Attenuation of Experimental Autoimmune Demyelination in Complement-Deficient Mice

Serge Nataf, Steven L. Carroll, Rick A. Wetsel, Alexander J. Szalai and Scott R. Barnum
J Immunol November 15, 2000, 165 (10) 5867-5873; DOI: https://doi.org/10.4049/jimmunol.165.10.5867

Abstract

The exact mechanisms leading to CNS inflammation and myelin destruction in multiple sclerosis and in its animal model, experimental allergic encephalomyelitis (EAE) remain equivocal. In both multiple sclerosis and EAE, complement activation is thought to play a pivotal role by recruiting inflammatory cells, increasing myelin phagocytosis by macrophages, and exerting direct cytotoxic effects through the deposition of the membrane attack complex on oligodendrocytes. Despite this assumption, attempts to evaluate complement’s contribution to autoimmune demyelination in vivo have been limited by the lack of nontoxic and/or nonimmunogenic complement inhibitors. In this report, we used mice deficient in either C3 or factor B to clarify the role of the complement system in an Ab-independent model of EAE. Both types of complement-deficient mice presented with a markedly reduced disease severity. Although induction of EAE led to inflammatory changes in the meninges and perivascular spaces of both wild-type and complement-deficient animals, in both C3−/− and factor B−/− mice there was little infiltration of the parenchyma by macrophages and T cells. In addition, compared with their wild-type littermates, the CNS of both C3−/− and factor B−/− mice induced for EAE are protected from demyelination. These results suggest that complement might be a target for the therapeutic treatment of inflammatory demyelinating diseases of the CNS.

Numerous studies have documented deposition of complement components in multiple sclerosis (MS)3 lesions (1, 2, 3). Antimyelin Abs, which are potent complement activators, were also recently demonstrated in situ in MS, suggesting that a subset of MS patients may present with Ab-mediated myelin damage (4, 5). Beside direct lysis of oligodendrocytes, multiple other functions of complement can contribute to the pathophysiology of inflammatory demyelinating diseases. For example, the complement anaphylatoxins C3a and C5a and their receptors are involved in the recruitment of both blood-derived leukocytes and glial scar-forming cells during experimental allergic encephalomyelitis (EAE) and MS (6, 7, 8). Furthermore, myelin phagocytosis is enhanced by the deposition of C3b on myelin and the subsequent interaction of C3b with its receptors on macrophages and microglia (9, 10). Finally, the cell cycle and metabolism of oligodendrocytes in vitro is significantly altered by the deposition of the sublytic membrane attack complex (11, 12). However, despite this large body of ex vivo and in vitro evidence, the exact role of complement in vivo in models of autoimmune demyelination remains controversial. Injection of cobra venom factor (CVF), which induces complement depletion, is a widely used technique and has been used in studies of EAE (13, 14, 15, 16, 17), but suffers from important limitations. CVF is toxic and strongly immunogenic, and depletion of C3 by CVF is accompanied by massive generation of C3a and C5a. In fact, depending on the species, strain, and immunization protocols used to induce EAE, complement depletion by CVF treatment has led to conflicting results ranging from delayed onset of the disease to a lack of any clinical or histopathological effect (13, 14, 15, 16, 17). More recent studies using the complement activation inhibitor, sCR1, have demonstrated clearly that complement is crucial in an Ab-dependent form of acute EAE in the Lewis rat (18). In contrast, there is abundant literature showing that MS and EAE are primarily cell-mediated diseases initiated by CD4+ T cells (19, 20); thus, the relevance of the Ab-dependent model to the clinical situation observed in a large majority of MS patients is questionable.

To overcome the limitations of exogenously administered complement inhibitors and Ab-dependent disease models, we chose to study myelin oligodendrocyte glycoprotein (MOG)-induced EAE in mice deficient for C3 (C3−/−) or factor B (FB−/−). MOG-induced EAE has been shown to be Ab-independent in the C57BL/6 mouse strain when induced using the encephalitogenic peptide 35-55 (21, 22). Here, we show that C3−/− and FB−/− mice are largely protected from myelin damage and develop less severe clinical signs of the disease. This effect is accompanied by an unusual distribution of inflammatory cells, characterized by a minimal intraparenchymal infiltration of the CNS. In particular, both C3−/− and FB−/− mice induced for EAE present with decreased numbers of infiltrating macrophages, T cells, and ICAM-1+ cells in the CNS compared with complement-sufficient mice. These results show unequivocally that complement contributes to the pathogenesis of MOG-induced EAE in mice and supports the notion that complement inhibitors may be useful in the treatment of MS.

Materials and Methods

C3−/− and factor B−/− mice

The C3−/− and FB−/− mice used have been described previously (23, 24) and were originally generated in the 129SVJ/C57BL/6 (H2-Db/H2-Db) background. The C3−/− mice produce no serum C3 and consequently lack the ability to generate the anaphylatoxin C3a and the opsonin C3b, and have no serum complement lytic activity (23). The FB−/− mice produce no serum FB and thus cannot form the alternative pathway C3 convertase, rendering them devoid of alternative pathway lytic activity (24). These mice also have reduced classical pathway activity (24). The C3−/− and FB−/− mice were backcrossed to C57BL/6 for at least five generations before the induction of EAE, and their complement-sufficient littermates served as controls. It should be noted that the 129SVJ strain is EAE resistant (25); thus, the clinical presentation of EAE that we observed in this study is a trait of the susceptible C57BL/6 strain. Additional experiments (n = 22 mice) performed for another study confirmed a 100% incidence of EAE in C3−/− and FB−/− mice bearing the C57BL/6-backcrossed background (data not shown). Screening of complement-deficient mice was performed by PCR amplification of the targeted DNA sequence from samples of DNA extracted from tail biopsies (23, 24) and by assessment of serum C3 and FB levels by ELISA (24, 26).

EAE induction and evaluation

In all EAE experiments, C3−/− and FB−/− mice were compared with and immunized at the same time as their wild-type littermates expressing one or two copies of the normal gene. All mice used in this study were females between 8 and 12 wk of age at the time of immunization. Mice were immunized with the MOG peptide 35-55 as previously described (27). Briefly, MOG peptide was synthesized by standard 9-fluorenyl-methoxycarbonyl chemistry and was shown to be >95% pure as determined by reversed phase-HPLC (Research Genetics, Huntsville, AL). Mice were then injected s.c. on days 0 and 7 with 150 μg of peptide emulsified in CFA. In addition, on days 0 and 2 postimmunization (p.i.), mice were given pertussis toxin (500 ng) i.p. Clinical signs of EAE were assessed daily using a standard scale of 0–6 as follows: 0, no clinical signs; 1, loss of tail tone; 2, flaccid tail; 3, incomplete paralysis of one or two hind legs; 4, complete hind limb paralysis; 5, moribund; and 6, death. Animals showing clinical signs of grade 5 for >2 consecutive days were sacrificed and assigned a score of 6. For each animal immunized for EAE, a mean cumulative disease index (CDI) was calculated from the sum of the daily clinical scores observed between day 1 p.i. and day 21 p.i. The average maximum clinical score was calculated for each phenotype group from the sums of the highest clinical score for each mouse.

Histological assessment

At day 21 p.i. or between days 30 and 34 p.i., mice were sacrificed by CO2 inhalation, and spinal cords were removed and either fixed with 4% paraformaldehyde and 2% glutaraldehyde or snap frozen and kept at −80°C until examination. Five to seven animals were randomly chosen in each experimental group (C3- and FB-sufficient controls, FB−/−, and C3−/−), and their spinal cords were assessed for inflammation and demyelination. First, 8-μm frozen sections were stained with hematoxylin and eosin for initial assessment of inflammation. In parallel, the extent of demyelination was evaluated by toluidine blue staining on 1-μm sections of lumbo-thoracic spinal cords embedded in Epon. The presence or absence of demyelination was further confirmed by Luxol fast blue-cresyl violet stains for myelin, and the demyelinated nature of identified lesions was verified by demonstrating axonal integrity within the lesions using modified Bielschowsky stains of adjacent sections. For each technique, four to six sections were evaluated blindly for demyelination and inflammation by three examiners (S.N., S.R.B., and S.L.C.). Demyelination was scored from 0 (normal white matter) to +++ (extensive demyelination), and inflammation was evaluated in different anatomical compartments (meninges, parenchyma, and vessels). Inflammation was scored using the following scale: for meninges and parenchyma: 0, no infiltrating cells; +, few infiltrating cells; ++, numerous infiltrating cells; and +++, widespread infiltration; for vessels: 0, no cuffed vessel; +, one or two cuffed vessels per section; ++, three to five cuffed vessels per section; and +++, more than five cuffed vessels per section.

Immunohistochemistry

C3−/− mice (n = 3) and FB−/− mice (n = 3) and their complement protein-sufficient littermates (n = 3 and 2, respectively) were immunized for EAE and sacrificed on day 30 p.i. to collect lumbo-thoracic spinal cords for immunohistochemical analysis. Immunohistochemistry was performed on 10-μm-thick frozen transversal sections using the Vectastain avidin-biotin complex kit (Vector Laboratories, Burlingame, CA). Acetone-fixed sections were incubated for 60 min with mouse anti-CD11b mAb (BD PharMingen, San Diego, CA), rat anti-mouse CD3 monoclonal Ab (Serotec, Kidlington, U.K.), or hamster anti-mouse ICAM-1 monoclonal Ab (BD PharMingen, San Diego, CA). Biotin-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Baltimore, MD) or a biotin-conjugated “universal” Ab (Vector Laboratories) was then applied. Sections were then rinsed in PBS, incubated in a solution of 1% hydrogen peroxide for 15 min, and washed. Finally, the sections were treated with avidin-peroxidase for 50 min at room temperature (Vectastain avidin-biotin complex kit; Vector Laboratories) followed by addition of 0.04% diaminobenzamadine (Sigma, St. Louis, MO) in PBS with 0.01% H2O2 for 10 min. Semiquantitative analysis of CD11b, CD3, and ICAM-1 was performed by two examiners, and cells were counted as described: three to six sections per animal were examined at low magnification (×20), and the three lesions showing the strongest staining were analyzed at higher magnification (×100) to count labeled cells. Data obtained from C3-and FB-sufficient controls (n = 3 and 2, respectively) were pooled and are referred to as results from EAE control mice. These results were compared with those obtained from C3−/− (n = 3) and FB−/− mice (n = 3).

Electron microscopy

For one animal in each experimental group, ultrathin sections of lumbo-thoracic spinal cords were stained with toluidine blue and white matter tracts were examined by electron microscopy.

Statistical analysis

Results were analyzed for statistical significance using Student’s t test with p < 0.05 considered to be significant.

Results

EAE clinical signs in C3−/− and FB−/− mice

All immunized controls (n = 18) presented with clinical signs of EAE starting on average at day 11 p.i. and with the clinical peak of disease occurring at days 17–21 (Fig. 1⇓). In contrast, the incidence of disease did not reach 100% in complement-deficient animals as one of six of the C3−/− and two of eight of the FB−/− mice did not show any clinical signs of EAE during the 30-day survey period. Furthermore, the clinical severity of EAE was attenuated in both C3−/− and FB−/− mice compared with their wild-type littermates (Fig. 1⇓). FB−/− mice had a lower maximum clinical score (2.7 vs 3.9; p = 0.03) and a lower CDI (15.6 vs 27.4; p = 0.03) compared with those of controls. Similarly the maximum clinical score was significantly lower in C3−/− mice compared with controls (2.75 vs 4.7; p = 0.04) (Table I⇓). The mean clinical scores (Fig. 1⇓) were significantly reduced in both C3−/− (2.1 vs 3.45; p = 0.01) and FB−/− mice (1.96 vs 2.83; p = 0.04).

FIGURE 1.
FIGURE 1.

Clinical course of MOG-induced EAE in complement-deficient mice. A, EAE was induced and scored in C3−/− mice (n = 6) and their C3-sufficient littermates (n = 9). B, Same as in A except FB−/− mice (n = 8) and their FB-sufficient littermates (n = 9) were analyzed. Results are shown as an average curve obtained from three independent experiments.

View this table:
Table I.

EAE clinical features in control, C3−/− and FB−/− mice

Histological analysis and electron microscopy

With the exception of one C3−/− mouse, all mice immunized for EAE demonstrated CNS inflammation as assessed by hematoxylin and eosin and toluidine blue staining (Table I⇑). On examination of the different CNS compartments (meninges, Virchow-Robin spaces, vessels, perivascular space, and parenchyma), we found cellular infiltration throughout the CNS in C3- and FB-sufficient controls (Figs. 2⇓, A and C, 3⇓, A and C, and Table I⇑). In contrast, although there was significant meningeal infiltration and perivascular cuffing in both C3−/− and FB−/− mice, we observed virtually no infiltration in the parenchyma (Figs. 2⇓B and 3⇓B; Table I⇑). We also evaluated the extent of demyelination in control and complement-deficient mice by toluidine blue staining of lumbo-thoracic spinal cord sections obtained from three animals per experimental group. In addition, Luxol fast blue-cresyl violet stains were performed on spinal cord sections of two FB−/− and two FB control animals immunized for EAE and sacrificed at day 34 p.i. In control groups, extensive areas of subpial, perivascular, and parenchymal demyelination were observed associated with parenchymal infiltrating cells (Figs. 2⇓, C and E, 3, C and E, and Table I⇑). In contrast, in both C3−/− and FB−/− mice immunized for EAE, only a few areas of limited subpial demyelination were noticed in all sections examined (Figs. 2⇓D and 3⇓D; Table I⇑). Interestingly, in these animals, the loss of myelin sheaths in areas surrounding severe meningeal and/or perivascular infiltration appeared very mild or negligible (Figs. 2⇓F and 3⇓F). These data were supported by electron microscopy analysis of spinal cord sections obtained from EAE control animals which showed extensive myelin destruction along with astrogliosis and parenchymal infiltration by multivacuolar macrophage cells containing lipid droplets (Fig. 4⇓, A and B). In contrast, C3−/− and FB−/− mice, sacrificed at the same time point after induction of EAE, had only mild or no myelin damage (Fig. 4⇓C).

FIGURE 2.
FIGURE 2.

Neuropathology of lumbo-thoracic spinal cord lesions during MOG-induced EAE in C3−/− mice (right panel) and their wild-type littermates (left panel) sacrificed 21 days (A and B) or 30 days (C–F) after immunization. A and B, Hematoxylin staining of representative spinal cord sections from a wild-type mouse (A) shows extensive infiltration of the parenchyma while, in C3−/− mouse (B), inflammatory cells are predominantly located in the meninges and Virchow-Robin space (original magnification, ×100). C–F, Toluidine blue staining of representative spinal cord sections from a wild-type mouse (C and E) and C3−/− mouse (D and F). Multiple inflammatory cells (arrows in C) and naked axons (arrowheads in E) are found in the parenchyma of a C3 control mouse. Myelin sheaths are preserved in a C3−/− mouse (D and E). Original magnification: ×100, C and D and ×500, E and F.

FIGURE 3.
FIGURE 3.

Neuropathology of lumbo-thoracic spinal cord lesions during MOG-induced EAE in FB−/− mice (right panel) and their wild-type littermates (left panel) sacrificed 34 days after immunization. A and B, Luxol fast blue-cresyl violet stains of representative spinal cord sections from a wild-type mouse (A) show a widespread parenchymal infiltration (arrows), whereas in the FB−/− mouse (B), inflammatory cells are only found in the meninges and Virchow-Robin space (original magnification, ×200). C–F, Toluidine blue staining of representative spinal cord sections from a wild-type mouse (C and E) and FB−/− mouse (D and F). Multiple macrophages containing lipid droplets (arrows in E) as well as naked axons (a) are observed in the parenchyma of an FB control mouse. Myelin sheaths located in the vicinity of an infiltrated vessel are preserved in an FB mouse (D). The border of an infiltrated vessel (double-headed arrow) and the adjacent white matter of an FB−/− mouse is shown in F. Original magnification: ×100, C; ×200, D; and ×500, E and F.

FIGURE 4.
FIGURE 4.

Electron micrographs of the spinal cord of a C3-sufficient (A and B) and a C3−/− mouse induced for EAE. A, Demyelinated axons (a) along with infiltrating macrophage cells (M) containing lipid droplets (arrows) are observed in the control mouse immunized for EAE and sacrificed at day 30. Original magnification, ×4,000 B, In the same animal, axons (a) show obvious loss of myelin. Original magnification, ×12,000. C, Normal-appearing white matter in a C3−/− animal sacrificed at day 30 p.i. Original magnification, ×3,000.

Immunohistochemical analysis

To determine whether there were differences in the composition of the cellular infiltrate among control, C3−/−, and FB−/− mice, we performed immunohistochemical analysis. In control EAE mice, numerous CD3+ T cells were detected in the meninges, perivascular space, and parenchyma of the spinal cord (Fig. 5⇓A). Compared with control mice, C3−/− mice presented with remarkably fewer CD3+-infiltrating cells (Fig. 5⇓B and Table II⇓). Comparable results were obtained using FB−/− mice (data not shown). Infiltrating cells expressing the macrophage marker CD11b were also detected in the spinal cords of control EAE mice and most of these cells displayed a round morphology (Fig. 5⇓C). It has been demonstrated that during EAE, CD11b+ round cells comprise macrophages and ameboid microglia with high phagocytic activity whereas CD11b+ process-bearing cells are putative microglial cells with low phagocytic activity (28). Interestingly, although there was a decrease in CD11b+ cells in C3−/− EAE mice compared with control EAE mice, the number of CD11b+ round cells compared with CD11b+ process-bearing cells was significantly reduced (Fig. 5⇓D and Table II⇓). Similar results were obtained using FB−/− mice (data not shown). We also observed a reduction in ICAM-1+ staining in C3−/− or FB−/− mice on both infiltrating cells and putative endothelial cells (Table II⇓).

FIGURE 5.
FIGURE 5.

CD3 and CD11b expression in the spinal cords of control and C3−/− mice during EAE. Spinal cords from EAE control or C3−/− mice (sacrificed on day 30 p.i.) were analyzed by immunohistochemistry using anti-CD3 Ab or an anti-CD11b Ab as described in Materials and Methods. A, Microphotograph showing a massive and widespread parenchymal infiltration by CD3+ T cells in a representative control animal. A higher magnification is presented in the inset. B, Only a few CD3+ T cells (arrows) are detected in the spinal cord of a representative C3−/− mouse. C, In a representative control animal, numerous CD11b+ round cells are found in an inflammatory lesion of the spinal cord. Note that very few process-bearing CD11b+ cells are detectable in the adjacent white matter (arrow). D, In an inflammatory lesion of a C3−/− mice, the majority of CD11b+ cells are process-bearing putative microglial cells (arrows). Original magnification: ×20, A; ×100, inset in A; ×50, B; and ×100, C and D.

View this table:
Table II.

Semiquantitative analysis of infiltration control, C3−/−, and FB−/− mice

Discussion

In the present study, we show that both C3−/− and FB−/− mice are protected from EAE. Although the main immunological abnormality in C3−/− mice is a decreased Ab response to T cell-dependent Ags (reviewed in Ref. 28), this does not account for the protective effect we observed in C3−/− mice since the EAE model we used was shown by others to fully develop even in the absence of Abs (21, 22). FB−/− mice have no demonstrated immunological deficiency and are partially protected from lupus nephritis (29). Moreover, we consistently observed CNS inflammation in both FB−/− and C3−/− mice induced for EAE, albeit limited compared with wild type, also making it unlikely that the protective effect was due to a defective T cell response in the periphery. Rather, our combined data indicate that complement-mediated events that occur in the CNS during the effector phase of EAE are blocked or impaired in C3−/− and FB−/− mice. The fact that few infiltrating cells were found in the parenchyma of the complement-deficient animals suggests that their protective defect might be reduced production of C3a and/or C5a, which would otherwise direct the recruitment of inflammatory cells from the meninges and/or the perivascular cells to the parenchyma. In fact, it has been suggested that leukocyte migration to these different CNS compartments is regulated separately and that parenchyma-derived chemokines might control the trafficking of cells from a perivascular location into the adjacent white matter (30, 31). Our data raise the possibility that C3a and C5a may play a major role in this phenomenon, since both molecules could act through a direct chemotactic effect on monocytes and T cells (32), induction of chemokine synthesis by parenchymal resident cells (33, 34) and/or by increasing the expression of adhesion molecules on leukocytes and endothelial cells (reviewed in Ref. 35). In particular, studies have shown that both C5a and the membrane attack complex are able to induce ICAM-1 expression on endothelial cells (36, 37, 38). Thus, in the complement-deficient animals reduced production of C5a would lead to decreased ICAM-1 expression which might be responsible, in part, for decreased T cell extravasation toward the CNS parenchyma (39). In addition, C5a is chemotactic for T cells and since the C5aR is expressed on parenchymal T cell in EAE (7, 32), it is also possible that reduced C5a levels might reduce T cell trafficking into the CNS of C3−/− and FB−/− mice through an ICAM-1-independent pathway. As astrocytes and microglia are chemoattracted by C5a (40, 41, 42), low C5a levels would limit movement of both astrocytes and microglia to developing lesions and subsequently reduce demyelination and glial scar formation. Results from recent preliminary studies from our laboratory demonstrate significantly reduced disease incidence and delayed disease onset and severity in C5aR−/− mice, supporting a significant role for C5a in EAE (43).

In addition to altered cellular trafficking, we observed a remarkable reduction in demyelination in FB−/− and C3−/− mice compared with controls. Demyelination involves macrophage- and microglia-mediated removal of myelin sheaths and engulfment of myelin debris, a process considered to be largely complement dependent (9, 10, 44, 45). Furthermore, it is established that the complement receptor type 3 (CR3, CD18/CD11b) plays a pivotal role in myelin phagocytosis during EAE, with ligand binding resulting in increased phagocytosis as well as TNF-α and NO release by macrophages and microglia (45, 46). Obviously the limited complement activation potential of FB−/− and C3−/− mice would partially or completely inhibit the generation of the complement-derived opsonin C3b. As we observed little to no demyelination in both types of complement-deficient mice, our data demonstrate an essential role for C3 and factor B in this process. The role C3a plays in glial cell chemoattraction is unclear, but may be less important than C5a as microglia are not chemoattracted by C3a in vitro (42).

It is noteworthy that despite the absence of key complement components, FB−/− and C3−/− mice developed EAE (albeit in an attenuated form). C5a, and perhaps C3a, may still contribute to disease development and progression in the complement-deficient mice, despite their reduced levels, as it is possible that proteases other than the C3 and C5 convertases might generate small amounts of these potent inflammatory mediators. For example, it has been shown that trypsin, α-thrombin, plasmin, elastase, and kallikrein can generate C5a or C5a-like fragments with functional activity (47, 48, 49). Whether C3a or C3a-like molecules can also be generated by similar mechanisms is not established. Thus, the possibility that complement anaphylatoxins, especially C5a, might be released through bypass mechanisms in C3−/− and FB−/− mice during EAE cannot be excluded.

We have recently shown that CNS-targeted expression of a soluble complement inhibitor, sCrry, prevents or delays the onset of MOG-induced EAE (50). sCrry transgenic mice with a C57BL/6 background have delayed disease onset, but then develop EAE with similar severity to that observed in their nontransgenic littermates. In contrast, sCrry-transgenic mice with a SJL background (a strain which develops a milder form of EAE) had no clinical signs of disease. We report here that for MOG-induced EAE in FB−/− and C3−/− C57BL/6 mice, disease onset was identical to that of complement-sufficient controls, but disease severity was significantly reduced. The combined data from both the sCrry-transgenic mice and the complement-deficient mice demonstrate that inhibiting complement-mediated functions at multiple levels in the complement activation pathways provides significant protection from MOG-induced EAE. Moreover, our data indicate that the alternative pathway might be the predominate pathway involved in complement-mediated inflammation and demyelination in MOG-induced EAE. Preliminary studies from our laboratory show that treatment with the chimeric recombinant complement inhibitor Crry-Ig (51) also provides significant protection from MOG-induced EAE (data not shown). Although these data indicate that inhibition of complement activation provides protection in the effector phase of disease, it is likely that complement-mediated functions are important in the inductive phase of disease as well. The increased expression of the C5aR on CNS endothelium and on infiltrating cells before clinical signs of disease (7), and the marked attenuation of EAE severity in C5aR−/− mice (43) support this notion. Taken together, these data strengthen the concept that intervention in the complement system provides a potentially beneficial therapeutic avenue for the treatment of MS and other CNS inflammatory diseases.

Acknowledgments

We thank Ed Philipps from the High Resolution Imaging Facility (University of Alabama at Birmingham) for technical assistance in electron microscopy studies. We thank Martin Pierre Charlie Nataf and his mother for continuing inspiration throughout the course of this study. The continuing inspiration of F.J.B. is acknowledged.

Footnotes

  • ↵1 This study was supported by National Institutes of Health Grants NS29719 (to S.R.B.), AI25011 (to R.A.W.), and AI142183 (to A.J.S.) and by National Multiple Sclerosis Society Advanced Postdoctoral Fellowship FA 1306-A (to S.N.).

  • ↵2 Address correspondence and reprint requests to Dr. Scott R. Barnum, Department of Microbiology, University of Alabama, Birmingham, 701 19th Street South, LHR/141, Birmingham, AL 35294. E-mail address: sbarnum{at}uab.edu

  • ↵3 Abbreviations used in this paper: MS, multiple sclerosis; EAE, experimental allergic encephalomyelitis; CVF, cobra venom factor; MOG, myelin oligodendrocyte glycoprotein; FB factor B; p.i., postimmunization; CDI, cumulative disease index.

  • Received June 13, 2000.
  • Accepted August 17, 2000.

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