Autoantibody-Mediated Demyelination Depends on Complement Activation but Not Activatory Fc-Receptors

Autoantibody-mediated demyelination depends on complement activation but not activatory Fc-receptors

Eduard Urich*, Ilona Gutcher*, Marco Prinz†, and Burkhard Becher*‡

*Neurology Department, Division of Neuroimmunology, University of Zurich, Y44-J38͞J42, Winterthurerstrasse 190, 8057 Zurich, Switzerland; and †Department of Neuropathology, Georg-August-University, University of Go¨ttingen, Robert-Koch-Strasse 40, 37075 Go¨ttingen, Germany

Edited by Michael Sela, Weizmann Institute of Science, Rehovot, Israel, and approved October 3, 2006 (received for review August 23, 2006) The precise mechanisms leading to CNS inflammation and myelin ␥-chain, which bears the immunoreceptor tyrosine-based acti- destruction in both multiple sclerosis (MS) and experimental autoim- vation motif sequence required for surface expression and signal mune encephalomyelitis (EAE) remain the subject of intense debate. transduction. Therefore, FcR␥ deletion results in the combined In both MS and EAE, autoantibodies (autoAbs) are thought to be deficiency of activating-type FcRs (15). involved in tissue destruction through recruiting Fc receptor (FcR)- To study the mechanistic underpinnings of Ab-mediated damage bearing cells or direct cytotoxic effects through the activation of the in the CNS in vivo, we chose the C57BL͞6 myelin oligodendrocyte complement pathway. Whereas intrathecal immunoglobulin (Ig) pro- glycoprotein (MOG)-EAE model. The advantage here is that Abs duction and Ig deposition in inflammatory lesions is a hallmark of MS, raised by the immunization with MOG peptide do not facilitate the mice deficient in B cells and Igs develop severe EAE. Paradoxically, disease process, because mice deficient in B cells develop severe mice of the same genetic background but deficient in FcR␥ are EAE (16–21). Our aim was to determine the effector mechanism EAE-resistant. We found that the functional expression of FcR␥ on invoked by systemically administered demyelinating anti-MOG Abs systemic accessory cells, but not CNS-resident cells, appears to be vital by using mice deficient in either the classical complement or the for the development of CNS inflammation, independent of antigen- Fc␥R pathway. A dilemma is caused by the fact that FcR␥-deficient Ϫ Ϫ presenting cell function or Ab involvement. On the other hand, we mice (FcR␥ / ) are resistant to the induction of EAE through found that the injection of antimyelin oligodendrocyte glycoprotein- immunization with MOG peptide emulsified in complete Freund’s Abs drastically worsens disease severity, inflammation, and demyeli- adjuvant (CFA), which is in apparent conflict with the EAE- nation. Using FcR␥؊/؊ and C1q؊/؊ mice, we could definitively estab- susceptibility of B cell-deficient mice. Here, we provide clear lish that the demyelinating capacity of such autoAb in vivo relies evidence that the observed protective effect of FcR␥ deficiency in entirely on complement activation and is FcR-independent. EAE is completely independent of Abs and IC binding by FcR- bearing cells. In light of the apparent role of FcR␥, we demonstrate autoimmunity ͉ multiple sclerosis ͉ neuropathology ͉ B cells ͉ that the contribution of FcR␥ signaling in EAE is linked to an humoral immunity accessory leukocyte residing within the systemic immune compartment and that the lack of FcR␥ diminishes the encephalitogenic potential of adoptively transferred pathogenic lymphocytes. Al- ultiple sclerosis (MS) is the most common inflammatory though our data corroborate the notion that the lack of B cells or disease of the CNS, characterized by large, sharply de- M Abs does not have an impact on the natural course of EAE marcated areas of demyelination associated with deposition of pathogenesis in C57BL͞6 mice, inoculation of anti-MOG Abs complement-activation products, macrophage infiltration, accu- drastically increases clinical EAE in an FcR␥-independent yet mulation of T cells and progressive astrocytic proliferation complement-dependent fashion. (gliosis) (for review, see refs. 1–3). It is widely held that MS is a T cell-mediated autoimmune disease, yet the most important Results diagnostic hallmark for MS is the presence of oligoclonal Igs and Deficiency in FcR␥ Delays Onset and Decreases Severity of EAE IMMUNOLOGY plasma cells in the cerebrospinal fluid (CSF) (4–7). CNS- Independent of Ab Binding. It is well documented that the FcR- infiltrating B cell clones are thought to produce monoclonal associated signaling subunit, termed the common ␥-chain (FcR␥), autoAbs that damage myelin membranes. The first evidence is important in the development of EAE, because FcR␥Ϫ/Ϫ mice are demonstrating functional capacity of antimyelin Abs to induce EAE resistant (21–24), even though they developed comparable demyelination in vivo, was provided by an animal model for MS, levels of MOG-specific Abs (22, 23). To assess whether this EAE experimental autoimmune encephalomyelitis (EAE), whereby it resistance is due to the absence of FcR-bearing cells that cause could be demonstrated that administration of antimyelin Abs demyelination of Ab-coated myelin structures, we initially immu- enhances demyelination in rats and nonhuman primates (8, 9). nized FcR␥Ϫ/Ϫ and WT mice (both on the C57BL͞6 background) However, how such autoAbs could potentially mediate tissue with MOG35-55 in CFA and evaluated clinical disease development. destruction and demyelination remains a subject of intense Consistent with the findings by Lock et al. (24) in BL͞6–129PF2 debate. Abs can mediate their effector function either through strain, we found that, whereas WT mice developed progressive recruitment of the classical complement cascade or through Fc receptor (FcR)-mediated Ab-directed cell-mediated cytotoxicity (ADCC) (10). As to the former, C1q is the first subcomponent Author contributions: E.U. and B.B. designed research; E.U. and I.G. performed research; of the C1 complex and initiates the complement cascade’s I.G. contributed new reagents͞analytic tools; E.U. and M.P. analyzed data; and E.U. and B.B. classical pathway by binding to the FcR-portion of IgG engaged wrote the paper. in immune complexes (ICs) (11, 12). The affinity to IgG The authors declare no conflict of interest. subclasses is comparable between C1q and Fc␥R, and both This article is a PNAS direct submission. classes of molecules interact with the same C␥2 domain of the Abbreviations: ADCC, Ab-directed cell-mediated cytotoxicity; Ag, antigen; APC, antigen- IgG heavy chain (13). ADCC, on the other hand, requires that presenting cell; BM, bone marrow; DC, dendritic cell; dpi, days after inoculation; EAE, experimental autoimmune encephalomyelitis; FcR, Fc receptor; IC, immune complex; LN, Ab-opsonized structures are recognized by Fc receptor-bearing lymph node; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis; TCR, T cell cells, (e.g., macrophages, granulocytes, and NK cells) binding the receptor; Tg, transgenic. constant region of Igs (14). Activatory FcRs (FcRI, Fc␥RIII, ‡To whom correspondence should be addressed. E-mail: burkhard.becher@neuroimm. Fc␧RI, and Fc␣RI) are composed of a ligand-binding ␣-chain, unizh.ch. which confers ligand specificity and affinity, and an associated © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607283103 PNAS ͉ December 5, 2006 ͉ vol. 103 ͉ no. 49 ͉ 18697–18702 Downloaded by guest on September 30, 2021 A B

Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ Fig. 1. The loss of FcR␥ attenuates EAE development independent of B cell function. EAE was induced in FcR␥ (triangles) and WT (circles) (A)orJH ͞FcR␥ (squares) and WT (circles) (B) mice by immunization with MOG35-55 in CFA, and the mice were scored daily for clinical symptoms. Tables 1 and 2, which are published as supporting information on the PNAS web site, correspond to the graphs shown in A and B, respectively. The ﬁgure is a representative of at least three individual experiments (total n Ն 18 per group). Statistical signiﬁcance was determined by using Student’s t test (*, P Ͻ 0.05; #, P Ͻ 0.01).

paralysis, FcR␥Ϫ/Ϫ mice were relatively resistant to EAE and function of FcRs expressed by CNS-resident cells, we generated developed a significantly (P Ͻ 0.01) milder disease with delayed a series of BM chimeric mice and induced EAE through active onset (Fig. 1A and Table 1). The relative EAE resistance of immunization with MOG35-55. The absence of FcR␥ from the FcR␥Ϫ/Ϫ mice is in clear conflict with the finding that mice lacking radio-resistant (microglia) compartment (WT 3 FcR␥Ϫ/Ϫ) did all Abs develop severe EAE (16–21), raising the question as to not alter the disease course when compared with control mice whether the FcR␥-deficiency functions independently of Abs. To (WT 3 WT) (Fig. 2A and Table 3, which is published as Ϫ/Ϫ Ϫ/Ϫ this end, we crossed B cell-deficient JH mice with FcR␥ mice. supporting information on the PNAS web site), whereas the Ϫ/Ϫ JH mice, which, in contrast to the widely used ␮Mt mice deletion of FcR␥ from the systemic immune compartment (targeted deletion of ␮-heavy chain), are completely deficient in (FcR␥Ϫ/Ϫ 3 WT) resulted in reduced clinical disease (Fig. 2B mature B cells and Igs (25, 26). After induction of EAE, we found and Table 4, which is published as supporting information on the Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ that, similar to FcR␥ mice, FcR␥ ͞JH mice displayed a PNAS web site). We conclude that FcR␥ expression by CNS- delayed disease onset and decreased severity (Fig. 1B and Table 2), resident cells does not influence the course of clinical disease or demonstrating that the EAE resistance of FcR␥Ϫ/Ϫ mice functions histopathological changes (data not shown) associated with CNS independently of B cells and their products. Immunohistochemical inflammation. analysis revealed significantly reduced pathological changes in the Considering the potential direct involvement of FcR␥ signaling Ϫ/Ϫ Ϫ/Ϫ Ϫ/Ϫ FcR␥ and FcR␥ ͞JH when compared with WT mice. The in lymphocyte activation (34–37), we wanted to assess whether Ϫ/Ϫ Ϫ/Ϫ FcR␥ ͞JH mice developed only mild demyelination, as as- lymphocyte function in vivo is directly affected by the loss of FcR␥ sessed by Luxol-fast blue staining, and a substantial decrease in or whether accessory cells require FcR␥ for the development of cellular infiltration (H&E staining) when compared with WT mice autoimmunity. To narrow down the FcR␥-bearing cell influencing (Fig. 6, which is published as supporting information on the PNAS EAE, we constructed mixed BM chimeras in which the FcR␥ lesion web site). is restricted to either lymphocytes or to all other leukocytes. To restrict genetic deficiency to lymphocytes only, the BM inoculum Peripheral Leukocytes but Not CNS-Resident Cells Are Required for consisted of 25% RAG1Ϫ/Ϫ BM cells supplemented with 75% BM FcR␥-Mediated Disease Development. CNS-resident microglia are cells from FcR␥Ϫ/Ϫ mice. Control groups received either 100% WT, widely held to be central to the development of CNS lesions 100% RAG1Ϫ/Ϫ, or 100% FcR␥Ϫ/Ϫ BM cells. As expected, the (27–29), and their expression of Fc␥Rs (Fc␥RI, Fc␥RII, and groups reconstituted with 100% RAG1Ϫ/Ϫ or 100% FcR␥Ϫ/Ϫ were Fc␥RIII) is thought to mediate Ab-induced tissue destruction resistant or developed mild EAE, respectively (Fig. 2B and Table (30, 31). The use of bone marrow (BM) chimeric mice allows 4). Mice bearing FcR␥Ϫ/Ϫ lymphocytes but carrying normal acces- selective manipulation of the genotype of the peripheral hema- sory leukocytes (FcR␥Ϫ/Ϫ ϩ RAGϪ/Ϫ 3 WT) were fully susceptible topoietic immune system while sparing host-derived CNS- to EAE and indistinguishable from the WT 3 WT control group resident cells such as CNS parenchymal microglia, astroglia, and (Fig. 2B and Table 4). Our data demonstrate that FcR␥ is vital for macrophages, which are not significantly repopulated after BM the development of EAE, and FcR␥ must exert its primary effect reconstitution (28, 32). In contrast to the CNS parenchyma, the through accessory cells such as mono- and polymorphonucleated genotype of the systemic immune compartment and that of phagocytes, dendritic cells (DCs) or NK cells but not through perivascular cells can be exchanged with that of donor-derived lymphocytes directly. hematopoietic cells (28, 29, 33). To determine the role and To determine whether loss of FcR␥ decreases the capacity of

Fig. 2. FcR␥-bearing accessory cells restore EAE susceptibility in BM chimeras. (A) BM chimeric mice in A B 4 3.5 which the FcR␥ deficiency was restricted to CNS- resident cells were generated by transferring 2 ϫ 107 3 2.5 WT-BM cells into lethally irradiated FcR␥Ϫ/Ϫ (WT 3 Ϫ Ϫ FcR␥ / , open circles) and WT mice (WT 3 WT, open 2 1.5 squares) as a control group. (B) Irradiated WT mice were reconstituted with BM cells from RAG1Ϫ/Ϫ mice 1 * # ## mean clinical score 0.5 * Ϫ/Ϫ 3 ␥Ϫ/Ϫ ␥Ϫ/Ϫ mean clinical score (RAG WT, open triangles), FcR mice (FcR * * * ### 0 0 * * * 3 3 * WT, filled circles), WT mice (WT WT, open circles), 1 3 5791113 15 17 1357911131517 Ϫ Ϫ Ϫ Ϫ and a 4:1 mixture of FcR␥ / and RAG1 / BM cells days post immunization days post immunization (FcR␥Ϫ/Ϫ ϩ RAGϪ/Ϫ 3 WT, open diamonds). Eight weeks later, reconstituted BM chimeras (Ն6 per group) were actively immunized with MOG35-55 in CFA, and clinical disease development was assessed daily. Tables 3 and 4 correspond to the graphs shown in A and B, respectively. The figure is representative of at least two separate experiments (total n Ն 10). Statistical significance was determined by using Student’s t test (*, P Ͻ 0.05; #, P Ͻ 0.01).

18698 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607283103 Urich et al. Downloaded by guest on September 30, 2021 Fig. 3. Encephalitogenicity but not 12000 lymph nodes spleen Ag proliferation and cytokine pro- A C MOG35-55/CFA MOG1-121/CFA MOG35-55/CFA duction depend on FcR␥.(A)WT 10000 CFA CFA CFA (black) and FcR␥Ϫ/Ϫ (white) mice 8000 were immunized with MOG35-55

emulsiﬁed in CFA. Regional draining cpm 6000

ers wt-BL/6 LNs and spleens were removed at 7 b 4000 dpi, and a single-cell suspension was

restimulated with MOG35-55 (50 ␮g͞ 2000

-/ cell num cell

ml), rMOG1-121 (50 ␮g͞ml), or ConA relative FcR ␮ ͞ in vitro Medium MOG rMOG Medium Con.A γ (5 g ml) in 96-well plates. T cell pro- restimulation 35-55 1-121 Con.A MOG35-55 rMOG1-121 liferation was determined by lymph nodes spleen 3[H]thymidine incorporation in trip- CFSE fluorescence intensity licate wells for 48 h. Representative B data from at least three experiments 12000 3.5 40 D 4 are presented as mean cpm of tripli- 3 10000 cate cultures. (B) Lymphocytes were 30 3 2.5 prepared from WT (ﬁlled circles) and 8000 ␥Ϫ/Ϫ 2 FcR (open squares) mice immu- 2 5/6 ␥ cpm 6000 20 nized with MOG35-55 at 7 dpi. IFN- 1.5 and IL-2 secretion was elicited with IL-2 [pg/ml]

4000 IFN γ [ng/ml] 1 1 different doses of MOG35-55 for 24 h 10 3 2000 mean clinical score and measured by [H]thymidine in- 0.5 * # # # # ### ## corporation and ELISA. Data shown 0 0/6 2 46810 12 14 16 18 represent the mean of two individ- 0 0.5 5 50 0 0,5 5 50 0 0,5 5 50 MOG35-55 µg/ml MOG35-55 µg/ml MOG35-55 µg/ml days post cell transfer ual experiments Ϯ SEM. (C) To assess the expansion of MOG-specific Tg (2D2 Tg) T cells in the context of FcR␥Ϫ/Ϫ or WT host, 25 ϫ 106 lymphocytes from naı¨ve2D2 TCR Tg mice were labeled with 10 ␮M carbofluorescein diacetate succinimidyl ester and injected i.v. Recipients were subsequently immunized s.c. with MOG35-55, MOG1-121 emulsified in CFA, or CFA alone. FACS analysis of spleen and draining LNs was performed at 4 dpi. Gating on Tg V␣3.2ϩ cells allows specifically following the proliferation of the adoptively transferred T cells. Shown is a representative of two individual experiments (n Ն 2). (D) FcR␥Ϫ/Ϫ (filled circles) and WT (open circles) mice received a single injection of 30 ϫ 106 encephalitogenic MOG-reactive WT-derived lymphocytes (prepared as described above). After 3 h, the mice received 200 ng of pertussis toxin i.p. The mice were examined daily for clinical disease. Data show the mean EAE score, and the figure is representative of three separate experiments (total n ϭ 18). Statistical significance was determined by using Student’s t test (*, P Ͻ 0.05; #, P Ͻ 0.01).

antigen-presenting cells (APCs) to drive the expansion of enceph- 24 h, proliferation was measured by 3[H]thymidine incorporation. alitogenic T cells, FcR␥Ϫ/Ϫ and WT mice were immunized with No significant difference in T cell priming was observed between MOG, and,7dafterinoculation (dpi), lymphocytes were chal- the groups regardless of the degree of DC maturation (data not lenged with either cognate antigen (Ag) or mitogen. Interestingly, shown). These results suggest that FcR␥ is not involved in APC we found that FcR␥ deficiency does not affect cognate Ag-driven maturation or Ag presentation and that priming and expansion of lymphocyte proliferation (Fig. 3A) or cytokine production (e.g., autoreactive T cells is undisturbed. IFN␥͞IL-2) (Fig. 3B). To corroborate the capacity of FcR␥Ϫ/Ϫ mice ͞ Loss of FcR␥ Diminishes Inflammation During the Effector Phase of to develop a protein peptide Ag-driven immune response, we ␥ adoptively transferred carbofluorescein diacetate succinimidyl es- EAE. To assess the role and function of FcR during the effector phase of EAE, we determined the susceptibility of FcR␥Ϫ/Ϫ mice to ter-labeled TcR-Tg (2D2) T cells specific for MOG peptide (38) 35-55 EAE induced by adoptive transfer of a population of encephalito- into either WT or FcR␥Ϫ/Ϫ mice. The mice were subsequently

genic lymphocytes. We immunized WT mice with MOG35-55 in IMMUNOLOGY immunized with their cognate peptide (MOG35-55)orMOG1-121 CFA and harvested splenocytes and LN cells 11 d after immuni- protein emulsified in CFA. After 4 d, proliferation of TcR Tg-T zation. The encephalitogenic MOG-restricted population was ex- cells in spleen and lymph nodes (LNs) was determined by flow panded in vitro before injection into either WT or FcR␥Ϫ/Ϫ recip- cytometry. As shown in Fig. 3C, all groups displayed similar ients. Fully primed and activated encephalitogenic WT T cells proliferation in vivo after antigen challenge. In a complementary induced EAE in WT recipients, yet, surprisingly, they were inca- Ϫ Ϫ approach to determine the APC capacity of FcR␥ / DCs, we pable of inducing clinical EAE in FcR␥Ϫ/Ϫ hosts (Fig. 3D). This exposed mature BM DCs, derived from WT or FcR␥Ϫ/Ϫ mice, to finding shows that FcR␥ signaling is crucial for the actions of the MOG35-55 and cocultured them with TcR Tg T cells in vitro.After accessory cell compartment during the effector phase of EAE.

A B 3.5 # 3.5 # # # 3 * * * 3 * # # 2.5 # # # 2.5 * # # # * 2 * 2 * 1.5 * * 1.5 * 1 1 *

mean clinical score 0.5 0.5 * * * * * * mean clinical score 0 0 135791113151719 13579111315 17 19 days post immunization days post immunization

Fig. 4. Demyelinating Abs drive the disease process in an FcR-independent but complement-dependent fashion. WT (circles), FcR␥Ϫ/Ϫ (squares) (A), and C1qϪ/Ϫ (triangles) (B) mice were immunized with MOG35-55 in CFA, and, at days Ϫ1, 3, 5, and 9 of immunization, animals were given 150 ␮g of either ␣-MOG mAb (filled symbols) or an equal concentration͞volume of isotype control Ab or PBS (open symbols) by i.v. injection into the tail vein. Mice were examined daily for clinical disease. Tables 5 and 6 correspond to the graphs shown in A and B, respectively. The figures are representative of at least two separate experiments (total n Ն 10) and were analyzed for statistical significance by using a Student’s t test (*, P Ͻ 0.05; #, P Ͻ 0.01).

Urich et al. PNAS ͉ December 5, 2006 ͉ vol. 103 ͉ no. 49 ͉ 18699 Downloaded by guest on September 30, 2021 Fig. 5. Histological analysis of CNS tissue from ␣-MOG mAb-treated C1qϪ/Ϫ and FcR␥Ϫ/Ϫ mice. Spinal cords were isolated at 19 dpi from randomly selected and perfused WT, FcR␥Ϫ/Ϫ, and C1qϪ/Ϫ mice and embedded into paraffin. Representative lumbar sections were analyzed for demyelination and cell infiltration. Demyelination is depicted by Luxol-fast blue (LFB) staining, and cellular infiltration is shown by H&E staining. Infiltration of T cells (CD3), macrophages͞monocytes (MAC-3), and B cells (B220) were also compared. (Scale bar: 200 ␮m.)

Exacerbation of EAE After Injection of Demyelinating Anti-MOG mAbs developed EAE with the same onset and severity as the WT control Is Complement-Dependent but Not Driven by ADCC. After discon- group after immunization with either encephalitogenic MOG35-55 necting the actions of FcR␥ from the function of anti-MOG Abs or recombinant protein (data not shown), and, importantly, the generated through immunization with MOG, we were pressed to injection of the anti-MOG mAb did not lead to disease exacerbation consolidate our findings with the fact that ␣-MOG Abs have been in C1qϪ/Ϫ compared with WT or FcR␥Ϫ/Ϫ mice (Fig. 4B and Table shown to exacerbate EAE (39–41). We observed that the systemic 6, which is published as supporting information on the PNAS web application of anti-MOG mAb (8–18C5) starting at the time of site). Histopathological analysis revealed that anti-MOG mAb- immunization rapidly increased clinical signs of EAE in both WT induced demyelination completely depends on the activity of and FcR␥Ϫ/Ϫ mice, clearly demonstrating that Ab-mediated exac- complement. Macrophages are visible in areas of demyelination erbation of EAE is independent of FcR␥ (Fig. 4A and Table 5, and constitute the majority of the infiltrated cells (Fig. 5). In which is published as supporting information on the PNAS web contrast to FcR␥Ϫ/Ϫ or WT mice, injection of anti-MOG mAb in site). C1qϪ/Ϫ mice did not affect the degree of demyelination or inflam- We noticed that this exacerbation of clinical EAE did not affect mation (Fig. 5). Although the complement cascade appears to be the delayed onset of clinical symptoms characteristic for FcR␥Ϫ/Ϫ critical for the demyelination mediated by the injection of anti- mice, again supporting the notion that the function of FcR␥ in EAE MOG mAb, lack of complement per se did not alter the disease is unrelated to Ab binding, and FcR␥ signaling may contribute to course, as evidenced by comparing untreated MOG-immunized the preclinical phase of the disease. The anti-MOG-mAb caused C1qϪ/Ϫ and WT mice (Fig. 4B), emphasizing again that MOG- strong demyelination and increased cellular infiltration of T cells, induced EAE in C57BL͞6 mice develops completely independent macrophages, and B cells, when compared with mice treated with of anti-MOG Ab raised by the immunization. either isotype control Ab or PBS alone (Fig. 5). Numerous studies have documented deposition of complement Discussion components in MS lesions (42–44). Antimyelin Abs, which are Although MS is considered to be primarily mediated by autoreac- potent complement activators, were also recently demonstrated in tive T cells, recent developments in the field suggest that humoral situ in MS (45, 46), and it was shown that injection of anti-MOG Abs immunity is involved in MS lesion formation in a subgroup of into animals with acute EAE results in massive activation of patients and that antimyelin Abs have predictive value for the rapid complement in areas of demyelination (39). We therefore investi- development of relapses after the first clinical episode of disease (3, gated the relevance of the IC-dependent portion of the complement 47). However, the study by Berger et al. (47) is hotly debated, and system in the Ab-mediated worsening of EAE by injection of it is unclear whether antimyelin Abs actually contribute to MS ␣MOG mAbs into MOG-immunized C1qϪ/Ϫ mice. C1qϪ/Ϫ mice pathogenesis or merely represent an epiphenomenon that develops

18700 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607283103 Urich et al. Downloaded by guest on September 30, 2021 in response to continuous myelin breakdown. By using EAE, it was inoculation of demyelinating Abs (39, 40, 58, 59). On the other shown that, in some models, antimyelin Abs do have the capacity hand, others (54, 55) could demonstrate that key components of the to enhance myelin phagocytosis and contribute to ongoing CNS complement system, such as C3 and C4, are not involved in inflammation and demyelination (40, 48). MOG35-55-induced EAE pathogenesis, challenging the notion that The effector mechanism driving Ab-mediated demyelination, complement activation drives demyelination. however, remains unclear, because antimyelin Abs may contribute The capacity of antimyelin Abs to exacerbate clinical signs of to local CNS inflammation by enhancing cell-independent com- EAE is mostly restricted to Abs directed against MOG, and, indeed, plement activation or cell-dependent ADCC trough recruiting the Abs that could fix complement most efficiently had the highest FcR-bearing cells. demyelinating capacity (39). Studies have demonstrated that com- In studying the role of ADCC in EAE, we had to initially resolve plement depletion cannot (60), or not completely (48), prevent the dilemma that a vital role for Ig-binding FcRs for EAE devel- acute exacerbation of EAE by anti-MOG Abs, implicating other opment is in apparent conflict with the EAE susceptibility of B effector mechanisms such as ADCC. Although these findings are cell-deficient mice (16–20). To resolve this paradox, we generated indicative of a role of the complement system, they by no means mice deficient in both B cells and FcR␥ and demonstrated that the provide conclusive evidence. FcR␥ deficiency decreases immunity independently of B cells or Igs Our study of anti-MOG Ab-exacerbated EAE in complement- ␥Ϫ/Ϫ and that the EAE resistance of FcR mice cannot be attributed deficient mice definitively implicates the complement system as the ␥ to humoral immunity. The notion that FcR functions indepen- dominant (if not only) effector cascade invoked by demyelinating dently of FcR-mediated Ab recognition is supported by the recent Abs. We could show that application of anti-MOG Abs in C1q- report by Szalai et al. (34), which demonstrated that the Ab-binding deficient mice does not induce increased disease severity when ␣ FcR units are not required for EAE development. compared with WT or FcR␥Ϫ/Ϫ mice. Also, the fact that comple- ␥ Evidently, FcR is not only critical for FcR-mediated signaling, ment-deficient mice are fully EAE susceptible again supports the but this immunoreceptor tyrosine-based activation motif- notion that humoral immunity is not relevant for EAE development ␥ containing signaling -chain is further involved in the signal trans- induced by active MOG immunization. However, we show that duction of several other FcR-unrelated receptor complexes on inoculated pathogenic demyelinating Abs mediate their disease- different cell types, including NKR-P1 on NK, NK T, and DCs and promoting function exclusively through the complement cascade. Pir-A on DCs, macrophages, B cells, and platelets. Furthermore, ␥ ␨ The data presented here may be relevant for the pathology and FcR is closely related to the -chain of the T cell antigen receptor treatment of MS. Enhanced levels of antimyelin Abs have been (TCR)͞CD3 complex, and, in fact, FcR␥-␨ heterodimers and ␥ ␥ described in serum and CSF of MS patients and in a subgroup of FcR –FcR homodimers can participate in TCR formation and patients with a clinically isolated syndrome where, specifically, the function (34–37, 49–52). Indeed, Szalai et al. (34) have even levels of anti-MOG Abs have been suggested to be useful as a suggested that FcR␥ signaling on ␥͞␦ T cells could explain the prognostic marker (5–7). Not much is known about the origin of resistance of FcR␥Ϫ/Ϫ mice to EAE. They implied that expression complement proteins in MS lesions. Enhanced expression of of FcR␥ by ␥␦ T cells, probably in conjunction with the TCR͞CD3 mRNA for C1q and, to a lesser extent, C3 in MS lesions demon- complex, is potentially required for full development of EAE. Our strates that at least part of the complement proteins are produced data, however, dismiss any vital FcR␥ function in B or T lympho- locally in areas of active demyelination (61, 62). Gene-microarray cytes and point toward a crucial role of FcR␥ in the accessory cell compartment. Reconstitution of FcR␥-deficient mice with WT analysis of active MS lesions had also shown enhanced mRNA accessory cells renders them fully EAE susceptible, even when expression for C1r (24), a protein that forms the C1 complex with lymphocytes remain FcR␥-deficient. C1q and C1s. In a subgroup of early MS patients, deposition of IgG Several independent studies support the notion that CNS- and complement was detected together in active demyelination resident cells, in particular microglia, express activatory Fc␥R lesions in situ (3, 63, 64), suggesting that Ab-mediated activation of (Fc␥RI and Fc␥RIII), which recognize IC and Ab deposits within complement is involved in the demyelinating process in a subpopu- the CNS, leading to phagocytosis of opsonized target cells (30). lation of MS patients. The fact that administration of anti-MOG However, deletion of FcR␥ from the CNS parenchyma (and thus Abs exacerbates EAE is in agreement with the conclusions drawn IMMUNOLOGY all microglia) did not impact on EAE susceptibility, clinical disease, from this histopathological analysis of MS tissue, whereas we could or demyelination, whereas the absence of FcR␥ in the peripheral not establish any role for ADCC-mediated tissue damage. Hence, immune compartment conveys protection. We suspected that the targeting of the Ab–complement pathway poses a viable therapeu- loss of FcR␥ impairs APC capacity and, thus, the expansion of tic strategy for a subgroup of patients suffering from MS. encephalitogenic T cells. However, our data dismiss the involve- Materials and Methods ment of FcR␥ in APC function or T cell expansion. In this context, ͞ we found that FcR␥ does not affect the induction of DC maturation, Mice and Reagents. C57BL 6 mice were obtained from Harlan ␥Ϫ/Ϫ Ϫ/Ϫ Ag-processing, or presentation. In support of these findings, Laboratories (Zeist, The Netherlands) and FcR and JH FcR␥Ϫ/Ϫ mice do not develop EAE even when an expanded mice from Taconic Farms (Germantown, NY). MOG-specific TCR Tg C57BL͞6 (2D2) mice were provided by V. J. Kuchroo population of fully primed encephalitogenic lymphocytes is adop- Ϫ Ϫ tively transferred, again underlining that FcR␥-mediated accessory (Harvard Medical School, Boston, MA) (38). C1qa / mice (12) cell activation is required for the effector phase of EAE. The were obtained through A. Aguzzi (University of Zurich) and Ϫ Ϫ mechanistic underpinnings of the FcR␥-mediated effects in EAE Rag1 / were provided by R. Zinkernagel (University of Zurich) are under investigation. Murine models for autoimmune disease (65). All mice were bred at the animal facilities of the University indicate the indispensable roles of the inhibitory FcRs in the of Zurich under specific pathogen-free conditions. Animal ex- suppression of such disorders, whereas activating-type FcRs are periments and breeding were approved by the Swiss Veterinary crucial for the onset and exacerbation of the disease (15). It is Office (#69͞2003 and #70͞2003). BM chimeric mice were feasible that the EAE resistance of FcR␥Ϫ/Ϫ mice is caused by generated as described (33). unbalanced steady-state signal transduction, skewing the response Murine His6-tagged recombinant mouse MOG (amino acids toward attenuated status. The role of complement activation in 1–121) was expressed in Escherichia coli and purified by using metal EAE has been hotly debated over the years (53–55). Loss of C3 chelate chromatography (66). MOG35-55 peptide (MEVGWYR- apparently attenuates EAE (56), and expression of the complement SPFSRVVHLYRNGK) was synthesized by GenScript (Piscataway, inhibitor sCrry prevents EAE (57). Also, complement activation NJ). Mouse monoclonal anti-MOG Ab 8-18C5 (IgG1) was provided appears to be crucial in the context of EAE exacerbated by the by N. Goebels (University of Zurich) (67).

Urich et al. PNAS ͉ December 5, 2006 ͉ vol. 103 ͉ no. 49 ͉ 18701 Downloaded by guest on September 30, 2021 Induction of EAE and Clinical Assessment. EAE was induced by s.c. (PerkinElmer, Wellesley, MA). For cytokine analysis, sister cul- flank injection of 200 ␮gofMOG35-55 peptide emulsified (1:1) tures were harvested 48 h after culture, and supernatants were in CFA (Difco, Detroit, MI) on day 0, supplemented by i.p. analyzed by commercially available murine ELISA for IFN-␥ and injections of 200 ng of pertussis toxin (Sigma, St. Louis, MO) on IL-2 (BD Pharmingen, Franklin Lakes, NJ). days 0 and 2. For in vivo proliferation assays, mice were immunized with 200 For adoptive transfer studies, donor mice were immunized as ␮gofMOG35-55 peptide, rMOG1-121 protein emulsified in CFA, or described above. At 11 d later, the mice were killed, spleens and CFA alone. The mice were injected i.v. with carbofluorescein LNs were removed and homogenized, and red blood cells were diacetate succinimidyl ester-labeled splenocytes (10 ␮M) (Invitro- lysed. Cells are subsequently cultured for4dinRPMI medium 1640 gen–Molecular Probes, Eugene, OR) obtained from TcR Tg mice. ͞ supplemented with 10% FCS and 1% penicillin streptomycin and At4dlater, spleens and LNs were isolated from immunized mice, ␮ ͞ ͞ in the presence of 20 g ml MOG peptide and 2.5 ng ml IL-12. and proliferation of Tg CD4 T cells was performed by FACS MOG35-55-reactive lymphocytes were then washed and injected into analysis using specific antibodies to TCR V␣3.2. recipient mice (20–30 ϫ 106 cells per mouse). Animals also received 200 ng per mouse of pertussis toxin on days 0 and 2 after transfer. Histology. Mice were euthanized, bled, and transcardially perfused The mice were evaluated daily for clinical signs of disease on with 20 ml of PBS and, subsequently, 4% paraformaldehyde (PFA) a scale of 0–5, with gradations of 0.25 for intermediate scores: in PBS. The spinal column was removed and postfixed in 4% PFA 0, no clinical signs; 1, loss of tail tone; 2, limp tail and hind limb in PBS. The spinal cord was then dissected and paraffin-embedded weakness (waddling gait); 3, hind limb paralysis; 4, hind and fore before staining with either H&E and anti-MAC-3 or CD3 and B220 limb paralysis (score Ͼ4 to be killed); 5, death. Each time point shown is the average disease score of each group. Statistical antibodies (BD Pharmingen and Serotec, Ontario, Canada) to significance was assessed by using an unpaired Student t test. assess infiltration of inflammatory cells or Luxol-fast blue to determine the degree of demyelination. T Cell Expansion Assays. Mice were immunized by flank injections of ͞ We thank Dr. M. Botto (Imperial College London, U.K.) for providing CFA alone or MOG35-55 CFA. After 7 d, the axillary and inguinal Ϫ/Ϫ LNs and spleens were removed and homogenized. Cells (2 ϫ 105) C1q mice and Drs. M. Greter, A. Fontana, N. Goebels, and T. Buch for critical review of this manuscript and helpful discussions. This work were seeded as triplicates in a 96-well plate and pulsed with 0.5, 5, was supported by grants from the Swiss National Fond (to B.B.) and and 50 ␮g͞ml MOG or 5 ␮g͞ml concanavalin-A (ConA; Fluka, ␮ ϭ Serono Pharmaceuticals Geneva (to B.B.), by the Thyssen (M.P.), Buchs, Switzerland). After 24 h, 1 Ci (1 Ci 37 GBq) of Meyerhof (I.G.), and Roche Research Foundations (E.U.), the Center 3 [H]thymidine (Fluka) was added and incubated for an additional for Molecular Physiology of the Brain (M.P.), the National Center for 24 h before cells were harvested by using a Filtermate harvester Competence in Research (B.B.), and the German Academic Exchange (Inotech, Dottikon, Switzerland), and thymidine incorporation was Service (to E.U.). B.B. is a Harry Weaver Neuroscience Scholar of the assessed by using a Wallac MicroBeta TriLux scintillation-counter National Multiple Sclerosis Society, New York, NY.

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