolic inhibition of ganglioside biosynthesis
The glucosylceramide synthetase analogue, 1-phenyl-2-hexadecanolamino-3-morpholino-1-propanol (P4) (Matreya), an inhibitor of biosynthesis of all glycosphingolipids including gangliosides (Abe et al., 1995), was used to block all ganglioside biosynthesis in the NG108-15 cell line. These cells were pre-treated with 2.5 µM P4 for 48 h at 37°C in serum-containing medium as described previously (Vyas et al., 2002), and then washed gently before plating in serum-free medium in a 96-well plate for cytotoxicity assays. The change in NG108-15 ganglioside profile after P4 treatment was confirmed by immunocytochemistry with cholera toxin and TLC analysis of extracted gangliosides.
Determining the role of complement in cytotoxicity assays
Complement was depleted in the rabbit serum by heat inactivation (56°C for 30 min), and this serum was used as a source of complement in the cytotoxicity assays with both mAbs and GBS sera. Leupeptin (100 µM) (Sigma, St Louis, MO), a protease inhibitor that also inhibits C1 activation (Takada et al., 1978), was included in the cytotoxicity assays to inhibit initiation and activation of the classical pathway of complement activation. The availability of GD1a mAbs with similar reactivity but complement-fixing and non-fixing Fc fragments allowed their direct comparison in cytotoxicity assays.
Immunocytochemistry
For immunocytochemistry, NG108-15 and NG-CR72 cells were plated on glass coverslips in 24-well plates (30 000 cells/well) coated with poly-L-lysine in DMEM containing 5% FBS and HAT, and incubated overnight to allow cells to adhere to coverslips, after which the cells were fixed in 4% paraformaldehyde at room temperature for 30 min. The cells were then incubated with 2.5% normal horse serum and 2.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature to block non-specific binding. These cells were incubated with mAbs (10 µg/ml) overnight and developed with FITC- or Texas red-conjugated specific anti-mouse IgG (1 : 200) for 1–2 h at room temperature. FITC-labelled cholera toxin B subunit (2.5 µg/ml) was also used for GM1 staining and screening of the NG-CR72 cell line. The immunostained coverslips were mounted in anti-fade medium (Biomeda, Foster City, CA), and images were acquired with an LSM510 confocal microscope (Carl Zeiss, Jena, Germany).
Western blotting with GD1a/GT1b-2b mAb
Lysates were made rat sciatic nerves and from NG108-15, P4-treated NG108-15, and NG-CR72 cells in 1% sodium dodecylsulphate (SDS) buffer, and the protein concentration in the samples was determined by the Bradford assay according to the manufacturer’s instructions (Pierce, Rockford, IL). The cell and nerve lysates were heat-denatured in Laemmli buffer and run on 10% SDS–polyacrylamide gels, transferred to PVDF membranes, and blot-probed with GD1a/GT1b-2b mAb (10 µg/ml overnight at 4°C) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG
(Southern Biotechnology, Birmingham, AL) at a dilution of 1 : 5000 for 1 h at room temperature. The blot was developed with enhanced chemiluminescense according to the manufacturer’s protocol (Amersham, Piscataway, NJ).
Use of IVIg in the cytotoxicity assays
Dialysed IVIg (ZLB, Bioplasma, Glendale, CA, lot 02696-00136) was used in different concentrations (5–20 mg/ml) in the cytotoxicity assays to determine whether it was protective and to generate a dose–response curve. The optimal protective dose of IVIg was determined to be 
10 mg/ml and this dose was used in cytotoxicity assays with GD1a-reactive mAbs (GD1a/GT1b-2b and GD1a-2a) and GBS sera with predominant GD1a (HB-94-6, HB-93-8, HB-98-10 and JHH-17) and GM1 (HB-93-18, HB-94-18 and HB-95-2) reactivity. IgG was purified from a control serum by protein G affinity chromatography and its concentration was determined by the Bradford assay according to the manufacturer’s instructions (Pierce). An equal concentration of this IgG was used as a control for IVIg in cytotoxicity assays instead of an irrelevant protein such as serum albumin.
Mechanism(s) of IVIg protection
Two possible mechanisms of IVIg-mediated protection, anti-idiotypic Abs and inhibition of complement activation, were examined by TLC immuno-overlay and immunocytochemistry. TLC assays were used for both GBS sera and mAbs, whereas immunocytochemistry was done only with mAbs, not with GBS sera, because of non-specific binding of normal human sera to nerve sections and cell lines. BSA in equal concentration to IVIg was used as a control for non-specific protein-mediated inhibition in solid phase assays. Because patients administered an IVIg dose of 2 g/kg can achieve serum IgG levels up to 50 mg/ml (Sekul et al., 1994), maximal concentrations of IVIg used in the various assays described below never exceeded 50 mg/ml. Most solid phase assays were done with 50 mg/ml of IVIg to detect both qualitative and quantitative effects.
Anti-idiotypic Abs
Selected anti-ganglioside mAbs (GD1a/GT1b-2b, GM1-2b and GD1a-2a, 2–4 µg/ml) or GBS sera (HB-93-8, HB-94-6, HB-98-10 and JHH-17, 1 : 100) were pre-incubated with IVIg (0.5 or 50 mg/ml) for 1–2 h at room temperature. This mixture was then used for TLC immuno-overlay as previously described (Lopez et al., 2000, 2002) and compared with binding of mAbs or GBS sera without IVIg pre- or co-incubation. Immunocytochemistry was also done with the above mAbs (10–20 µg/ml) in the presence (20– 50 mg/ml pre- and co-incubation) or absence of IVIg on the NG108-15 cell line and fresh frozen rat sciatic nerve sections according to the above and previously described methods (Gong et al., 2002), respectively, to determine whether IVIg treatment decreased binding of mAbs to gangliosides in cells or nerve sections. The images were collected by confocal microscopy and quantified for immunostaining of mAbs by using the image analysis software Openlab according to the manufacturer’s instructions (Improvision Inc., Lexington, MA).
Complement inhibition
Two separate assays were adapted to determine the inhibition of complement activity by IVIg that was independent of its blocking effects on anti-ganglioside Abs. Fresh frozen rat sciatic nerve sections and ganglioside immobilized on TLC plates were used to show complement activation and its inhibition with IVIg. The deposition of human C1q and C3d (the activation product of complement component C3) was measured and compared by immunocytochemistry, and only C3d deposition was examined by TLC.
The complement deposition assay on nerve sections was adapted from the method described by Yan et al. (2000). Briefly, fresh frozen rat sciatic nerve sections were prepared according to previously described methods (Gong et al., 2002). These sections were blocked with 5% BSA and 0.1% Triton X-100 in PBS for 1 h at room temperature. After blocking, the nerve sections were incubated with complement-fixing (GD1a/GT1b-2b, GD1a-2a and GM1-2b) and non-complement-fixing (GD1a-1) Abs (10–20 µg/ml) for 2 h at room temperature or 1 h at 37°C. These sections were washed thoroughly and then incubated with normal human serum (1 : 10), as a source of complement, with or without IVIg (20–50 mg/ml) for 2 h at room temperature or 1 h at 37°C. Controls included use of ganglioside mAbs alone, normal serum alone (1 : 10), heat-inactivated serum with mAbs, irrelevant Ab (HB-94) and human serum, and addition of EDTA (5 mM). C1q and C3d deposition was then detected with rabbit anti-human C1q (1 : 200) or C3d (1 : 500) overnight at 4°C. The sections subsequently were incubated with the Texas red-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG (1 : 200) for 1 h at room temperature to detect ganglioside mAb binding and C1q or C3d deposition simultaneously. The images were collected by confocal microscopy, and C1q and C3d immunostaining was quantified by the image analysis software Openlab.
The gangliosides were applied to TLC plates and subjected to a TLC immuno-overlay with mAbs or patient sera according to the previously described methods (Lopez et al., 2000, 2002), with minor modifications as follows. The TLC plates were incubated with mAbs (GD1a/GT1b-2b or GM1-2b, 2–4 µg/ml) or serum (HB-93-8, HB-94-6, HB-98-10 or JHH-17, 1 : 100) overnight at 4°C in 1% BSA in PBS containing 0.05% Tween-20, after which the plates were washed thoroughly and incubated with normal human serum as a source of complement (1 : 150) pre-incubated with or without IVIg (0.5 or 50 mg/ml) for 90 min at 4°C. Controls included heat-inactivated complement, complement with or without EDTA (5 mM) and primary Abs only. C3d deposition was then detected by serial incubations with (i) rabbit anti-human C3d (1 : 2000 for 1 h at room temperature); (ii) biotin-conjugated anti-rabbit IgG (Jackson Immunoresearch) (1 : 1500, 1 h at room temperature); and (iii) alkaline phosphatase–strepavidin (Jackson Immunoresearch) (1 : 2000, 1 h at room temperature). The plates were developed with nitroblue tetrazolium/bromochloroindolyl phosphate, the resulting bands were scanned, and the signal was quantified as described previously (Lopez et al., 2000).
In view of a recent report (Jacobs et al., 2003) that IVIg can displace the anti-ganglioside Abs already bound to target antigens in enzyme-linked immunosorbent assays (ELISAs), a series of experiments were done with mAb (GD1a/GT1b-2b) and GBS sera (HB-94-6, HB-94-18, HB-95-2 and JHH-17) to address this issue in TLC immuno-overlay assays and to determine the basis of complement inhibition observed in this assay. TLC plates were incubated sequentially with GBS sera (1 : 100) or mAb (2–4 µg/ml) overnight at 4°C, washed with PBS, and then IVIg for 1 h at 4°C and compared with TLC plates not treated with IVIg. A dose–response curve was also generated with a GBS serum (JHH-17, 1 : 100) comparing pre- and co-incubation and post-incubation with IVIg (1–50 mg/ml) for 1 h at 4°C.
Statistical analysis
The Student’s t test and ANOVA were used to ascertain the significance of differences within groups. Differences were considered statistically significant if P < 0.05.
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Results |
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Our key findings are that GBS sera containing anti-ganglioside Abs or mAbs with anti-ganglioside reactivity cause complement-dependent cytotoxicity in neuronal cells that express gangliosides and that treatment with IVIg protects from this anti-ganglioside Ab-mediated neuronal cytotoxicity.
Cytotoxicity assays
The LDH release, reverse LDH release and Live-Death vital dye assays correlated well. All cytotoxicity experiments used at least one LDH assay that was confirmed by the Live-Death kit, and vice versa. LDH release was done preferentially with mAbs, whereas reverse LDH release was performed with GBS sera. GD1a/GT1b-2b Ab was used as a prototype because it binds not only to gangliosides but also to a glycoprotein, and other Abs were compared with this mAb. The results with monoclonal Abs are described first and the findings with GBS sera are summarized at the end of this section.
Gangliosides as target antigens
The cytotoxicity assays showed that the extent of NG108-15 and NG-CR72 cell lysis correlated with their profiles of ganglioside expression and the specificity of anti-ganglioside Abs used in these assays. None of the ganglioside mAbs used in this study caused significant lysis (8–13%) of the complex ganglioside-deficient mutant cell line NG-CR72 (Fig. 1A). Among the mAbs, those with GD1a reactivity and complement-fixing isotype (GD1a-2a and GD1a/GT1b-2b) caused significant lysis of NG108-15 cells compared with GM1, GT1b or control Abs, and this lytic effect was dose dependent (Fig. 1A and B). Maximal cell lysis seen with the anti-GD1a mAbs was in the range of 83–95%, GM1-2b 19–22%, and GT1b and HB-94 <10% (Fig. 1B). There was a remarkable increase in the lysis of both cell lines after reconstitution with GD1a and GT1b, and only a modest but significant increase with GM1 (Fig. 1C). Neuraminidase treatment of NG108-15 cells (which converts GD1a to GM1) caused significantly more lysis by GM1-2b Ab compared with GM1-reconstituted cells (Fig. 1D). Further, the lysis of neuraminidase-treated GD1a-reconstituted NG-CR72 cells was significantly increased by GM1-2b compared with NG-CR72 cells reconstituted with GM1 with or without neuraminidase treatment (Fig. 1E). In contrast, neuraminidase treatment of NG108-15 cells almost completely abolished the cytolytic effects of GD1a-reactive mAbs (Fig. 1F). Finally, the P4-treated NG108-15 cells were completely protected from injury mediated by GD1a-reactive mAbs (Fig. 1F).
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Quantitative TLC showed that the total amount and efficiency of reconstitution of the three complex gangliosides GM1, GD1a and GT1b in these cell lines were comparable. Further, the total GM1 content in NG108-15 cells reconstituted with exogenous GM1 was comparable with the content of those treated with neuraminidase (Fig. 2 and Table 3). The total amount of GM1 in NG-CR72 cells after reconstitution with GM1 or GD1a followed by neuraminidase treatment was also not significantly different (Table 3). P4 treatment almost completely eliminated all gangliosides in NG108-15 cells (Fig. 2, Table 3).
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We have previously reported that GD1a/GT1b-2b binds protein(s) in nervous tissues (Gong et al., 2002
). We further characterized protein binding of this mAb to cell lines used in this study and compared it with rat sciatic nerve by western blotting. The availability of this Ab allowed us to address the issue of whether gangliosides or cross-reactive proteins are target antigens for the cytotoxicity seen in our assays. Our western blotting data clearly showed that this mAb binds to a protein band that migrated just above the 56 000 Da molecular weight marker in rat sciatic nerves, both cell lines and P4-treated NG108-15 cells, and there were additional minor bands that appeared in cell lines only (Fig. 3). The expression of the cross-reactive proteins was not significantly altered in the NG-CR72 and P4-treated NG108-15 cell lines, indicating that the differences in susceptibility do not correlate with the expression of cross-reactive protein.
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Role of complement
The role of complement was assessed in the cytotoxicity assays by (i) comparing the lytic capacity of complement-fixing and non-fixing isotypes; (ii) depletion of complement by heat; and (iii) inhibition of the classical pathway. In the cytotoxicity assays, only complement-fixing isotypes caused lysis, and this was best exemplified by comparison of complement-fixing and complement non-fixing isotypes of GD1a-reactive mAbs (Fig. 4A). Heat inactivation of complement in the serum also completely eliminated the Ab-mediated cytotoxicity (Fig. 4B). Further, leupeptin effectively inhibited cell lysis by anti-ganglioside mAbs (Fig. 4C), suggesting that activation of the classical pathway of complement was necessary for cell lysis in these assays.
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Cytotoxicity induced by patient sera
The GBS sera caused significantly more lysis of NG108-15 cells (38–94%) than did control sera (5–18%) (Fig. 5A). GBS sera with predominant GD1a reactivity caused significantly more lysis (75 ± 17.4) than did sera with predominant GM1 reactivity (42 ± 4.7). There was no significant difference in the NG-CR72 lysis induced by GBS (31–44%) or control (29–42%) sera; because of the increase in baseline lysis with control sera, this cell line was not used for further reconstitution experiments. The cytotoxicity (NG108-15 cell line) induced by GM1 and GD1a Abs in GBS sera was significantly enhanced by treatment with neuraminidase (Fig. 5B) or GD1a reconstitution, respectively (data not shown). Conversely, the cytotoxicity of sera with only GD1a reactivity was significantly decreased after neuraminidase reactivity (Fig. 5B). There was a significant increase in the control sera-mediated cell lysis of P4-treated NG108-15 cells (46 ± 5.3) compared with untreated cells (15 ± 4.5); based on this observation, only five GBS sera with high cytotoxicity (>70%) were selected to demonstrate protection by P4 treatment. There was a significant decrease in GBS sera-mediated cytotoxicity of P4-treated NG108-15 cells (53 ± 5.4) compared with untreated cells (84 ± 8.2). The lysis by GBS sera was also complement dependent, and heat inactivation or leupeptin treatment virtually eliminated the cytotoxicity (data not shown).
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Protection by IVIg and its mechanism of action
IVIg treatment significantly reduced the GBS sera- and mAb-mediated cytolytic injury by 50–60% (Fig. 6A and B). IVIg provided a dose-dependent protection from cytotoxicity caused both by mAbs and GBS sera (Fig. 6C). Among the possible mechanisms responsible for IVIg-mediated protection, anti-idiotypic Abs and inhibition of complement activation were explored. The use of the term anti-idiotypic herein broadly denotes the ability of IVIg to inhibit binding of mAbs or anti-ganglioside Abs in GBS sera. IVIg decreased the binding of anti-ganglioside Abs in GBS sera to their respective antigens in TLC immuno-overlay by 30–40% at low dose and 82–100% at high dose of IVIg (Fig. 7A and B). This IVIg-mediated decrease in binding to ganglioside antigens was much less pronounced with the ganglioside mAbs by TLC immuno-overlay and was in the range of 10–16% with 50 mg/ml of IVIg treatment (Fig. 7A and B). IVIg (50 mg/ml) significantly decreased the GBS sera- and ganglioside mAb-mediated complement activation, as assessed by the C3d deposition on TLC plates, by 30–74 and 88–98%, respectively, compared with controls (Fig. 7B and C). The post-incubation experiments with IVIg showed that this treatment displaced 70–90% binding of sera with GD1a (HB-94-6 and JHH-17) (Fig. 7A) and GM1 (HB-94-18 and HB-95-2) reactivity, whereas the binding of mAb GD1a/GT1b-2b was not significantly affected. Comparison of IVIg pre- and co-incubation with post-incubation with the same GBS serum (JHH-17) showed that IgG binding to target ganglioside was inhibited in a dose-dependent manner; however, this inhibition reached a plateau at a lower dose (5 mg/ml) with pre- and co-incubation compared with post-incubation (10–25 mg/ml) (Fig. 7D).
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In the TLC assay, almost all IgG anti-ganglioside Ab binding in GBS sera was displaced after post-incubation with IVIg; however, in comparison, C3d deposition with co-incubation of IVIg was much less reduced. This discrepant finding was investigated further by examining the following two possibilities: (i) immununoglobulin M (IgM) anti-ganglioside reactivity present in GBS sera might be less susceptible to displacement by post-incubation with IVIg and is responsible for complement fixation; and (ii) complement deposition might lead to stabilization of the immune complex and therefore displace less IgG anti-ganglioside Abs by post-incubation with IVIg. The IgM issue was examined by post-incubation of IVIg (50 mg/ml) in GBS serum (JHH-17, 1 : 100) with anti-GD1a IgG and IgM reactivity, for displacement of IgM, and the IgM binding was probed with specific anti-human IgM secondary Ab (Jackson Immunoresearch). The issue of complement deposition leading to stabilization of IgG–ganglioside immune complexes was investigated by purifying IgG from GBS serum (JHH-17) by a protein G column according to the manufacturer’s instructions (Pierce) and this purified IgG was examined further by comparing the IVIg-mediated (50 mg/ml) displacement of GBS IgG with and without complement incubation as described above.
Post-incubation with IVIg displaced 50% of IgM GD1a and 90% of IgG GD1a Abs in the GBS serum (JHH-17). Comparison of anti-GD1a IgG displacement without (70–90% inhibition) or with (75–80% inhibition) complement deposition showed no significant differences with post-IVIg treatment, suggesting that complement deposition does not stabilize IgG–ganglioside binding in this solid phase assay. The effects of IVIg on anti-ganglioside Ab binding or C3d deposition in different TLC immuno-overlay experimental paradigms are summarized in Table 4.
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Image analysis of immunostaining on NG108-15 cells (data not shown) or sciatic nerve sections stained with mAbs showed minimal (9–12%) reduction in Ab binding by co-incubation with 50 mg/ml of IVIg (Fig. 8). Immuno cytochemistry analysis with ganglioside mAbs and high dose IVIg showed that C1q deposition on tissue sections was only decreased by 12–18% (Fig. 8A), whereas C3d deposition was decreased by
90% compared with controls (Fig. 8B). Heat inactivation of complement, EDTA, non-complement-fixing isotypes of mAbs, and leupeptin prevented complement deposition in TLC and immunocytochemistry assays (data not shown).
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Discussion |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionConflict of interest statementReferences |
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Our results demonstrate that anti-ganglioside Abs can target gangliosides directly to mediate cellular injury in a complement-dependent manner in the model used in this study. Our findings strongly support the notion that in GBS cases that are associated with anti-ganglioside Abs, the pathogenicity of these Abs involves targeting of gangliosides enriched in the nerves. IVIg, now a standard immunomodulatory therapy for GBS, protects from anti-ganglioside Ab-mediated cytotoxicity by two mechanisms: (i) idiotypic/blocking Abs against anti-ganglioside Abs; and (ii) direct inhibition of complement activation. Further, the results obtained with IVIg demonstrate that this treatment not only involves binding inhibition of the (Fab)2 portion of anti-ganglioside Abs, but also interference with Fc effector functions such as complement activation. The differential cytotoxicity that occurs with the endogenous versus exogenous GM1- and GD1a-derived GM1 raises the possibility that the Ab-mediated injury resulting from targeting of complex gangliosides in the cell membrane requires them to be in a ‘susceptible membrane compartment’. The observation that gangliosides in different membrane pools are differentially susceptible to Ab-mediated injury provides one potential explanation for selective injury to different nerve fibres (despite similar ganglioside expression) in some forms of GBS.
The current study focused on the GD1a and GM1 antigens because of our previous observation in northern China that motor axonal GBS is associated with IgG anti-GD1a Abs (Ho et al., 1999), and anti-GM1 Abs are frequently associated with GBS, particularly in Japanese patients with the axonal form of the disease (Kuwabara et al., 1998; Ogawara et al., 2000). The availability of complement- fixing and complement-non-fixing GD1a-specific mAbs and NG108-15 cells and their mutant clone NG-CR72 allowed pairing of these mAbs with the cell lines expressing or lacking GD1a. Moreover, ganglioside mAbs and matched GBS sera with GD1a and/or GM1 reactivity provided direct comparison of experimental and human disease-associated Abs, and the results obtained with ganglioside mAbs were validated by similar findings with the patient sera.
Gangliosides as target antigens
Alteration of cell surface ganglioside expression by genetic and enzymatic modulation and direct reconstitution convincingly demonstrate that cytotoxicity mediated by GBS sera or mAbs is due to targeting of these surface glycolipids. The Ab-mediated injury was not restricted to GM1 or GD1a Abs, but could also be seen with GT1b Ab after reconstitution. Although the immune response against GT1b is not commonly seen in GBS, the availability of GT1b mAbs allowed establishment of the cytotoxicity in the presence of this antigen, thus raising the possibility of broader applicability of this assay and its extension to other gangliosides implicated as target antigens in GBS and other neurological disorders.
The finding that GBS sera with anti-GM1 reactivity caused more efficient lysis of NG108-15 cells with or without reconstitution compared with GM1-2b mAb probably reflects higher affinity/avidity of anti-GM1 Abs in GBS sera. An implication of this finding is that this cytotoxicity assay, at least in the context of GM1, can functionally distinguish the relative affinities/avidities of Abs and possibly pathogenic and non-pathogenic Abs given that the properties/expression of target ganglioside in the cells remain constant. Further, normal sera known to contain low affinity anti-GM1 Abs (Mizutamari et al., 1994) did not cause significant cytotoxicity in this assay. The tools used in our experiments allowed us to establish firmly the ganglioside nature of target antigens in these cytotoxicity assays, and these findings extend previous observations showing that patient sera with anti-GM2 Abs cause cytotoxicity (O’Hanlon et al., 2000; Cavanna et al., 2001). This simple, reproducible and functional (cytotoxicity) assay can potentially be used in conjunction with the solid phase assay currently being used for screening of anti-ganglioside Abs in patient sera.
The availability of the GD1a/GT1b-2b Ab allowed us to address the issue of whether anti-ganglioside Ab binding to cross-reactive glycoprotein(s) plays a role in the cytotoxicity. GD1a/GT1b-2b recognized a protein that migrated just above a 56 kDa marker in rat sciatic nerves, P4-treated and untreated NG108-15, and NG-CR72 cell lines. Notably, the expression of this protein was unchanged with P4, despite almost complete elimination of complex gangliosides. The reversal of Ab-induced toxicity by P4 indicates that the cross-reactive protein is not involved. The protein is not unique to NG108-15 and NG-CR72 cells, in that it is also expressed in sciatic nerves of wild-type and genetically engineered mice lacking complex gangliosides recognized by this GD1a/GT1b-2b (K. Sheikh, unpublished observations). Taken together, these findings argue that although the 56 kDa protein carries a cross-reactive epitope for GD1a/GT1b-2b unrelated to ganglioside biosynthesis, the cytotoxicity of the Ab is entirely dependent on its recognition of gangliosides. Currently, it is not firmly established whether anti-GM1 and anti-GD1a Abs from GBS sera cross-react with glycoproteins and whether this cross-reactivity has any relevance to nerve fibre injury; however, it has been demonstrated that in transgenic mice lacking complex gangliosides, neuromuscular transmission is resistant to the paralytic effects of Fisher syndrome sera containing anti-GQ1b Abs (Bullens et al., 2002).
Compartmental hypothesis
The GM1 mAb-mediated cytotoxicity after reconstitution with exogenous GM1 was less than that after neuraminidase treatment of GD1a-expressing cells (naïve NG108-15 and GD1a-reconstituted NG-CR72). This suggests that the two ganglioside pools are distributed differently in the cell membranes of NG108-15 and NG-CR72 cells. Analytical studies preclude the hypothesis that the enhanced susceptibility of neuraminidase-generated GM1 compared with exogenously added GM1 is due to quantitative differences in the total levels of ganglioside expression. Since exogenously added bovine brain GD1a supported lysis by anti-GD1a Abs and, after neuraminidase treatment, anti-GM1 Abs, one cannot evoke the well-established differences in ceramide structures between bovine brain and NG108-15 gangliosides (Dahms and Schnaar, 1983) to explain the differential susceptibility of cells having exogenously added versus endogenously generated GM1. The data are consistent with the speculation that exogenously added GM1 partitions into a ‘lysis-resistant’ membrane subdomain, where it is less capable of supporting Ab-induced lysis than its endogenous counterpart. Exogenously added GD1a, unlike GM1, appears to partition into a lysis-susceptible subdomain, supporting the notion of differential susceptibility. Different biological effects of endogenous versus exogenously added GM1 in NG108-15 cells have been reported previously (Wu et al., 2001b), data that are consistent with those in the current study. If gangliosides distribute into different membrane subdomains in vivo, different cells with similar ganglioside expression may not necessarily be equally susceptible to injury by anti-ganglioside Abs, thus providing a potential explanation for the preferential motor nerve fibre injury seen in acute motor axonal neuropathy (AMAN).
Our results indicate that GBS sera with GM1 Abs lysed NG108-15 cells much more efficiently than GM1 mAb, and we speculate that this reflects relatively higher affinity of anti-GM1 Abs in the sera. It seems that the concept of ganglioside compartmental susceptibility is relative rather than absolute. Higher affinity Abs can target gangliosides in other compartments. Conversely, lower affinity Abs can cause injury if the target ganglioside is enriched in the susceptible compartment. We postulate a model of anti-ganglioside Ab-mediated cytotoxicity in which the extent and distribution of injury is dictated by three factors: (i) the specificity of the Ab response; (ii) the affinity of the Ab; and (iii) the distribution of target gangliosides in low versus high susceptibility membrane subdomains. It is likely that in the disease setting, additional variables such as Ab accessibility, cellular expression of complement inhibitors, and other factors contribute to the distribution and extent of nerve injury.
Role of complement
The current study indicates that the cytolytic assays used in this study are complement dependent and activation of the classical pathway is required. These observations are in line with the previous findings of deposition of complement activation products in the nerves of GBS cases (Hafer-Macko et al., 1996a, b) and are consistent with the notion that membrano-lytic insult may play a prominent role in Schwann cell or axonal pathology in GBS. Previous experimental models have implicated both complement-dependent and -independent pathophysiological effects of anti-ganglioside Abs at motor nerve terminals (Buchwald et al., 1998a, b; Plomp et al., 1999; O’Hanlon et al., 2001). Our findings are consistent with previous reports of anti-ganglioside Ab-mediated complement-dependent destruction of motor nerve terminals, but these observations do not contradict anti-ganglioside Ab-mediated complement-independent pathophysiological effects, and both mechanisms may contribute to GBS pathophysiology.
Rabbit complement was most effective in the cytolytic assay used in this study. This finding is consistent with previous observations that rabbit complement is very efficient for nucleated cell lysis compared with other sources of complement (Young-Rodenchuk and Gyenes, 1975; Grant, 1976). Although the basis of this is not clear (Ong et al., 1996), one potential explanation is that homologous membrane complement inhibitors are much less effective against heterologous complement (Rollins et al., 1991; White et al., 1994).
Mechanism of IVIg protection
IVIg provided significant protection from the ganglioside Ab-mediated injury in our model. The results of TLC immuno-overlay indicate that the IVIg preparation used in this study had blocked the binding of anti-ganglioside Abs in GBS sera, confirming similar findings reported previously (Malik et al., 1996; Yuki and Miyagi, 1996). That the IVIg-mediated decrease in GBS sera binding to gangliosides is not due to stereotypic hindrance, but rather to binding inhibition by IVIg, is argued by the observation that IVIg does not affect mAb binding to gangliosides in the same solid phase assay. The blocking effect of IVIg is unlikely to be due to N-linked cross-reactive oligosaccharide moieties present on IgG, as previously reported for an anti-GM1 IgM monoclonal Ab (Thomas et al., 1989), because anti-ganglioside mAbs were not significantly inhibited by IVIg. Based on these findings, we propose that the anti-idiotypic reactivity against human ganglioside Abs in IVIg contributes to its protective effect in our assay. This observation is consistent with the findings of two recent reports (Buchwald et al., 2002; Jacobs et al., 2003) showing that IVIg blocks the pathophysiological effects of GBS sera with anti-ganglioside reactivity on the neuromuscular junction resulting from anti-idiotypic neutralization of anti-ganglioside Abs. The reason for the failure of human IVIg to block the binding of mouse anti-ganglioside mAbs is unclear, but similar findings were reported with anti-GM1 Abs produced in a rabbit (Lopez et al., 2000). This failure could reflect differences in anti-idiotypic Ab repertoire present in different species.
IVIg not only prevents the binding of GBS sera to target gangliosides with co-incubation, but it also displaces the already bound Ab with post-incubation in TLC immuno-overlay assay. The efficiency of displacement (elution/neutralization) with post-incubation was somewhat lower in the 1–5 mg/ml range compared with co-incubation experiments, but higher doses almost completely displaced the bound Ab. This finding is consistent with the study of Jacobs et al. (2003), who reported a similar phenomenon in ELISA in Fisher syndrome sera with anti-GQ1b reactivity. Another study did not observe this phenomenon in GBS sera with anti-GM1 reactivity (Lopez et al., 2000); however, that study used immunoglobulins pooled from a relatively small number (10) of donors. IVIg preparations contain variable amounts of IgG in dimeric form, whereas IgG isolated from an individual donor is monomeric; the amount of dimer increases with the number of donors contributing to the plasma pool from which IVIg is derived (Tankersley et al., 1988) and, importantly, it has been shown that in an Ab-mediated disease model of idiopathic thrombocytopenic purpura, the dimer content of IVIg correlated with protection (Teeling et al., 2001). It is possible that the discrepancy between our results and previous findings may be related to differences in the dimer content of the IVIg preparations.
It has been proposed that a likely mechanism of IVIg-mediated protection and recovery in GBS is due to inhibition of complement activation (Dalakas, 2002a, b), and our experimental findings with mAbs support this hypothesis. The mAb data demonstrate that IVIg significantly decreased the complement activation in TLC assays and on nerve sections, consistent with immunopathological findings seen in AMAN (Hafer-Macko et al., 1996a). The mechanism of IVIg-mediated complement inhibition in our model is at the C3 level, if not upstream in the classical pathway, as suggested by almost complete inhibition of C3d deposition in the presence of IVIg. The C1q deposition on nerve sections was not significantly decreased by IVIg, suggesting that the initiation of the classical pathway was not affected. These findings are consistent with the previous observations that IVIg does not affect the recognition phase of the classical complement pathway (Basta et al., 1991) and that IgG in IVIg preparations binds to C3 and interferes with the assembly of C5 convertase by inhibiting incorporation of C3b (Basta and Dalakas, 1994). The relevance of our findings is supported by clinical observations that there is increased in vitro uptake of C3b in GBS and dermatomyositis patient sera (Basta et al., 1996) and, when tested in dermatomyositis patients treated with IVIg, uptake of C3b was significantly decreased in the sera, and endomysial deposition of C3b and terminal complement complex was decreased in muscle biopsies (Basta and Dalakas, 1994). Our observation that higher doses of IVIg were more effective in inhibition of complement activation and anti-ganglioside Ab binding in different assays used in this study would suggest the need to achieve high concentrations of IVIg in patients for more effective/robust clinical response. Since there is some debate about the optimal dose of IVIg (2 g/kg versus 1 g/kg), this finding argues in favour of using a 2 g/kg dose for the treatment of autoimmune neuropathies.
In contrast to anti-ganglioside mAbs, the results obtained with the solid phase C3d inhibition assay on GBS sera do not clarify whether or not IVIg directly inhibits complement activation in the context of human anti-ganglioside Abs. Although IVIg caused a significant decrease in C3 activation by GBS sera on sequential TLC overlay studies, this decrease in complement deposition was paradoxically much less than IgG anti-ganglioside Ab displacement observed with the post-incubation of IVIg at the same concentration as used for the complement inhibition assay. The post-incubation IVIg experiment probing IgM displacement indicates that only 50% of IgM anti-ganglioside Abs were displaced compared with 90% of IgG anti-ganglioside Abs in the same serum. This finding suggests that the decrease in complement deposition in the TLC assay was mostly due to anti-ganglioside Ab displacement and that residual C3d staining after post-incubation with IVIg was mostly secondary to IgM anti-ganglioside Abs. IVIg post-incubation experiments with purified IgG from a GBS serum showed that C3d deposition does not stabilize the Ab–ganglioside immune complex in this assay. These findings are consistent with a recent report indicating difficulties in distinguishing between IVIg- mediated indirect and direct effects on anti-GQ1b Ab binding and complement activation (Jacobs et al., 2003).
We postulate that the addition of IVIg not only blocks/neutralizes the circulating anti-ganglioside Abs in GBS cases but, given the access to target tissues, it can also displace Abs already bound to target antigens and potentially reduce complement activation, thus decreasing the cumulative nerve fibre injury. It is likely that the IVIg-mediated Ab displacement and inhibition of complement activation is restricted neither to anti-ganglioside Abs nor to GBS, but that such a mechanism of action is more broadly applicable to other Ab-mediated neurological diseases.
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Acknowledgements |
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We wish to thank Dr Robert Ledeen for providing us with NG-CR72 cell line, and Dr Pamela Talalay for editorial assistance. This work was supported by NIH grant NS42888 and Johns Hopkins School of Medicine Clinician-Scientist award (K.A.S.).
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Conflict of interest statement |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionConflict of interest statementReferences |
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‘Under a licensing agreement between Seikagaku America and the Johns Hopkins University, K.A.S. and R.L.S. are entitled to a share of royalty received by the University on sales of products described in this article. The terms of this arrangement are being managed by the Johns Hopkins University in accordance with its conflict of interest policies.’
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