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The Journal of Immunology, 2002, 168: 3105-3112.
Copyright © 2002 by The American Association of Immunologists

Increased Severity of Experimental Allergic Encephalomyelitis in lyn-/- Mice in the Absence of Elevated Proinflammatory Cytokine Response in the Central Nervous System

Caigan Du and Subramaniam Sriram1

Department of Neurology, Multiple Sclerosis Research Center, Vanderbilt University Medical Center, Nashville, TN 37212


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
lyn, a member of the src kinase family, is an important signaling molecule in B cells. lyn-/- mice display hyperactive B-1 cells and IgM hyperglobulinemia. The role of lyn on T cell function and development of Th1-mediated inflammatory disease is not known. Therefore, we examined the effect of disruption of the lyn gene on the development of experimental allergic encephalomyelitis (EAE), a well-established Th1-mediated autoimmune disease. Following immunization with myelin oligodendrocyte protein (MOG) p35-55, lyn-/- mice had higher clinical and pathological severity scores of EAE when compared with wild type (WT). The increase in the severity of EAE in lyn-/- mice was not associated with a commensurate increase in the production of proinflammatory cytokines in the CNS. lyn-/- mice with EAE showed elevation in serum anti-IgM MOG Ab levels over that seen in WT mice, along with a modest increase in the mRNA levels of complement C5 and its receptor, C5aR, in the spinal cord. Transfer of serum from MOG-immunized lyn-/- mice worsened EAE in WT mice, suggesting a pathogenic role for anti-MOG IgM Abs in EAE. These observations underscore the potential role of lyn in regulation of Th1-mediated disease and the role of autoantibodies and complement in the development of EAE.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Experimental allergic encephalomyelitis (EAE)2 is a Th1 (CD4+)-mediated inflammatory demyelinating disease of the CNS, and shows many pathological and clinical similarities with human multiple sclerosis (1, 2, 3). The disease is induced in susceptible strains of animals from different species including primates, by either immunization with neural Ags (myelin basic protein (MBP), proteolipid protein (PLP), or myelin oligodendrocyte protein (MOG)) or the transfer of T cells that recognize neural Ags. EAE is considered a prototypic Th1-mediated autoimmune disease because adoptive transfer of neural Ag-specific encephalitogenic Th1 cells alone is sufficient to induce the disease. The mechanism by which CD4+ T cells mediate CNS demyelination is unclear because the target tissue (oligodendrocyte and myelin) does not express MHC class II Ags. It is believed that the presence of proinflammatory cytokines such as TNF-{alpha}, IL-12, and IFN-{gamma} may lead to damage and subsequent loss of myelin (4). The role of B cells and myelin-specific Abs in mediating myelin loss is controversial. In MOG Ag-induced EAE, B cells are required for development of EAE induced following immunization with whole protein Ag (human rMOG), but not following immunization with MOG p35-55 (5, 6, 7, 8). EAE in rats and marmosets induced following immunization with MOG Ag results in extensive primary demyelination of the CNS, which is dependent on the presence of anti-MOG Abs. These results indicate that B cells and autoantibodies play a role in the pathologic damage to the CNS tissue of EAE animals (9, 10, 11).

lyn, a nonreceptor tyrosine kinase of the src family, is predominantly expressed in hemopoietic cells (12, 13). lyn associates with a number of hemopoietic cell surface receptors, including B cell receptor, Fc{epsilon}RI, CD40, Fc{gamma}RI, CD14, and cytokine receptors (e.g., G-CSFR; IL-2R) (14, 15, 16). The physiological importance of lyn in B cell signaling has been established in lyn-/- mice by a number of groups (17, 18, 19). lyn knockout (KO) mice show IgM hyperglobulinemia, splenomegaly, and accumulation of lymphoblast-like cells (B-1 lineage) and IgM-secreting plasma cells. The increased state of activation of lyn-/- B cells and the association of lyn with the B cell receptor have suggested that lyn may act as a negative regulator of B cell function (20). The role of lyn on T cell function has not been fully explored. In particular, T cell proliferative response and cytokine secretion against Ag challenge have to date not been studied in lyn-/- mice. We predicted that if B cells were important in the development of EAE, the presence of a heightened autoantibody response would enhance the development of EAE. We examined the effect of the disruption of the lyn gene on T cell function, Th1 cytokine response, and the pattern of autoantibodies against MOG. We hypothesized that the increase in the autoantibody levels was likely to modify the expression of clinical and pathologic severity of EAE.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and reagents

lyn-/- mice were from inbreed of an established lyn-/- colony with C57BL/6 background, kindly provided by C. A. Lowell (University of California, San Francisco, CA). Wild-type (WT) C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were bred and maintained in the animal care facility at Vanderbilt University (Preston Research Building, Nashville, TN). The anti-murine IL-12 mAbs (C17.5 and C15.6), gifts from G. Trinchieri (Wister Institute, Boston, MA), were purified by quaternary aminoethyl gel and Sephadex G-25 M column (Amersham Biosciences, Uppsala, Sweden) from hybridoma ascetic fluid. C15.6 was biotinylated according to manufacturer’s protocol and used as a detecting Ab, whereas C17.5 was used as a capture Ab, for IL-12 ELISA. Murine rIL-12 was kindly provided by Genetics Institute (Cambridge, MA). MOG peptide (p35-55: MEVGWYRSPFSRVVHLYRNGK) was synthesized by Genemed Synthesis (South San Francisco, CA).

Ig assay

Anti-MOG p35-55 levels (IgG and IgM) in serum were determined by ELISA. The high binding enzyme immunoassay/RIA plate (Corning, Corning, NY) was coated with 100 µl/well 10 µg/ml MOG peptide along with known concentrations of appropriate mouse Ig type (Sigma-Aldrich, St. Louis, MO) in triplicate as Ig standards. Both were dissolved in 60 mM bicarbonate buffer (pH 9.6) and bound to the plate overnight at 4°C. After washing with PBST (PBS containing 0.05% Tween 20) three times, the plate was blocked with 3% BSA for 1 h at 25°C. Serial dilution of serum in 3% BSA was added to the coated wells in triplicate and incubated overnight at 4°C. After washing with PBST four times, 100 µl/well biotinylated goat anti-mouse IgG or goat anti-mouse IgM (µ-chain specific; Sigma-Aldrich) was added and incubated for 2 h at 25°C. The plate was washed with PBST four times, followed by incubation with avidin-alkaline phosphatase (1:10,000) for 1 h at 25°C. After washing again with PBST five times, the substrate p-nitrophenyl phosphate (Sigma-Aldrich) was added and then incubated for 30 min. The color in each well was measured by the absorbance at 405 nm in an ELISA plate reader (Bio-Tek Instruments, Winooski, VT), and was calculated to the amount of each Ig against MOG peptide in serum by interpolation from the standard curve.

Proliferation assay

T cell cultures were grown in RPMI 1640 complete medium (RPMI 1640 medium, 100 U/ml penicillin, and 100 µg/ml streptomycin, and 10% FBS; Life Technologies, Rockville, MD) in a 96-well microtiter plate under the atmosphere of 5% CO2 and 95% air at 37°C. A total of 2.5 x 105 MOG peptide-primed lymphocytes per well was cultured in the presence or absence of MOG peptide in triplicate for 72 h, or 2 x 105 splenocytes/well were cultured in the presence or absence of mitogen (Con A or anti-CD3 Ab) in triplicate for 12 h. The cultures were harvested using a Harvester96 (Tomtec, Orange, CT) after incubation with 0.5 µCi/well [3H]thymidine (DuPont, Boston, MA) for the last 18 h (with MOG peptide) or 10 h (with mitogen). The radioisotope incorporation as index of T cell proliferation was determined using a betaplate liquid scintillation counter (Wallac, Turku, Finland).

ELISA of cytokines

Levels of cytokine (IL-12 p40, IFN-{gamma}, IL-4, and TNF-{alpha}) were measured by ELISA, as described previously (21). The matched Ab pairs and standards for IL-4, IFN-{gamma}, and TNF-{alpha} were purchased from Endogen (Woburn, MA), and the Abs and standards used for IL-12 p40 ELISA were gifts from G. Trinchieri, as described above.

Induction, treatment, and evaluation of EAE

Active EAE was induced in female mice (6–8-wk old) by s.c. immunization with either 800 µg mouse spinal cord homogenate (MSCH) or 200 µg MOG peptide (residue 35-55) in CFA (Sigma-Aldrich) on days 0 and 7. In addition, pertussis toxin (350 ng in 0.5 ml PBS per animal; Sigma-Aldrich) was i.p. injected on days 0 and 2. To determine the role of anti-MOG Abs (IgM) in EAE, recipient WT mice were i.p. injected with anti-MOG hyperimmune sera from lyn-/- or WT mice on days 9 (200 µl), 11 (50 µl), and 13 (50 µl). Paralysis was graded as follows: 0, normal; 0.5, stiff tail; 1, limp tail; 1.5, limp tail with inability to right; 2, paralysis of one limb; 2.5, paralysis of one limb and weakness of one other limb; 3, complete paralysis of both hind limbs; 4, moribund state; 5, dead.

To assess the degree of inflammation and demyelination, mice with MOG-induced EAE were euthanized on day 25 and perfused by intracardiac injection of 4% paraformaldehyde and 1% glutaraldehyde in PBS. Transverse sections of the cervical, upper thoracic, lower thoracic, and lumbar region of the spinal cord were stained with Luxol Fast Blue or H&E. Each spinal cord section was further subdivided into an anterior, posterior, and two lateral columns. Each subdivided area displaying either lymphocyte infiltration or demyelination was assigned a score of one; thus, each animal had a potential maximum score of 16.

Flow cytometry analysis

Mice with EAE were euthanized on day 25 and perfused by intracardiac injection of cold PBS. The mononuclear cells from the CNS were isolated, as described previously (22). The mononuclear cells were resuspended in RPMI 1640 containing 2% FBS and incubated for 15 min with anti-CD16/32 Abs (eBioscience, San Diego, CA) to block FcR. The cells were either double stained with anti-CD11b/Mac-1-FITC and anti-CD3-PE-Cy5 (eBioscience) or triple stained with anti-CD3-PE-Cy5, anti-CD8-PE, and anti-CD4-FITC (eBioscience). Staining was accomplished with incubation of the cells with the respective Abs for 30 min at 4°C. The cells were then washed with PBS and fixed in 1% paraformaldehyde. The cells were analyzed using a FACScan (BD Biosciences, Franklin Lakes, NJ). The forward/side scatter gating was used to exclude dead cells.

Isolation of total RNA and semiquantitative RT-PCR

After perfusion with PBS, spinal cord from each MOG-induced EAE mouse was collected, and total RNA was extracted using TRI Reagent (Sigma-Aldrich) in accordance with manufacturer’s protocol. Total RNA (4 µg) was reverse transcribed to cDNA using GeneAmp RNA PCR kit with oligo(dT)16 primers (PerkinElmer, Branchburg, NJ). PCR amplification of each cDNA target was performed along with GAPDH, which served as an internal control for RNA quantity. Each PCR contained 5 µl cDNA, 2 µl 10x PCR buffer (PerkinElmer), 1 µl 25 mM MgCl2, 0.5 µl of each dNTP (10 mM), 0.5 µl of sense and antisense target gene-specific primers (50 pmol/µl), 0.25 µl AmpliTaq DNA polymerase (5 U/µl; Perkin-Elmer), and 13.75 µl nuclease-free H2O, and was conducted in PTC-200 Peltier Thermal Cycler (MJ Research, Watertown, MA). The specific sense and antisense oligonucleotide PCR primers for each cDNA target were listed in Table IGo. Amplification conditions, including annealing temperatures, number of cycles, and extension times, were optimized for each target. PCR products were resolved on a 1.5% agarose gel containing 0.5 µg ethidium bromide/ml and visualized under UV light. The band density was quantitated using a Digital Imaging System (IS-1000 version 2.0; Alpha Innotech, San Leandro, CA).


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  		Table I. Primers used in PCR amplification1

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
lyn-/- mice produce higher levels of anti-MOG IgM Abs

Following immunization with MOG p35-55, lyn-/- mice developed circulating anti-MOG IgM Abs that were higher than those in WT mice. In lyn-/- mice, mean serum level of anti-MOG IgM was 49.1 µg/ml, as compared with 15.58 µg/ml in WT (p < 0.01) (Fig. 1GoA). However, when the serum levels of anti-MOG IgG Abs were compared between these two groups, no significant difference was noted (Fig. 1GoB). These studies show a preferential expansion of anti-MOG IgM-producing cells in lyn-/- mice when compared with WT mice following immunization with MOG p35-55.



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  		FIGURE 1. Serum levels of anti-MOG Abs in lyn-/- and WT mice. Three mice from each group were immunized with MOG p35-55 (200 µg/animal) in CFA on days 0 and 7. Sera were collected on day 14, and IgM (A) and IgG (B) levels were measured by ELISA. The data are presented by the mean value of three separate measurements of each sample.

 
MOG-primed lymphocytes from lyn-/- mice show a reduced T cell proliferative response and Th1 cytokine secretion

To determine the effect of disruption of the lyn gene on T cell function, we examined the cytokine profile and T cell proliferative response of lymph node cells following immunization with MOG p35-55. The proliferative responses of lyn-/- T cells to all doses of MOG p35-55 (6.25–50 µg/ml) were reduced when compared with those of WT T cells (Fig. 2Go). For example, in cultures stimulated with 50 µg/ml MOG p35-55, the proliferative response of lyn-/- lymphocytes was 5042 ± 1649 cpm, equal to 47.5% of 9611 ± 1799 cpm in WT lymphocytes. This defect was not due to differences in TCR signaling, because T cell proliferative response of lyn-/- splenocytes was similar with that of WT counterparts following stimulation with either anti-CD3 Abs or Con A (Fig. 3Go).



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  		FIGURE 2. Proliferative response to MOG p35-55 in MOG-primed lymphocytes from lyn-/- and WT mice. Lymphocytes were isolated from regional draining lymph nodes of lyn-/- and WT mice (three mice in each group) on day 14 following immunization with MOG p35-55 (200 µg/mouse) in CFA. Lymph node cells were cultured with MOG p35-55 for 72 h, and T cell proliferation in each culture was determined using tritium-labeled thymidine incorporation assay.

 


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  		FIGURE 3. Proliferative response of naive splenocytes from lyn-/- and WT mice to anti-CD3 Abs and Con A. The splenocytes were isolated from spleens of lyn-/- and WT mice, and stimulated with Con A or anti-CD3 Ab for 12 h. T cell proliferation in each culture was determined using tritium-labeled thymidine incorporation assay.

 
We next examined whether the difference of Ag-specific proliferation was correlated with proinflammatory cytokine levels in the culture supernatants. As shown in Fig. 4Go, lyn-/- lymphocytes produced lower levels of IFN-{gamma}, IL-12 p40, and TNF-{alpha} than WT lymphocytes did. In cultures stimulated with 50 µg/ml MOG p35–55, lyn-/- lymphocytes produced 5.76 ± 1.5 ng/ml IFN-{gamma}, 5.61 ± 0.32 ng/ml IL-12 p40, and 130.35 ± 4.95 pg/ml TNF-{alpha}, respectively. WT lymphocytes cultured under the same conditions produced 13.85 ± 1.09 ng/ml IFN-{gamma} (41% increase over lyn-/-), 8.44 ± 0.61 ng/ml IL-12p40 (66.6% increase over lyn-/-), and 278.55 ± 17.56 pg/ml TNF-{alpha} (46% increase over lyn-/-). No measurable IL-4 was seen in either lyn-/- or WT cultures. These results suggest that lyn-/- mice show a reduction in not only T cell proliferation, but also proinflammatory cytokine production to MOG p35-55 when compared with WT mice.



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  		FIGURE 4. Measurement of cytokines in culture supernatants of MOG p35-55-primed lymphocytes from lyn-/- and WT mice. Lymphocytes were isolated from regional draining lymph nodes of lyn-/- and WT mice (three in each group) on day 14 following immunization with MOG p35-55 (200 µg/mouse) in CFA. A total of 4 x 106 cells/ml of MOG-primed lymphocytes was cultured in the presence of increasing concentration of MOG p35-55, and the culture supernatants were collected at 72 h. The Ag-stimulated production of cytokine (IFN-{gamma}, IL-4, IL-12 p40, and TNF-{alpha}) in the cultures of these lymphocytes was measured by ELISA. IL-4 was not detectable in both lyn-/- and WT cultures. The levels of other cytokines were reduced in the lyn-/- cultures (KO vs WT, p < 0.001).

 
lyn-/- mice show increase in severity of EAE

We next examined the effect of lyn gene disruption on the development of EAE (Fig. 5Go). When EAE was induced following immunization with MOG p35-55, every mouse in the lyn-/- group developed severe paralytic signs. Three lyn-/- mice died in the acute paralytic stage. The mean day of onset of symptoms was on day 11, and a maximal mean severity score was 3.86. In the WT group, EAE was less severe, with onset on day 12 and a maximal mean severity score 0.85. None of the WT mice died from the acute attack (lyn-/- vs WT, p < 0.001) (Fig. 5GoA). Following immunization with MSCH, a similar pattern of disease severity was noted (Fig. 5GoB). Among lyn-/- mice, five of seven developed clinical paralysis, with a maximal mean score of 1.43, while only three of seven mice in the WT group were paralyzed with a maximal score of 0.43 (lyn-/- vs WT, p < 0.05).



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  		FIGURE 5. Induction of EAE in lyn-/- and WT mice. EAE was induced following immunization with A, MOG p35-55 (200 µg/mouse, n = 10), or B, MSCH (800 µg/mouse, n = 7) in CFA on days 0 and 7, as described in Materials and Methods. The severity of EAE is presented as mean clinical score in each group.

 
To further show that clinical severity correlated with pathologic severity, transverse sections of the cervical, upper thoracic, lower thoracic, and lumbar region of the spinal cord were stained with Luxol Fast Blue-Periodic acid and H&E, and scored for the degree of inflammation and demyelination (Fig. 6Go). Areas of demyelination and inflammation were more widespread in lyn-/- mice when compared with those seen in WT mice (Fig. 6Go, A and B). The pathologic scores in the lyn-/- mice were 9.1 for demyelination and 11.3 for inflammation, while in the WT mice, the scores for demyelination and inflammation were 4 and 6.5, respectively. These observations showed a close association between the development of paralysis and the pathological severity of the disease.



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  		FIGURE 6. CNS inflammation (lymphocyte infiltration) and demyelination in lyn-/- and WT mice with EAE. Spinal cords were isolated from EAE mice randomly selected from lyn-/- (clinical score: 3, 3, 1.5) and WT (clinical score: 1, 1, 1, 1) mice on day 25. A representative section from each group showing A, demyelination (stained by Luxol Fast Blue-Periodic Acid Schiff), and B, lymphocyte infiltration (stained by H&E), and C, the mean pathologic score of demyelination and infiltration of each group.

 
The nature of the infiltrating lymphocytes was analyzed by flow cytometry. CNS tissues from two lyn-/- (clinical score 3) or WT (clinical score 1) mice with EAE were harvested on day 25 and stained, as described above. As shown in Fig. 7Go, the ratio of CD4-CD8 T cells in lyn-/- mice was 1.9, slightly higher than 1.34 found in WT mice. The number of macrophages was not different between the two groups.



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  		FIGURE 7. The population analysis of infiltrating lymphocytes in the CNS of lyn-/- and WT mice with EAE. The mononuclear cells isolated from the CNS of lyn-/- (clinical score: 3, 3) and WT (clinical score: 1, 1) mice on day 25 were stained for CD3 and CD11b (Mac-1) (left column) or for CD3, CD4, and CD8 (right column), and analyzed by flow cytometry. Percentage of subsets of CD4+ or CD8+ T cells was counted on gated CD3+ T cells. CD3-CD11b- cells were not defined, but could represent B cells or nonhemopoietically derived CNS glial cells.

 
lyn-/- mice with EAE have reduced levels of proinflammatory cytokines

RNA was isolated from spinal cords of mice with EAE, and levels of CD3-{gamma}, IL-4, IFN-{gamma}, IL-12 p40, and TNF-{alpha} were measured by a semiquantitative RT-PCR. As shown in Fig. 8Go, even though the clinical severity scores of the lyn-/- mice were higher than those of the WT mice, the relative amounts of all the targeted proinflammatory genes (with the exception of IL-4) were reduced in the CNS of lyn-/- mice when compared with those shown in the WT mice. In the WT group, all three animals with clinical score of 1 (paralyzed tail at the height of clinical paralysis) had increased levels of CD3-{gamma}, IFN-{gamma}, and TNF-{alpha} when compared with the two mice that did not show any paralytic signs. However, this pattern was not seen in the lyn-/- group with higher clinical score of 2, 3, 3, and 4, respectively. The levels of CD3-{gamma}, IFN-{gamma}, TNF-{alpha}, and IL-12 p40 in these lyn-/- mice were lower than those seen in the paralyzed WT animals (clinical score 1), and similar with those seen in the nonparalyzed WT mice. The presence of CD3-{gamma} mRNA in the CNS of naive lyn-/- mouse could be due to contamination from circulating T cells, resulting from incomplete perfusion. These results show a discordance between the clinical/pathologic severity and levels of proinflammatory cytokine mRNA in the CNS.



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  		FIGURE 8. The mRNA levels of CD3 (T cells) and proinflammatory cytokines in CNS of lyn-/- and WT mice with EAE, following immunization with MOG p35-55. Total RNA was isolated from PBS-perfused spinal cords of EAE mice randomly selected from lyn-/- (clinical score: 2, 3, 3, 4) and WT (clinical score: 1, 0, 0, 1, 1) mice on day 25 and naive mice. The mRNA levels of CD3-{gamma}, IL-4, IFN-{gamma}, IL-12 p40, TNF-{alpha}, and internal control GAPDH were determined by semiquantitative RT-PCR. A, PCR products were visualized in 1.5% agarose containing 0.5 µg/ml ethidium bromide; B, mRNA levels of each target were measured by a digital-image system, and normalized with the internal control GAPDH. N, naive mice. The data are a representative of three separate experiments.

 
The role of complement/Ab-mediated tissue damage in lyn-/- mice with EAE

Because lyn-/- mice showed an increase in anti-MOG IgM Abs in serum following immunization with MOG peptide, we predicted that the increase in the clinical and pathological scores in this group of mice might relate to complement-mediated tissue damage. We therefore first examined the expression of C5 and its receptor C5aR in the CNS of lyn-/- and WT mice with EAE (Fig. 9Go). The average level of C5 mRNA in WT mice was 1.2, while in lyn-/- mice it was 2.95 (a 2.5-fold increase over WT). When C5aR mRNA was examined, the level of C5aR mRNA was 3.09 in WT and 5.21 in lyn-/- mice (a 1.7-fold increase over WT). These studies show that when compared with WT mice, although the levels of proinflammatory cytokines were reduced, the expression of complement C5 and its receptor C5aR was not decreased in the CNS of lyn-/- mice.



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  		FIGURE 9. The mRNA levels of C5 and C5aR in the CNS of lyn-/- and WT mice with EAE. The C5 and C5aR mRNA in the CNS of lyn-/- and WT mice with EAE were analyzed by semiquantitative RT-PCR using procedures as described in Fig. 7Go. A, PCR products were visualized in 1.5% agarose containing 0.5 µg/ml ethidium bromide; B, mRNA levels of each target were measured by a digital-image system, and normalized with the internal control GAPDH. N, naive mice. The data are a representative of three separate experiments.

 
We next examined the role of anti-MOG Abs on the development of EAE. Hyperimmune anti-MOG antisera from lyn-/- mice were able to induce severe EAE in WT mice following immunization with MOG p35-55 (Fig. 10Go). Of five WT animals that received hyperimmune sera from MOG-immunized lyn-/- mice, four animals died from acute EAE (mean clinical score 4.5). In contrast, in WT mice that were injected with serum from MOG-immunized WT mice, no mortality was seen, and the mean clinical score was 1.6 (KO vs WT, p < 0.05). The higher titers of anti-IgM Ab in lyn-/- mice, when compared with controls, would argue for a pathogenic role of anti-MOG IgM Abs in the development of EAE in lyn-/- mice.



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  		FIGURE 10. Enhancement of clinical severity of EAE in WT mice following administration of MOG-primed lyn-/- serum. Anti-MOG antisera were collected from MOG peptide-immunized lyn-/- or WT mice on day 14, as described in Fig. 1Go. Active EAE was induced in WT mice by immunization with MOG peptide, as described in Materials and Methods. The mice with EAE were treated by i.p. injection with either lyn-/- serum (n = 5) or WT serum (n = 5) on day 9 (200 µl) and days 11 and 13 (50 µl on each day). The severity of EAE is presented as mean clinical score in each group (lyn-/- vs WT, p < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies demonstrated a role for lyn in the regulation of an autoimmune response and the development of EAE. Lyn-/- mice displayed increase in anti-MOG IgM Ab levels and a decrease in the proliferative response to MOG p35-55. The decrease in the proliferative response was associated with a lowered production of proinflammatory cytokines IL-12 p40, IFN-{gamma}, and TNF-{alpha} in the culture supernatants of MOG p35-55-primed lymphocyte cultures. Despite the apparent inability of lyn-/- mice to mount a Th1-mediated inflammatory response that was comparable with the WT, lyn-/- mice displayed an increased clinical and pathological severity of EAE. The clinical worsening in lyn-/- mice was seen in the presence of a paradoxical decrease in levels of proinflammatory cytokines and an increase of C5 levels in the CNS. In addition, treatment with MOG-primed lyn-/- serum worsened EAE in WT mice.

Although EAE is considered a prototypic Th1-mediated autoimmune disease, a number of studies have shown a role for B cells in the development of EAE (23). In the earlier studies, depletion of B cells led to the resistance of MBP-induced EAE (24, 25, 26). The resistance was overcome following administration of MBP-primed serum at the time of immunization (27). In MOG Ag-induced EAE, Lyons et al. (5) suggested that B cells were required for development of EAE induced following immunization with whole human MOG protein (human rMOG), but not for the EAE induced following immunization with MOG p35-55 (6, 7, 8). In transgenic mice that were engineered to produce MOG-specific autoantibodies, severity of EAE was increased following immunization with PLP peptide, suggesting an important role of neural Ag-specific Abs in EAE (28).

Unlike PLP or MBP, MOG is expressed on the cell surface, and therefore is a potential target for autoantibodies (29). Rats or marmosets show much severe EAE with histological evidence of large demyelinating areas when these animals received anti-MOG Abs (9, 10, 30). Also, EAE is accelerated with increased severity when high levels of MOG-specific Ab are produced genetically in mice (28). In the marmoset, administration of anti-MOG Abs causes a severe worsening of EAE after recovery from the acute attack of EAE (31).

A number of earlier studies have shown the role of lyn in regulating B cell response. lyn is physically associated with a number of hemopoietic cell surface receptors, including B cell receptor (32) and CD40 (33). lyn-/- mice show a reduced number of peripheral B cells and elevated levels of IgM Abs (17, 18, 19, 34), but earlier studies did not examine the role of lyn in T cell function and T cell-mediated autoimmune disease. Our studies suggest that lyn modulates the cytokine profile of T cells against soluble Ags and the development of Th1 immune response in vivo. One reason that lyn-/- mice show a reduced Th1 immune response against the neural autoantigens could be due to its association with CD40 (33). Disruption of lyn gene may interfere with CD40 signaling in APCs, and consequently decrease IL-12 production and indirectly affect the development of a Th1 response.

Although our studies underscore the role of anti-MOG IgM Abs in EAE, the molecular mechanism by which lyn acts as a negative regulator of IgM synthesis remains unclear. As would be expected, lyn-/- mice are more likely to be prone to the development of other autoimmune diseases, and they show a greater severity of autoimmune hemolytic anemia following immunization with red cell Ags (35).

The mechanism of tissue injury and in particular the development of inflammation and demyelination in EAE remains poorly understood. Oligodendrocytes and myelin membrane do not express MHC class II Ags; hence, a direct interaction between T cells and myelin appears unlikely. We had suggested that the final event of CNS demyelination is perhaps a multifactorial including Ab, complement activation, and direct effect of inflammatory cytokines on the oligodendrocyte-myelin unit (36). Studies on mechanism of autoantibody IgG-mediated demyelination in EAE indicate that these autoantibodies enhance inflammation in the CNS via activation of complement (10, 37, 38) or directly through Ab-dependent cell-mediated cytotoxicity (39). In the CNS, complement proteins are synthesized by astrocytes in response to inflammation (40). After proteolytic activation by C5 convertases, C5 is cleaved into C5a and C5b. Although C5b becomes a terminal component of the membrane-attack complex, the biological functions of C5a include recruitment of leukocytes, release of granule-bound proteolytic enzymes, and production of free oxygen radicals (41). Barnum and coworkers (42, 43) have demonstrated that its receptor C5aR expression is increased in demyelinating lesions, particularly macrophages/microglia and astrocytes, of EAE rats and in glial fibrillary acidic protein/IL-3 transgenic mice with spontaneous paralytic disease. Our studies show that in the CNS of lyn-/- mice with EAE and high titers of anti-MOG IgM, levels of all proinflammatory cytokines are reduced, while expression of C5 and C5aR is modestly elevated. Flow cytometric analysis did not show an increase in the number of macrophages, but a small increase in the number of CD4 cells in spinal cord of lyn-/- mice was seen. Because a recent study has shown that in B6 mice MOG-reactive CD8+ cells are pathogenic (44), the relevance of this increase in CD4+ T cells in lyn-/- mice with more severe EAE is not clear. The role of CD8+ MOG-reactive T cell in lyn-/- mice is not known. The most persuasive data on the mechanism of enhanced EAE are the studies showing the increased severity of EAE in WT mice following transfer of anti-MOG serum from MOG p35-55-immunized lyn-/- mice (Fig. 10Go). Given the known effect of anti-MOG autoantibodies in EAE, our data suggest anti-MOG IgM Abs and the activation of complement (e.g., C5a/C5aR and membrane-attack complex) as most likely responsible for the increased severity of EAE in lyn-/- mice.

In conclusion, our studies show that lyn plays an important role in the development of EAE. Although there is a tendency to compartmentalize autoimmune diseases into those that are T cell mediated and those that are B cell dependent (mediated), these boundaries are not really clear-cut. In EAE, the clinical and pathologic phenotype of the disease show considerable homogeneity, despite significant differences in the underlying immunopathological processes. Recognition of the immunological differences is important, because the Th1 paradigm of EAE is used as a model for the understanding of multiple sclerosis, and in the development of therapeutic strategies. Therefore, dismissing the role of autoantibody as a byproduct of the immune response in the disease pathogenesis may be misleading. Our studies show that T cells and B cells both play a role in EAE, and at least in the lyn-/- mice the lack of an inflammatory immune response in the CNS may be offset by the increase in the autoantibody levels. The development of these Abs may be sufficient to compensate for any decrease in the potency of the Th1 cells, and thereby sufficient to mediate EAE.


   Acknowledgments
 
We thank Sandy Watkin for administration assistance and Åsa Ljunggren-Rose for technical assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Subramaniam Sriram, Multiple Sclerosis Research Laboratory, 1222H Vanderbilt Stallworth Rehabilitation Hospital, 2201 Capers Avenue, Nashville, TN 37212. E-mail address: sirams{at}ctrvax.vanderbilt.edu Back

2 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; KO, knockout; MBP, myelin basic protein; MOG, myelin oligodendrocyte protein; MSCH, mouse spinal cord homogenate; PLP, proteolipid protein; WT, wild type. Back

Received for publication July 13, 2001. Accepted for publication January 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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