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The Journal of Immunology, 2007, 178: 3323-3331.
Copyright © 2007 by The American Association of Immunologists, Inc.

C3d Binding to the Myelin Oligodendrocyte Glycoprotein Results in an Exacerbated Experimental Autoimmune Encephalomyelitis1

Jean-François Jégou*, Philippe Chan*, Marie-Thérèse Schouft*, Mark R. Griffiths{dagger}, James W. Neal{dagger}, Philippe Gasque{dagger},{ddagger}, Hubert Vaudry* and Marc Fontaine2,*

* Institut National de la Santé et de la Recherche Médicale U413, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23, University of Rouen, Mont Saint-Aignan, France; {dagger} Brain Inflammation and Immunity Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, United Kingdom; and {ddagger} Laboratoire de Biochimie et Génétique Moléculaire, EA 2526, University of La Reunion, Saint-Denis, La Reunion, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The complement system is known to contribute to demyelination in multiple sclerosis and experimental autoimmune encephalomyelitis. However, there are few data concerning the natural adjuvant effect of C3d on the humoral response when it binds to myelin Ags. This study addresses the effect of C3d binding to the myelin oligodendrocyte glycoprotein (MOG) in the induction of experimental autoimmune encephalomyelitis in C57BL/6J mice. Immunization with human MOG coupled to C3d was found to accelerate the appearance of clinical signs of the disease and to enhance its severity compared with MOG-immunized mice. This finding was correlated with an increased infiltration of leukocytes into the central nervous system accompanied by increased complement activation and associated with areas of demyelination and axonal loss. Furthermore, B cell participation in the pathogenesis of the disease was determined by their increased capacity to act as APCs and to form germinal centers. Consistent with this, the production of MOG-specific Abs was found to be enhanced following MOG/C3d immunization. These results suggest that binding of C3d to self-Ags could increase the severity of an autoimmune disease by enhancing the adaptive autoimmune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Multiple Sclerosis (MS)3 is an autoimmune and inflammatory demyelinating disease of the central nervous system (CNS). It is now recognized that autoreactive T cells, B cells, and Abs against myelin Ags contribute to the pathogenesis of the disease. Evidence from the human disease and its animal model, experimental autoimmune encephalomyelitis (EAE), implicates the complement system in demyelination. Complement activation by Abs directed against myelin Ags was detected in MS patients (1, 2) and complement activation products are deposited in and around areas of demyelination in MS and EAE (3, 4). In an Ab-mediated model of EAE, complement fixation to anti-myelin oligodendrocyte glycoprotein (MOG) Abs was reported to be essential in inducing demyelination through the formation of the membrane attack complex at the surface of oligodendrocytes, causing their disruption (5). Moreover, myelin has been reported to directly activate complement through the classical pathway in the absence of myelin-specific Abs (6). Among myelin Ags involved in the etiology of these demyelinating diseases, MOG is considered to be a relevant auto-Ag responsible for both cell-mediated autoimmunity and the production of pathogenic auto-Abs in MS and EAE (7). MOG has also been shown to be a good candidate for complement activation. Located at the outermost surface of myelin sheath and directly accessible to an autoimmune attack, MOG was reported to bind C1q, an event that initiates the activation of the classical pathway of complement (8, 9).

The consequences of complement activation are numerous and vary according to the different bioactive fragments generated following the cascade of proteolytic cleavages undergone by complement precursors. Early studies demonstrated that C3 could play an important role in demyelination. In rats, the use of cobra venom factor, a C3-depleting agent, or soluble CD35 was found to reduce Ab-mediated demyelination (4, 10). More recently, the attenuation of EAE induced by the encephalitogenic peptide MOG 35–55 was described in C3-deficient mice that presented reduced infiltration and were protected from demyelination (11). Although the complement system mainly exerts its cytolytic effect on oligodendrocytes via the formation of the membrane attack complex, many functions mediated by C3-derived activation fragments were investigated to determine their contribution to the demyelinating process. Thus, the chemotactic peptide C3a was found to be involved in the recruitment of inflammatory cells into the CNS (12). Among the opsonic fragments generated after C3 proteolytic cleavage, C3b and iC3b were shown to promote myelin phagocytosis by macrophages and microglia (13). However, little is known about the natural adjuvant effect of C3d binding to the myelin Ag in the context of demyelinating diseases, although this final and stable product of C3 maturation was found to be widely deposited on myelin (14).

Although the complement system is considered to be a key component of the innate immune system, it has been shown for a decade to link innate immunity to acquired immunity through C3 cleavage products, including C3b, C3dg, and C3d. C3b, which can covalently bind an Ag, serves as a ligand for the complement receptor type 1 (CD35) whereas C3dg and C3d are both specific ligands for the complement receptor type 2 (CD21). All of these opsonins were shown to enhance the humoral response to both T-dependent and T-independent Ags (15). Thus, the covalent attachment of C3b to the Ag hen egg lysozyme (HEL) (16) through a thioester bond was reported to modulate the humoral response by enhancing HEL-specific Ab titers (17). Even though C3b-Ag complexes are representative of those generated in vivo, the effect could be attributed to several complement receptors such as CD35 or CD21, because C3b may be further cleaved into C3dg or C3d. The demonstration of a direct adjuvant effect of C3d was performed using chimeric recombinant proteins associating the Ag HEL to multimers of C3d (18). Following immunization, HEL coupled to three copies of C3d was found to increase dramatically the immunogenicity of the Ag. C3d exerts its adjuvant effect through its binding to CD21 expressed on B cells and follicular dendritic cells (FDC). The coligation of the BCR and the CD21/CD19/CD81 complex results in lowering the threshold for B cell activation (18, 19, 20). CD21 is also involved in C3d-opsonized Ag uptake and retention by FDC in splenic follicles for B cell selection and propagation (21, 22). Mice deficient for Cr2 (encoding both CD21 and CD35) or C3 have defects in mounting immune responses to T cell-dependent Ags and some T-independent Ags (23, 24, 25, 26, 27, 28). This attractive adjuvant property was further exploited in numerous DNA-based vaccine approaches using multimeric C3d. Several DNA vaccines expressing foreign Ags coupled to three copies of C3d were designed to promote enhanced Ab responses against a variety of pathogens, including the measles virus (29), HIV type 1 (30, 31), or the influenza virus (16). Likewise, Ab titers were higher in mice following immunization with pneumococcal capsular polysaccharide type 14 conjugated to C3d compared with mice immunized with the Ag alone (32).

Although there are a lot of studies on the effect of C3d when coupled to foreign Ags, little is known about its impact on B cell activation and the subsequent humoral response when it binds to self-Ags, especially in some autoimmune inflammatory diseases in which complement activation occurs. This study addresses the direct role of C3d in the pathogenesis of EAE and demyelination. To confirm the adjuvant property of C3d in the humoral response to a myelin Ag, we induced the demyelinating disease in C57BL/6J mice using either the recombinant human MOG protein or MOG coupled to mouse C3d. The clinical and histological severities of EAE were compared between MOG and MOG/C3d-immunized animals, as well as their cellular and humoral responses to MOG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Sf9 cells, originating from the Spodoptera frugiperda IPLB-Sf21-AE cell line (Invitrogen Life Technologies), were grown in Insect-XPRESS protein-free insect cell growth medium (Cambrex) with 100 U/ml penicillin and 100 µg/ml streptomycin (Cambrex). Insect cells were cultured either in a tissue culture flask or in suspension at 27°C.

MOG and MOG/C3d fusion protein constructs, expression, and purification

Total RNA was extracted from the human glioblastoma cell line T98G (American Type Culture Collection). The cDNA encoding for the extracellular Ig-like domain of the human MOG was obtained by transcription with Moloney murine leukemia virus reverse transcriptase (Promega). A 348-bp DNA fragment was amplified by PCR with the Platinum Pfx polymerase (Invitrogen Life Technologies). The forward primer sequence was MOG-HindIII-BglII (5'-CCCAAGCTTAGATCTGGGCAGTTCAGAGTGATA-3') and the reverse primer sequence was MOG-EcoRV (5'-CGGATATCTTCTACTTTCAATTCCATTGCT-3'). After digestion by the restriction endonucleases HindIII and EcoRV, MOG was subcloned into the vector pUCBM21 (Boehringer Mannheim) upstream from a stop codon. Mouse C3d cDNA was amplified by PCR from the vector pMLC3/1 (a gift from Dr. G. Fey, University of Erlangen, Erlangen, Germany) containing the sequence of mouse C3d (33) by the use of the following primer set (forward/reverse): C3d1-EcoRV (5'-CGGATATCGGATCTACCCCCGCAGGCTGTGG-3') and C3d2-EcoRV (5'-GCGATATCGCTGGGGAGGTGGAAGGA-3'). The 894-bp DNA fragment C3d, digested with EcoRV, was inserted in pUCBM21 vector in fusion with MOG. MOG and MOG/C3d were further subcloned into the baculovirus transfer vector pAcSecG2T (BD Biosciences), using the BamHI and EcoRI restriction sites, downstream from the GST gene separated by a sequence coding for a thrombin cleavage site.

Recombinant viruses were generated in Sf9 cells following the cotransfection step with pAcSecG2T/MOG or pAcSecG2T/MOG/C3d constructs and BD BaculoGold linearized baculovirus DNA (BD Biosciences) by the use of a Lipofectin transfection reagent according to manufacturer’s instructions. Initial virus stocks were further amplified by propagation in Sf9 cells grown in a 75-cm2 flask. Individual recombinant clones were screened for the amount of secreted products by using the technique of plaque assay. For each clone, culture supernatants were tested for the expression and secretion of recombinant proteins by Western blotting with an anti-GST Ab (GE Healthcare) and an anti-MOG 35–55 Ab (a gift from Dr. O. Costa, DIFEMA, University of Rouen, Rouen, France). Selected viral clones were stored at 4°C in the dark. Large suspension cultures (500–1000 ml) at 2 x 106 Sf9 cells/ml were infected by ~108 viral particles/ml. After 72 h of incubation time, culture supernatants were collected and immediately used for protein purification. Recombinant proteins were purified by affinity chromatography using GSTrap FF Columns (GE Healthcare). Fractions were eluted with 10 U of thrombin (GE Healthcare) in PBS per milligram of bound GST-tagged proteins. Fractions containing MOG or MOG/C3d were pooled, concentrated with Amicon Ultra-4 spin columns with a cut-off of 10 kDa (Millipore), and analyzed by SDS-PAGE. Protein concentrations were determined using the BCA kit (Pierce).

Bacterial recombinant mouse MOG, consisting of aa 29–145 of the extracellular Ig domain of mouse MOG, was also generated as described previously (a gift from Dr. H. H. Reid and Dr J. Rossjohn, Monash University, Clayton, Australia) (34).

Animals and immunization

Wild-type female C57BL6/J mice were purchased from The Jackson Laboratory. Animals were 5- to 8-wk old at the time of immunization. EAE was induced by a single s.c. flank injection of 200 µg of recombinant human MOG or MOG/C3d in CFA (Sigma Immunochemicals) supplemented with 5 mg/ml Mycobacterium tuberculosis (Difco Laboratories). Mice were injected i.p. with 300 ng of pertussis toxin (Calbiochem) on days 0 and 2. All animal use and protocols conformed to the French Ethical Committee guidelines.

Clinical disease scoring

Mice were monitored daily for clinical signs of EAE. Disease was scored following a scale of 0–5 as follows: 0, no clinical signs; 1, tail weakness or paralysis; 2, paresis or partial paralysis of the hind limbs; 3, total hind limb paralysis; 4, front limb paresis; and 5, total paralysis, moribund state, or death. Data are presented as mean clinical scores for each group of mice. Disease onset was determined as the average day of appearance of clinical signs. The cumulative disease index (CDI) for each group was calculated as follows: [(sum of the mean clinical scores)/(day of disease onset)] x 100. According to ethical practices, mice with a total paralysis or in a moribund state were euthanized.

Histological analysis

Mice were sacrificed 30 days after immunization. Spinal cords were removed and snap frozen in isopentane chilled on dry ice and stored at –80°C. Spinal cord sections (10 µm) were cut using a cryotome and mounted onto glass slides. After overnight drying, sections were fixed in ice-cold acetone for 5 min and left to dry at room temperature for 30 min. For immunohistochemistry experiments, sections were incubated with 0.3% H2O2 in PBS for 10 min to quench endogenous peroxidase activity. Then, biotin and avidin binding sites were blocked using a biotin/avidin kit from Vector Laboratories. Sections were incubated with 1/10 rat anti-mouse CD18 (Table I) in 1% BSA/PBS. Slides were washed three times in PBS. Sections were then incubated with 1/200 biotin-conjugated goat anti-rat (Bio-Rad). Following washing and incubation with ABC reagent (Vector Laboratories), sections were developed using an 3-amino-9-ethylcarbazol (AEC) substrate (Vector Laboratories) according to the manufacturer’s instructions. Sections were rinsed with tap water and counterstained in Harris hematoxylin for 1 min. Slides were mounted using Faramount aqueous mounting medium (DakoCytomation). Sections were viewed using a Leica microscope with digital camera and analyzed using Openlab software (Improvision). To observe demyelination, spinal cord sections were stained with H&E and Luxol fast blue and axonal loss was analyzed following Palmgren staining. For immunofluorescence experiments, sections were washed in PBS and blocked with 1% BSA/PBS before incubation with primary Ab for 1 h at 37°C. The Abs used for immunofluorescence experiments are listed in Table I. Following washing in PBS, sections were incubated with 1/200 FITC-labeled donkey anti-rat Ab, 1/1000 Alexa 594-labeled goat anti-rabbit Ab (Jackson ImmunoResearch Laboratories), and 4'-6-diamidino-2-phenylindole (0.1 µg/ml) in 1% BSA/PBS for 1 h at room temperature. Coverslips were then applied with Vectorshield (Vector Laboratories). Fluorescent signals were imaged using a fluorescent microscope (Leica DMLB).


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  		Table I. List of antibodies used for immunohistofluorescence and immunohistochemistry experiments

 
Lymph node cell proliferation assays

Lymph node cells (LNC) were isolated from inguinal and axillary lymph nodes of wild-type mice immunized 11 days previously with a single injection of 200 µg of human MOG protein in CFA. Cell cultures contained 5 x 105 cells per well in a volume of 200 µl in 96-well flat-bottom plates, together with MOG or MOG/C3d proteins (at a previously determined concentration of 20 µg/ml) in RPMI 1640 supplemented with 1% penicillin/streptomycin and 10% FBS. Con A (Sigma-Aldrich) at a concentration of 1 µg/ml was used as positive control and cells cultured in medium alone were used as negative control. Cultures were pulsed with 1 µCi of [3H]thymidine at 72 h and harvested 24 h later with a Tomtec harvester. Thymidine incorporation was measured using a Wallac MicroBeta 1450 liquid scintillation counter.

Measurements for anti-MOG IgG levels by ELISA

Microtiter plates (Costar) were coated overnight at 4°C with 100 µl of mouse MOG protein at a concentration of 5 µg/ml in 100 mM carbonate buffer (pH 9.6). Plates were washed three times with PBS containing 0.05% Tween 20 and blocked for 1 h with PBS containing 1% BSA and 0.1% gelatin. After washing, serum samples diluted in blocking buffer were applied to the wells (100 µl/well). A 1/100 serum dilution was found on the linear portion of the binding curve. MOG-specific Abs were detected with biotin-conjugated goat anti-mouse IgG, IgG1, IgG2a, IgG2b, or IgG3 secondary Abs (Caltag Laboratories). Plates were developed using streptavidin-HRP (Beckman Coulter) and 2, 2'-azino-bis-[3-ethyl-benzthiazolin-6-sulfonic acid] substrate (Boehringer Mannheim). Absorbance was read at 405 nm using a Titertek Multiskan Plus MK II microplate reader (Labsystems).

Germinal center (GC) B cell population analysis by flow cytometry

Axillary and inguinal lymph nodes from PBS-, MOG-, or MOG/C3d-immunized or naive animals were removed as described above for a LNC proliferation assay. Single-cell suspensions were washed with PBS supplemented with 1% FBS and 1 x 106 cells were stained on ice for 30 min with anti-B220-PerCP and anti-GL7-FITC (BD Pharmingen) diluted in PBS and 1% FBS. Flow cytometry was performed on a FACScalibur instrument (BD Biosciences) and data were analyzed using the CellQuest software.

Statistical analysis

Data are shown as means ± SE. For comparison between groups, Mann and Whitney U nonparametric test was used; a two-tailed p ≤ 0.05 was considered as the minimum threshold of significance. The correlation of specific anti-MOG IgG levels with clinical scores of EAE was assessed using Spearman’s correlation test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Analysis of recombinant MOG and MOG/C3d expression and purification

To determine whether C3d can act as a molecular adjuvant when coupled to a myelin Ag, we decided to produce and purify the encephalitogenic recombinant 1–125 extracellular domain of the human MOG alone and human MOG coupled to mouse C3d. Different clones of baculoviruses expressing MOG and MOG/C3d tagged to GST were generated and used for insect cell infection. Supernatants from cultures of infected cells were analyzed for the secretion of recombinant proteins. Analysis by Western blotting was conducted using an anti-GST Ab and a MOG 35–55-specific Ab that recognized both MOG-GST and MOG/C3d-GST at the expected molecular mass (Fig. 1A). Similar results were obtained by Western blotting using Z12 and 8-18C5 MOG-specific mAbs that bind to conformational determinants on the extracellular domain of MOG (35, 36) (data not shown). Recombinant proteins were purified by affinity chromatography, cleaved from the GST moiety as described in Materials and Methods, and assessed by SDS-PAGE. Two distinct bands at 17- and 18-kDa corresponding to human MOG and a 50-kDa band corresponding to the MOG/C3d fusion protein were observed (Fig. 1B). The presence of two bands for human MOG may be due to differences in glycosylations, because insect cells are able to process N-glycans. Both bands were recognized by anti-MOG Abs (not shown). The 15-kDa band corresponds to the bacterial recombinant unglycosylated 29–145 extracellular domain of mouse MOG.


Figure 1
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  		FIGURE 1. Production of recombinant human MOG and MOG/C3d. A, Western blot analysis of secreted recombinant proteins in culture supernatants from insect cells infected with baculoviruses expressing MOG-GST or MOG/C3d-GST probed with anti-GST and anti-MOG (35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 ) Abs. Different volumes of culture supernatants were loaded (0, 1, 10, or 100 µl). Both GST-tagged proteins were detected at the expected molecular mass. B, Purified recombinant human MOG (lane 1, doublet with molecular mass of 17 and 18 kDa), MOG/C3d (lane 2, molecular mass of 50 kDa), and bacterial mouse MOG (lane 3, molecular mass of 15 kDa) were analyzed by electrophoresis on a 12% SDS-polyacrylamide gel and stained with Coomassie blue. The molecular mass standard (Std) is indicated on the far left of the panel.

 
C3d accelerates and enhances the severity of clinical signs of EAE

Animals immunized with the recombinant MOG/C3d protein developed a more severe EAE disease compared with those immunized with MOG (Fig. 2). MOG/C3d-immunized mice had a higher maximum clinical score (2.9 vs 1.5; p = 0.004) and a higher CDI (134 vs 56.6; p = 0.001) compared with MOG-immunized mice (Table II). Moreover, the disease onset appeared earlier in the MOG/C3d group because the first clinical signs of EAE were observed (on average) on day 11, whereas the signs were observed (on average) on day 17 in the MOG group of mice (p = 0.001). To insure that these results were not due to a nonspecific effect of C3d, MOG/C3d and MOG immunizations were compared to immunization with MOG fused to GST, a protein that may act as a nonspecific carrier protein. MOG-GST did not affect the encephalitogenicity of MOG alone, with similar maximum clinical scores (1.3 vs 1.5) and CDI (60 vs 56.6) of EAE. However, MOG-GST clinical scores were significantly different from those observed with MOG/C3d (1.3 vs 2.9; p = 0.006). In addition, there was no significant differences concerning the day of onset of disease between MOG and MOG/GST-immunized animals (16 vs 17). Together, these results underline the specific effect of C3d in the enhancement of disease severity.


Figure 2
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  		FIGURE 2. MOG/C3d immunization results in enhanced clinical scores of EAE. Groups of C57BL/6J mice were immunized in two independent experiments with PBS ({blacksquare}; n = 9), MOG ({diamond}; n = 11), MOG/GST ({blacktriangleup}; n = 6), or MOG/C3d (•; n = 12) in CFA. Mice were evaluated for disease severity on a 0–5 scale for 30 days. Clinical signs of disease appear earlier and are higher in MOG/C3d-immunized animals compared with MOG-immunized animals.

 

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  		Table II. C3d enhances the encephalitogenicity of human MOG proteina

 
C3d promotes the infiltration of CD18-positive cells

The possibility that the clinical differences observed could be reflected in cellular infiltration and resident cell activation was evaluated by histological analyses. Spinal cord sections from mice immunized with MOG, MOG/C3d, or PBS were removed at day 30 postimmunization and assessed by immunohistochemistry for the presence of CD18+ cells. The analyses showed an important presence of CD18+ cells into the white matter following MOG/C3d immunization (Fig. 3A). The infiltration had perimeningeal localization, and perivascular cuffing was also observed in the white matter although the gray matter was affected at a lesser level (not shown). Animals that exhibited higher clinical signs of EAE presented infiltration in the brain, especially in periventricular spaces, the brainstem, and olfactory lobes (data not shown). In contrast, spinal cord from mice injected with MOG showed a weaker presence of CD18+ cells compared with MOG/C3d-immunized mice (p = 0.008) but higher than those observed in PBS-injected mice (p = 0.09) (Fig. 3B).


Figure 3
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  		FIGURE 3. Spinal cords from MOG/C3d-immunized mice exhibit an enhanced presence of inflammatory cells. A, Spinal cord sections from immunized mice were analyzed for the presence of CD18+ cells by immunohistochemistry. Sections from MOG/C3d-immunized mice show an extensive infiltration in the parenchyma, whereas MOG-immunized animals exhibit less infiltrating cells, essentially located close to the meninges. Original magnification was x200. B, Quantification of the area of CD18+ cells in spinal cord sections. Area is represented as relative units (RU).

 
The nature of CD18+ cells was assessed on spinal cord sections by immunofluorescence labeling. Staining using an Ab directed against allograft inflammatory factor 1 revealed the presence of activated microglial cells (not shown). Although no difference could be observed in the number of perivascular macrophages/dendritic cells stained for the macrophage mannose receptor (MMR), high levels of infiltrating monocytes and activated endothelial cells expressing the CD93 marker were observed particularly in MOG/C3d animals (Fig. 4A). With regard to the lymphocyte population, the infiltrate was composed of a majority of CD19+ B cells and T CD4+ cells (Fig. 4B). Of note, very few CD8+ cells were found in the infiltrate in MOG/C3d animals (not shown).


Figure 4
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  		FIGURE 4. Increased infiltration of monocytes, B and T cells but not dendritic cells in MOG/C3d-immunized mice. A, Spinal cord sections from PBS-, MOG-, or MOG/C3d-immunized mice were analyzed by immunofluorescence and compared for the presence of infiltrating monocytes and activated endothelial cells, both stained for CD93 (red fluorescence), and perivascular dendritic cells, stained for MMR (green fluorescence). Spinal cords from MOG/C3d-immunized mice presented a higher number of monocytes and activated endothelial cells compared with MOG-immunized ones, whereas no significant difference could be observed in the number of perivascular dendritic cells. B, Characterization of the lymphocyte population in MOG/C3d infiltrates by immunofluorescence. The CD18-positive infiltrate induced by MOG/C3d immunization was composed of a majority of CD19-positive B cells. CD4 T cells were also observed whereas a small number of CD8 T cells were detected (data not shown). All sections were counterstained with 4'-6-diamidino-2-phenylindole to reveal cell nuclei. Original magnification was x400.

 
C3d enhances T cell proliferation

A possible explanation for C3d contributing to a more severe and robust EAE is that CD21-expressing B cells are enhanced in their ability to process and to present human MOG to specific T cells. To verify this, mice were immunized with MOG 11 days before lymph node removal. LNC were then submitted to MOG or MOG/C3d protein stimulation at a previously determined concentration of 20 µg/ml. Positive control for T cell proliferation was achieved with Con A at a concentration of 1 µg/ml. Results showed an increased proliferative response when LNC were stimulated with the MOG/C3d protein compared with MOG stimulation, exhibiting a stimulation index (stimulated to unstimulated) of 19 for MOG/C3d vs 6 for MOG alone (p = 0.0001) (Fig. 5A). This observation demonstrates the higher capacity of B cells to function as key APCs when C3d is coupled to MOG and to promote Ag-specific T cell activation.


Figure 5
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  		FIGURE 5. A, LNC exhibit higher proliferative response following MOG/C3d stimulation. LNC from mice 11 days after human MOG immunization were cultured with human MOG and MOG/C3d proteins. LNC exhibited higher proliferative response to MOG/C3d stimulation compared with MOG stimulation. Data represent mean counts per minute of triplicate wells, representative of three independent experiments. ***, p < 0.001. B, Analysis of the B cell (B220+) and GC B cell (B220+, GL7+) population by flow cytometry. Percentages of B cells or GC B cells indicated in corresponding quadrants are the average (n = 6) of two independent experiments performed with three mice per group. C3d enhances GC formation in response to MOG/C3d immunization.

 
Effect of C3d on GC B cell frequency

B220+GL7+ B cells are considered as GC B cells and their frequency is representative of the number of activated B cells. In Cr2–/– mice, GC are still generated but present abnormalities in their development because they are diminished in size and number (22, 37), suggesting that C3d might influence both Ag retention by FDC and B cell activation and proliferation. So, to evaluate GC formation in draining lymph nodes, cells were isolated 11 days after immunization and analyzed for the frequency of B cells (B220+) and GC B cells (B220+GL7+) (Fig. 5B). The percentage of B cells (B220+ only) in the lymph nodes from nonimmunized mice was approximately half of those observed in mice that received CFA/PBS only. Moreover the proportion of GC B cells in mice immunized with CFA only was higher than in naive mice (0.7 vs 0.4%, respectively), suggesting that CFA only is partially involved in B cell activation and GC formation. However, the percentage of activated B cells was slightly but repeatedly higher in lymph nodes from MOG/C3d-immunized mice compared with MOG-immunized animals (1.2 vs 0.9%, respectively), even though there was no difference in the percentages of B cells between PBS-, MOG-, or MOG/C3d-immunized mice. These data suggested that C3d binding to the self-Ag might promote GC formation in follicles from draining secondary lymphoid organs.

C3d enhances the production of pathogenic MOG-specific antibodies

We assessed the level of the Ab response to mouse MOG by ELISA in sera from mice immunized with MOG or the MOG/C3d protein. MOG immunization resulted in a modest IgG production, essentially composed of IgG1 Abs (Fig. 6A). By comparison, MOG/C3d induced a significantly higher IgG response against MOG (p = 0.03). MOG/C3d immunization was found to enhance both IgG1 (p = 0.03) and IgG2b (p = 0.004) Ab levels, whereas no IgG2a or IgG3 was detected in the two groups. Levels of MOG-specific IgG were correlated with EAE clinical scores as determined by Spearman’s correlation test (r = 0.79; p = 0.0004), suggesting that these enhanced levels of MOG Abs may account for the enhanced severity of EAE following MOG/C3d immunization.


Figure 6
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  		FIGURE 6. MOG/C3d induces a strong humoral and pathogenic response to MOG. A, Sera drawn 30 days after immunization with either PBS, MOG, or MOG/C3d were assayed by ELISA for total IgG, IgG1, IgG2a, IgG2b, and IgG3 Ab responses to mouse MOG protein. Values represent the mean OD (±SEM) from five to six mice per group. *, p < 0.05; **, p < 0.01. MOG/C3d immunization resulted in increased production of IgG with enhanced levels of IgG1 and IgG2b Abs. B, Immunohistofluorescence experiments on spinal cord sections showed the deposit of mouse IgG at the periphery of the CD18-positive infiltrate after MOG/C3d immunization and the activation of the complement system through the detection of C3 immunoreactivity. C, Histological analysis of CNS. Luxol fast blue/hematoxylin staining showed areas of demyelination at the site and the periphery of perimeningeal infiltrates (upper panel corresponding to the ventral cord) and perivascular cuffs (middle panel corresponding to the dorsal cord). Infiltration sites were increased in size and number in MOG/C3d-immunized mice. The perivascular cuff with demyelination (arrows) observed following MOG/C3d immunization corresponded to the area of axonal loss as assessed by Palmgren staining on consecutive spinal cord sections (lower panel). No demyelination and axonal loss were observed in control mice. Original magnification was x40.

 
Interestingly, the analysis of spinal cord sections from MOG/C3d-immunized mice by immunofluorescence reveals the deposit of mouse IgG in the white matter at the periphery of CD18+ infiltrates (Fig. 6B). Complement activation was also detected as assessed by the presence of C3 immunoreactivity at the same localization. Histological analyses of spinal cord sections stained by Luxol fast blue show the presence of large areas of demyelination, particularly in MOG/C3d animals. Demyelinating plaques were detected at the edges of infiltrating cells that corresponded to the sites of IgG deposition and complement activation (Fig. 6C). Furthermore, demyelination areas also contained axonal damage as assessed by the loss of Palmgren staining in MOG/C3d animals. These observations strongly suggest that C3d is able to enhance humoral responses to MOG by increasing the level of mouse MOG-specific Abs that contribute to demyelination and axonal injury.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The role of C3 in inducible autoimmune disease models has been highlighted in animal models by the use of C3-deficient mice. C3–/– mice were shown to be resistant to collagen-induced arthritis (CIA) with a decreased collagen type II-specific IgG Ab response (38). An attenuated form of EAE with reduced infiltration and demyelination of the CNS was also observed in C3–/– mice following MOG 35–55 peptide immunization (11). However, another study failed to observe any difference between the same strain of C3–/– mice and their wild-type littermates, suggesting that C3 is not required for the development of MOG peptide-induced EAE but not excluding its possible involvement in the Ab-mediated demyelination in EAE induced by whole protein (39, 40). In addition, C3 depletion by cobra venom factor led to a less severe disease in several models of autoimmune disorders such as glomerulonephritis, EAE, or CIA (4, 41, 42). Nevertheless, these findings did not discriminate among the effects mediated by the different fragments generated from C3 cleavage such as the anaphylatoxin C3a or ligands for CD35 and CD21. In particular, there are few data concerning self-Ag opsonization by C3d and its natural adjuvant effect in autoimmune pathologies, although C3d has been widely studied in the context of the humoral response to foreign Ags. In a CIA model in DBA/1 mice, a recent study showed that multimeric C3d attachment to collagen type II is sufficient to induce the disease in the absence of mycobacterial components of CFA (43). The B cell participation in the inductive phase of the pathology was demonstrated by enhanced levels of specific auto-Abs and an increased ability to form GC consistent with joint pathology. This previous study suggested that C3d could play an important role in the inductive phase of autoimmune inflammatory diseases.

In this study we assessed the direct role of C3d in a model of MOG-induced EAE in C57BL/6J mice. Unlike other studies that link artificially multimeric C3dg or C3d to the Ag, we decided to link a monomer of C3d to the activating auto-Ag, because its precursor C3b preferentially binds to an Ag according to a 1:1 stoichiometry under physiological conditions (17, 44). The 1–125 extracellular domain of the human MOG used as encephalitogenic auto-Ag has been shown to induce an Ab-dependent EAE in C57BL/6J mice (40, 45, 46). Several observations indicate that MOG is the best candidate for initiating cellular and humoral autoimmune responses and for being opsonized by C3 products. First, MOG differs from other myelin auto-Ags because of its immunodominance and its ability to trigger both cellular and pathogenic Ab responses (7, 47, 48). Second, MOG is located at the outermost surface of myelin sheaths and is directly accessible for the autoimmune attack (49), thereby constituting a privileged target for C3b fixation. Third, it has been reported that myelin was able to activate the complement system, particularly to cleave C3, in the presence or in the absence of specific Abs (6). Finally, MOG has been reported to activate complement through the classical pathway by binding C1q (8). Our results show that immunization with the human MOG protein from an insect cell expression system was responsible for moderate clinical signs of EAE that allowed us to test for the C3d adjuvant effect in the modulation of EAE pathology when the self-Ag is coupled to mouse C3d. To explain the moderate encephalitogenicity of our human MOG, we should consider the expression system used to produce recombinant proteins. The insect cell expression system allows proteins to be glycosylated unlike bacterial expression systems. According to t Hart et al. (50), the glycosylation of native MOG might be a protective factor in EAE, whereas unglycosylated human MOG is a much more potent trigger of cellular and humoral autoimmunity. As glycosylations by insect cells are not complete compared with native human MOG, this would explain the mild clinical scores of EAE following insect recombinant human MOG. Nevertheless, a significant difference in the onset and severity of EAE is observed between MOG and MOG/C3d immunizations, suggesting that monomeric C3d binding to MOG is sufficient to increase its encephalitogenicity. To verify the specificity of C3d in increasing the encephalitogenicity of MOG, we fused the same auto-Ag to GST, a protein that has a molecular mass similar to that of C3d (26 vs 33 kDa, respectively) and is known to be highly immunogenic. No differences were observed when mice were immunized with MOG/GST compared with MOG alone, which agues for a specific effect of mouse C3d independent of a simple protein carrier role. This result also dismisses the hypothesis that C3d might help in the refolding of the recombinant MOG by modifying its conformational structure, which was demonstrated to be important in eliciting a pathogenic Ab response (51, 52). These clinical observations raise the possibility that C3d plays a direct role in the pathogenesis of the disease by targeting the auto-Ag to B cells and to the FDC that express CD21.

In our study, MOG/C3d was found to increase the proportion of activated GC B cells in draining lymph nodes compared with MOG immunization. GC represent a specialized microenvironment within B cell follicles of the lymphoid compartment where B cell activation occurs, leading to B cell proliferation and Ab-secreting cells. C3d-Ag complexes targeting B cells results in their direct activation and proliferation through the coligation of the BCR and the CD21/CD19/CD81 complex. This coligation provides an additional transduction signal that amplifies the initial signal generated by the fixation of the Ag on the surface Ig (15). In addition to this direct activation of B cells, C3d can influence Ag retention by FDC in lymphoid organs. Although FDC express CD21 without any possibility of transducing an activation signal, they are able to retain Ag-C3d complexes for B cell activation and propagation. Several lines of evidence point to the importance of CD21 expression by FDC. Thus, the absence of CD21 expression in mice affected GC development, supporting that CD21 was involved in B cell entry, retention, and survival in these specialized structures (22). CD21 expression on FDC has also been shown to be of importance in the maintenance of a B cell memory (53). This enhanced activation and proliferation of B cells is in agreement with the large number of B cells found in the CNS infiltrate. Different compositions of the cellular infiltrate have been described depending on the genetic background of immunized animals or the Ag used to induce EAE, but little is known about the composition of infiltrating cells in the model of human MOG-induced EAE in C57BL/6J mice. It is known that a large proportion of these infiltrating cells are monocytes and macrophages, but no study has evaluated the relative importance of B cells and T cells in EAE lesions. Given the effect of C3d on B cell proliferation, it is not surprising to find them in high proportion into the CNS.

B cells could also contribute to the pathogenesis of EAE by promoting Ag presentation to MOG-specific T cells. It has been reported that B cells are not critical as APC in MOG-induced EAE because LNC from B cell-deficient mice were able to proliferate in response to the human MOG protein (46). However, our results from lymph node cell proliferation assays are in agreement with other studies that reported an involvement of CD21 in C3d-conjugated Ag uptake and presentation (54, 55). The increased T cell proliferation observed following MOG/C3d stimulation could also be explained by the up-regulation of costimulatory molecules, because cross-linking CD21 has also been shown to increase the expression of CD80/CD86 on APC (56).

The human MOG-induced EAE is a B cell-driven pathology that leads to the production of pathogenic Abs. In this study, we show that MOG/C3d immunization was responsible for an increased production of IgG, confirming the adjuvant property of C3d observed when it was coupled to foreign Ags. The Ab response was enhanced for IgG1 and IgG2b isotypes, which are able to fix complement and mediate demyelination in vivo. Several lines of evidence support the contention that these Abs might be pathogenic. Notably, an area of IgG deposit has been observed in the white matter of spinal cords from MOG/C3d-immunized animals that was associated with complement activation. This finding was also correlated with the presence of demyelination and axonal damages evidenced by histological analyses. Finally, there was a strong correlation between the level of IgG and the clinical scores of EAE determined from immunized mice. Consistent with these findings, C3d appears to modulate the MOG-specific Ab response and directly contribute to pathogenicity in the EAE model.

The results reported here confirm the adjuvant property of C3d when it is coupled to a self-Ag. The originality of this study was to bind a monomer of C3d to the auto-Ag to form a 1:1 complex that is more representative of those that are formed in vivo, and it has been shown that it was sufficient to exacerbate the autoimmune pathology. MOG opsonization by C3d induced a robust activation and proliferation of B cells which, in turn, acted as APC promoting Ag presentation to MOG-specific T cells. Finally, MOG-specific IgG were increased following MOG/C3d immunization and contributed, with complement activation, to exacerbate both the demyelination and the axonal loss that are responsible for the clinical signs of the CNS disease.


   Acknowledgments
 
We thank the Developmental Studies Hybridoma Bank of the University of Iowa, Dr. Yann Dean, and Dr. Odile Costa for providing us with Abs and Dr. George Fey, Dr. Jamie Rossjohn, and Dr. Hugh Reid for DNA plasmids.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the Agence Nationale de Recherches sur le SIDA, Institut National de la Santé et de la Recherche Médicale (U413), the European Institute for Peptide Research (Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23), and the Conseil Régional de Haute-Normandie. J.-F.J. was a recipient of a fellowship from the French Ministry of Education. Back

2 Address correspondence and reprint requests to Dr. Marc Fontaine, Institut National de la Santé et de la Recherche Médicale U413, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 23, Faculté des Sciences et Techniques, Université de Rouen, Place Emile Blondel, Mont Saint-Aignan Cedex, France. E-mail address: marc.fontaine{at}univ-rouen.fr Back

3 Abbreviations used in this paper: MS, multiple sclerosis; CIA, collagen-induced arthritis; CDI, cumulative disease index; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; FDC, follicular dendritic cell; GC, germinal center; HEL, hen egg lysozyme; LNC, lymph node cell; MMR, macrophage mannose receptor; MOG, myelin oligodendrocyte glycoprotein. Back

Received for publication September 26, 2006. Accepted for publication December 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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