Increased expression of B cell-associated regulatory cytokines by glatiramer acetate in mice with experimental autoimmune encephalomyelitis

Journal of Neuroimmunology 219 (2010) 47–53

Contents lists available at ScienceDirect

Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Increased expression of B cell-associated regulatory cytokines by glatiramer acetate in mice with experimental autoimmune encephalomyelitis Sakhina Begum-Haque a,b,⁎, Alok Sharma a,b, Marc Christy a,b, Tim Lentini a,b, Javier Ochoa-Reparaz a,b, Islam F. Fayed a,b, Daniel Mielcarz a,b, Azizul Haque a,b,c, Lloyd H. Kasper a,b a b c

Department of Medicine, Dartmouth Medical School, Lebanon, NH 03756, USA Multiple Sclerosis Center at Dartmouth College, Lebanon, NH 03756, USA Chercheur au CNRS, UPRES EA 3610, Faculté de Médecine de Lille, France

a r t i c l e

i n f o

Article history: Received 2 September 2009 Received in revised form 6 November 2009 Accepted 23 November 2009 Keywords: Experimental autoimmune encephalomyelitis (EAE) Glatiramer Acetate (GA) B cell-activating factor (BAFF) A proliferation-inducing ligand (APRIL) IL-13 production

a b s t r a c t B cells are of increasing importance as a target for multiple sclerosis treatment. Here we show that GA treatment of mice with experimental autoimmune encephalomyelitis (EAE) biases cytokine production by B cells towards cytokines associated with regulation in MS including interleukin (IL)-4, -10 and -13 and reduces pro-inflammatory IL-6, IL-12, and TNF alpha levels. GA also down-regulates expression of B cellactivating factor (BAFF) of the TNF family and a proliferation-inducing ligand (APRIL), as well as the BAFF receptor in mice with EAE. Thus, GA impacts both B cell survival and B cell cytokine production during CNS inflammatory disease in an EAE model. © 2009 Elsevier B.V. All rights reserved.

1. Introduction The chronic autoimmune disease, multiple sclerosis (MS), is characterized by central nervous system (CNS) nerve damage that presents in varying degrees of disability over the lifetime of the patient. Experimental autoimmune encephalomyelitis (EAE), in mice is used to study inflammatory demyelination characteristic of MS and is associated with production of the pro-inflammatory cytokines, interferon gamma (IFNγ) and IL-17 (Kuchroo et al., 1993; Park et al., 2005). Whereas, anti-inflammatory cytokines, such as IL-4 and IL-10, are associated with recovery (Matsushita et al., 2008; Raine, 1994; Yanaba et al., 2008). The disease modifying drug (DMD), Glatiramer Acetate (GA), is primarily regarded as a T cell-directed immunotherapeutic agent (Arnon and Aharoni, 2007; Arnon and Sela, 2003), and was initially believed to interfere with MS pathology by acting as a peptide ligand that blocked the T cell receptor, since GA binds to class II major histocompatibility complex promiscuously in vitro (Aharoni et al., 1999; Wiesemann et al., 2001). Later GA was shown to inhibit EAE using bystander suppression, whereby GA-specific regulatory CD4(+) and CD8(+) T cells were induced that led to a T helper (h)1-Th2 shift with increased secretion of anti-inflammatory cytokines, for review see (Schrempf and Ziemssen, 2007). GA also increases the level of ⁎ Corresponding author. Department of Medicine, 1 Medical Center Drive, Rubin Bldg 710, Lebanon, NH 03756, USA. Tel.: +1 603 653 9946; fax: +1 603 653 9949. E-mail address: [email protected] (S. Begum-Haque). 0165-5728/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2009.11.016

regulatory T cells in mice with EAE (Kasper et al., 2007; Jee et al., 2007). Recent studies by Weber et al. (2007) demonstrate that GA treatment of EAE mice leads to the polarization of monocytes that direct differentiation of naïve T cells into Th2 and regulatory T cells, independent of antigen specificity. Furthermore, transfer of type II monocytes into mice with EAE reverses paralysis and reduces CNS infiltration (Weber et al., 2007). We recently demonstrated that GA treatment leads to a reduction in IL-17 expression in brains of EAE mice and to a down-regulation of the production of Th17 cells throughout the immune system (Begum-Haque et al., 2008). The role of B cells in EAE and MS is controversial. The presence of oligoclonal Ig bands within the cerebrospinal fluid that hallmark MS and the clonally expanded B cell accumulation in chronic MS lesions point to a significant role of B cells in MS pathogenesis, for review see Meinl et al. (2008). However, B cells may be involved in both an inflammatory as well as regulatory response in the disease process. During disease induction in EAE or perhaps relapse in MS, B cells may be an important fulcrum for mediating the inflammatory and subsequent demyelinating process. In this inflammatory scenario B cells may act as either antigenpresenting cells or interact with T cells to enhance production of IFNγ as has been shown in an experimental model of inflammatory bowel disease (Menard et al., 2007). Studies in double-transgenic mice with MOG-specific T and B cell antigen receptors showed that B cells can function as antigen-presenting cells during EAE initiation (Bettelli et al., 2006; Krishnamoorthy et al., 2006). More than 50% of the doubletransgenic mice developed inflammatory demyelinating lesions in the

48

S. Begum-Haque et al. / Journal of Neuroimmunology 219 (2010) 47–53

CNS, while disease incidence was 5% in MOG-specific T cell receptortransgenic mice. In contrast, B cell-deficient mice and CD19-deficient mice with reduced B cell function develop a severe non-remitting form of EAE (Fillatreau et al., 2002; Matsushita et al., 2006; Wolf et al., 1996). These studies led to the identification of IL-10-producing CD1dhiCD5+ regulatory B cells (Matsushita et al., 2008; Yanaba et al., 2008). More directly relevant to human disease, recent clinical trials in relapsing MS have refocused interest on B cells as important modulators involved in the CNS inflammatory process, for review see Dalakas (2008). Although the role of B cells in regulating the disease process and immune homeostasis in MS has not yet been fully established, experimental data in the EAE model as described above provide reasonable support for this to occur. Once B cells have crossed the endothelial blood brain barrier (BBB), factors involved in maintaining them are essential for their survival. These include BAFF (B cell-activating factor of the tumor necrosis factor (TNF) family) and APRIL (a proliferation-inducing ligand), two TNF family members with shared receptors (Ng et al., 2005; Sutherland et al., 2005). Indeed, the presence of increased BAFF and APRIL levels in MS lesions (Krumbholz et al., 2005; Thangarajh et al., 2007) points to B cell survival in facilitating inflammatory disease progression. Mature B cell survival depends on signaling from the BAFF receptor, which promotes NF-kappa β activity (Patke et al., 2004). Since GA interferes with neuroinflammatory disease progression in both mice and humans by affecting multiple immune cell compartments, we speculated that its effects on B cells were also multi-factorial. Here we demonstrate that GA's capacity to reduce expression of B cell survival factors and bias B cell-specific cytokine expression towards anti-inflammatory cytokines contributes to the beneficial effects of this DMD. 2. Materials and methods 2.1. Materials Glatiramer Acetate (GA) from batch 28704270 and 147245929 was a generous gift from Teva Pharmaceuticals (Petach Tikva, Israel). Myelin oligodendrocyte glycoprotein (MOG)35–55 peptide was purchased from Peptide International (Kentucky, USA), Mycobacterium tuberculosis and Complete Freund's Adjuvant (CFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pertussis Toxin (PT) was purchased from Biological Laboratories, Inc (Campbell, CA, USA). B cell and CD4+ cell sorting kits were purchased from Stem Cell Technologies Inc (Vancouver, BC). For real-time PCR (RT-PCR), control β-actin, all primers (TNFα, IL-13, IL-4, IL-6, IL-12, BAFF, APRIL) and probes were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). For flow cytometry assays, antibodies against CD19, CD5 as well as the corresponding isotype controls were obtained from eBiosciences and BioLegend (CA, USA). For Luminex immunoassays, antibodies against IFNγ, IL-6, IL-4, IL-13, and IL-10 were purchased from Bio-Rad (Hercules, CA, USA). 2.2. Mice Female 6–7 week old C57BL/6 mice were purchased from Jackson Lab (Bar Harbor, ME, USA) and maintained in the pathogen-free animal facility at Dartmouth Medical School, Hanover, NH. All animal study procedures were approved by the Dartmouth Institutional Animal Care Review Committee.

21. The clinical assessment of EAE used the following criteria: 0, no disease; 1, decreased tail tone; 2, hind limb weakness or partial paralysis; 3, complete hind limb paralysis; 4, forelimb and hind limb paralysis; 5, moribund state. 2.4. Collection and processing of brain, spleen and lymph node samples Mice were perfused via heart/aortic root with Dulbecco's calcium/ magnesium-free PBS. For ex vivo and in vitro assays tissues were collected from all groups of mice on 19 days after EAE induction or GA treatment. Tissues were suspended in RNA Stabilization Reagent (Invitrogen Life Technologies) and kept at − 80 °C until RNA extraction. A typical spleen has approximately 40–44% B cells, while lymph nodes have approximately 18–22%. Thus, the ratio of spleen B cells to lymph node B cells is approximately 1:0.4. 2.5. Cell isolation, cell culture, and cytokine profiles from CD19+ cells 2×106 cells/ml of CD19+ B cells sorted using a B cell Enrichment kit (Stem Cell Technology) were cultured with 100 ng/ml LPS at 37 °C, in humidified 10% CO2. Culture supernatants were collected after 2 to 4 h and stored at −20 °C. Cytokine levels were determined in triplicate using the Luminex assay according to the manufacturer's guidelines (R&D, Minneapolis, MN, USA). The detection threshold was less than 1 pg/ml. 2.6. Flow cytometry-intracellular cytokine staining Single cell suspensions from lymph nodes and spleens were prepared and stained with CD5 (clone 53-7.3, FITC) and CD19 (clone 1D3-APC) for 40 min at 4 °C. After washing, cells were fixed in 1% paraformaldehyde. Flow cytometry was performed on a FACS caliber (Becton Dickinson) running CellQuest software (BD Biosciences). For intracellular molecule detection, cells were washed, fixed, and permeabilized using Cytofix/Cytoperm and Perm/Wash buffer according to BD Biosciences' specifications. All viable lymphocytes were gated on and analyzed by flow cytometry. Data analysis was performed using FlowJo software (TreeStar, Inc.). 2.7. Real-time PCR (RT-PCR) RT-PCR was performed as described elsewhere (Begum-Haque et al., 2008). Expression was normalized to the expression of β-actin as described previously (Minns et al., 2006) and was expressed using the ΔCT method, where relative expression = 2−(exp − actin) ⁎ 1000. 2.8. Statistical analysis Clinical score, Cumulative Clinical Score, proliferation assays, and real-time expression between the various treatments groups were analyzed using a 2-tailed “t” test with 2 sample unequal variance. Significance was established at a p value of 0.05. Statistical significance is indicated in the figures by asterisks (*). A single asterisk identifies a statistically significant difference between the diseased vs. naïve groups (EAE vs. naïve mice) and diseased mice treated with GA and naïve mice (EAE/GA vs. naïve mice), while a double asterisk identifies a statistically significant difference between treated and diseased groups (EAE/GA vs. EAE mice). 3. Results

2.3. Induction of EAE, assessment, and GA treatment EAE was elicited via sub-cutaneous (SC) base of tail injection with 250 µg MOG 35–55 emulsified in CFA supplemented with 4 mg/ml of M. tuberculosis, which was also injected intraperitoneally on days 0 and 2 with 400 ng PT. GA treatment (150 µg/mouse by s.c. injections) was started at day 1 after MOG injection and continued daily until day

3.1. The distribution of CD19 and CD5 positive B cells in EAE-induced mice is unaffected by GA treatment B cells are important for encephalitogenic T cell activation (Bettelli et al., 2006; Krishnamoorthy et al., 2006) and for antigen-specific T cell proliferation in other disease models (Bouaziz et al., 2007;

S. Begum-Haque et al. / Journal of Neuroimmunology 219 (2010) 47–53

Crawford et al., 2006). We assayed for the effects of GA on B cells during EAE induction and progression following daily GA administration. EAE mice exhibited decreased tail tone by day 12 post-immunization (clinical score = 1). Hind limb weakness and some paralysis presented in EAE mice by day 15 (clinical score = 2), and animals had complete hind limb paralysis by day 16 post-immunization (clinical score= 3). Treatment of MOG-immunized animals with GA interfered with disease presentation at all stages of assessment, so animals had no clinical score (Supplemental data). Fig. 1 depicts the CD5+/CD19+ B cells isolated from both the spleen and lymph nodes from C57BL/6 mice that were either naïve controls (NM), had EAE or had EAE and then were treated with GA (EAE/GA). B cells were also phenotyped for co-expression of CD19+ and CD5+, markers for regulatory B cells (Matsushita et al., 2008). EAE was associated with a substantial decrease in the population of cells that expressed both CD19 and CD5 molecules compared to naïve mice. The number of cells expressing CD5 dropped 46.5% in EAE animals relative to naïve controls, CD19 positive cells dropped 34.5%, and double-positive cell levels dropped 72% in EAE mice relative to controls. GA appeared to have little effect on absolute numbers of B cells expressing CD19+ or CD5+ in EAE mice. 3.2. GA treatment affects the cytokine repertoire expressed by CD19+ splenic B cells in EAE mice Although B cell numbers are not significantly influenced by GA treatment, we hypothesized that the cytokines they express might be affected by this drug. Fig. 2A shows a comparison of the expression levels of IL-6, -12, -13 and -4 in CD19 positive B cells from naïve, EAE (day 19 post-induction, PI) and EAE/GA mice (day 19 post-induction/treatment)

49

determined using RT-PCR. All of the cytokines examined were affected by GA treatment, with statistically significant effects on IL-13, -6, and -12 (Fig. 2A; all pb 0.001). IL-6 levels increased approximately 7-fold during disease. Conversely, GA treatment reversed the up-regulation of IL-6 expression (Fig. 2A). GA had a similar effect on the IL-12 levels that decreased 4-fold due to this DMD. EAE doubled IL-13 levels and GA increased levels another 2.5-fold (pb 0.001). Thus, GA treatment polarized cytokine expression by CD19+ B cells in EAE mice significantly. This effect was also observed at the protein level. Fig. 2B shows a comparison of the levels of IFNγ, IL-6, -13, -10, and -4 in the culture medium from spleen B cells. The most striking effects of GA treatment on the CD19+ population of B cells from EAE mice was the 76% increase in IL-13 levels and 66% increase in IL-10 levels secreted by cells isolated from EAE mice. Thus, GA treatment reduces the expression of pro-inflammatory cytokines and concomitantly enhances production of anti-inflammatory cytokines by B cells. 3.3. GA reduces TNFα expression by CD19+ cells in EAE mice We previously demonstrated that levels of tumor necrosis factor alpha (TNFα) in brains of EAE mice are sensitive to GA treatment (Begum-Haque et al., 2008). However, whether or not B cell production of this inflammatory cytokine is affected by this DMD has not been specifically addressed. The level of TNFα mRNA was compared in freshly isolated splenic CD19+ cells (ex vivo) and in CD19+ cells that were cultured in vitro in the presence of LPS (Fig. 3, A–B). Ex vivo CD19 cells from EAE animals (19 days PI) expressed TNFα at only 40% of the level seen in age-matched controls (Fig. 3A) while TNFα mRNA was undetectable in ex vivo CD19 cells from EAE mice that were treated

Fig. 1. The expression profile of B cells in EAE mice is independent of GA treatment. FACS of spleens and lymph node cells from C57BL/6 stained with antibodies against CD19 and CD5. Naïve (n = 4): uninfected mice; EAE (n = 4): EAE-induced mice; EAE/GA (n = 4): EAE-induced mice treated with GA. (A). Representative results showing percentage of CD5+ cells (y-axis) and CD19+ cells (x-axis). (B). Histogram of results (representing the pooled data of three independent experiments) shown in A. Asterisks (*) indicate that the number of CD19+, CD5+ and CD19+CD5+ cells was significantly (p b 0.001) decreased in diseased mice and in diseased mice after GA treatment compared to naïve mice. Results shown (Fig. 1A) are one representation of 4 independent experiments.

50

S. Begum-Haque et al. / Journal of Neuroimmunology 219 (2010) 47–53

Fig. 2. GA administration increases both mRNA and protein levels of regulatory cytokines in splenic CD19+ cells from EAE mice. Relative mRNA levels are shown in A, while secreted protein levels are shown in B. A) Relative IL-6, IL-12, IL-13, and IL-4 mRNA levels in fresh CD19+ spleen cells, purified by B cell enrichment kit. NM (naïve C57BL/6 mice, N = 4, light blue); EAE (EAE-induced mice, N = 4, plum); EAE/GA (EAE-induced mice treated with GA, N = 4, yellow). Results represent four independent experiments and data depict the mean of 4–6 different mice ± SEM. B) Sorted CD19 cells were cultured for 4 h in the presence of LPS (100 ng/ml). Culture supernatants were collected for measurement of IFNγ, IL-6, IL-4, IL-13 and IL-10 levels. Data are shown as pg/ml and depict the mean of four different experiments ± SEM. For both A and B, asterisks (**p b 0.01) indicate statistical significance of EAE/GA vs. EAE groups.

The effect of GA on the expression of regulators that control B cell homeostasis in EAE mice was determined. Interference with the B cell

survival factors, BAFF and APRIL activity inhibits inflammatory disease progression in a variety of animal models for autoimmunity (Mackay et al., 2005; Olsson, 1995), possibly by reducing auto reactive B cell survival and/or limiting T cell activation and differentiation. Thus, RNA levels of BAFF and APRIL in brains from naïve, EAE and EAE/GA mice were compared. As shown in Fig. 4, EAE induced a 20% increase in BAFF mRNA levels and treatment of GA caused a marked, 3.5-fold, reduction in transcript levels compared with the naïve control. For the other B cell survival factor, APRIL, neither EAE nor GA appeared to impact transcript levels relative to those present in naïve brains. This finding suggests that GA treatment selectively suppresses the expression of BAFF mRNA in brains of EAE mice. Whether the expression of APRIL in spleen cells was affected by GA treatment of EAE mice was also assessed (Fig. 5). Spleen cells from naïve, EAE and EAE/GA mice were sorted into CD19 expressing (CD19+) and

Fig. 3. GA suppresses TNFα expression in splenic CD19+ cells from C57BL/6 mice. Relative expression of TNFα mRNA in B cells determined by RT-PCR. Results represent four independent experiments and data depict the mean of 4–6 different mice ± SEM. Asterisks (**p b 0.001) indicate statistical significance of GA treatments vs. EAE groups. (A) Fresh CD19+ cells and (B) CD19+ cells cultured for 2 h with 100 ng/ml of LPS. NM (n = 4), naïve mice; EAE (n = 5), EAE-induced mice; EAE/GA (n = 5), EAE-induced mice treated with GA.

Fig. 4. GA suppresses expression of BAFF in brains from EAE mice. Quantitative realtime PCR results using BAFF (left panel), and APRIL (right panel) specific primers on brain RNA from NM, naïve mice, EAE, EAE-induced mice, and EAE/GA, EAE-induced mice treated with GA. BAFF expression was significantly (p b 0.001) decreased after GA treatment compared to EAE-induced mice. However, APRIL CNS levels did not differ due to EAE or GA treatment vs. naïve controls. Results shown are one representative of 3 independent experiments and depict the mean of 4 different mice ± SEM. Asterisks (**p b 0.001) indicate statistical significance of GA treatments vs. EAE groups.

with GA (19 days PI). This indicates that after GA treatment, the initial inflammatory response is down-regulated in EAE mice. TNFα expression was up-regulated in CD19 cells of all three groups following 2 h of culture in the presence of LPS. However, the differences between EAE/ GA treated and normal groups and between EAE/GA treated and EAE mice still remained significant (p b 0.001) (Fig. 3B). These results suggest that prior exposure of CD19 cells to GA in vivo may affect the behavior of these cells ex vivo, whether freshly isolated or cultured.

3.4. Brain BAFF mRNA levels are down-regulated due to GA treatment of EAE mice

S. Begum-Haque et al. / Journal of Neuroimmunology 219 (2010) 47–53

51

CD19 positive spleen cells that express either receptor was determined in disease free animals, EAE animals or EAE/GA animals. CD19 expression of the BAFF-R was highly sensitive to both EAE induction and GA treatment (Fig. 6). The number of cells that coexpressed CD19 and the BAFF-R increased 6-fold due to EAE relative to naïve mice, whereas GA treatment caused a statistically significant 2.5-fold drop (p b 0.01) in the number of CD19 cells that expressed this receptor (Fig. 6). In contrast, TACI expression was relatively insensitive to GA. 4. Discussion

Fig. 5. GA treatment suppresses APRIL expression in spleen cells. Relative APRIL mRNA levels in CD19 positive spleen cells (left panel) and CD19 negative spleen cells (right panel) in NM, naïve mice, EAE, EAE-induced mice and EAE/GA, EAE-induced mice treated with GA. GA treatment significantly (p b 0.001) reduced the expression of APRIL in both CD19+ and CD19− cells in EAE-induced mice. Data are one representative of 5 independent experiments and depict the mean of 4–5 different mice ± SEM. Asterisks (**p b 0.001) indicate statistical significance of GA treatments vs. EAE groups.

non-expressing (CD19−) populations, and APRIL RNA levels were quantitated by PCR. CD19+ spleen B cells from naïve animals expressed relatively low levels of APRIL. EAE induced an approximately 35% increase in expression, while GA treatment reduced transcript levels by approximately 5-fold. In non-CD19+ spleen cells, EAE was associated with a 5-fold increase in APRIL expression and GA treatment of EAE mice virtually knocked out APRIL expression in these spleen cells (Fig. 5). Taken together, these results point to a highly significant effect of GA on the expression of B cell survival factors. 3.5. GA treatment significantly suppresses the number of spleen cells that express the BAFF receptor Next, whether GA affects the expression of receptors that bind either the BAFF or APRIL ligands was assessed. The BAFF receptor (BAFF-R) is specific for BAFF, while the TACI receptor can bind to either BAFF or APRIL, for review see Ng et al. (2005). The number of

Fig. 6. GA treatment suppresses the number of spleen B cells that express the BAFF-R. The number of BAFF-R positive cells (left) and TACI positive cells (right) in NM, naïve mice (light blue), EAE, EAE-induced mice (plum) and EAE/GA, EAE-induced mice treated with GA (yellow). BAFF-R+ cell numbers (left panel) were significantly (p b 0.001) decreased after GA treatment compared to EAE-induced mice. The absolute number of CD19+ TACI+ or BAFF-R+ cells/spleen was calculated by multiplying their percentage within the spleen (determined using FACS) by the total number of cells per spleen. Results shown represent the mean of two experiments and data are one representative of 5 independent experiments and depict the mean of 4–5 different mice ± SEM. Asterisks (**p b 0.001) indicate statistical significance of GA treatments vs. EAE groups and asterisks (*p b 0.01) indicate statistical significance of EAE vs. naïve animals.

Immune system deregulation is intrinsic to the progression of autoimmune diseases, including EAE and MS. Recent clinical studies in MS have demonstrated an important role for B cells in disease pathogenesis. Thus, currently approved DMDs for the treatment of MS such as Glatiramer Acetate would be anticipated to correct this dysregulation. GA appears to significantly modulate the immune compartment by enhancing the expression of anti-inflammatory cytokines and reciprocally down-regulating expression of pro-inflammatory cytokines by B cells. Moreover, GA treatment suppresses the expression of BAFF, APRIL, and the BAFF receptor in EAE mice. The data presented are consistent with published reports showing that regulatory B cells limit EAE disease progression (Matsushita et al., 2008). As mentioned previously, the role of B cells in neuroinflammatory disease is probably multi-factorial. In addition to producing antibodies, a number of animal model studies suggest that B cells participate in EAE pathogenesis using antibody-independent mechanisms (Chan et al., 1999a, 1999b; Chan and Shlomchik, 2000; O'Neill et al., 2005). In their role as antigen-presenting cells, B cells have significant effects on EAE disease progression. B cells regulate CNS-infiltrating CD4+ T cell numbers and activation during EAE development. Furthermore, B cells regulate MOG-specific CD4+ T cell expansion (Matsushita et al., 2008). Thus, in addition to being antibody-producing cells, B cells also present antigen, produce cytokines, and regulate the activities of T cells. These ‘secondary’ functions are critical to our appreciation of how B cells may be involved in inflammatory diseases, including EAE and probably multiple sclerosis. As reported by others, a novel population of IL-10-producing CD1dhiCD5+ B cells has been implicated in limiting neuroinflammatory disease progression (Matsushita et al., 2008; Yanaba et al., 2008). The data presented here indicate that GA biases the production of cytokines by B cells towards anti-inflammatory cytokines with a concomitant reduction in pro-inflammatory B cell cytokine production. The specific cytokine combination expressed at different stages of inflammatory disease progression dictates the overall outcome in terms of tissue damage. When the levels of pro-inflammatory cytokines exceed those of anti-inflammatory cytokines, inflammation proceeds unabated and tissue damage continues. Indeed, when antibodies against the combination of IL-4, IL-10, IL-13, and TGF beta were administered, they completely neutralized the protective effect of 1F1, a Th2 clone that prevented adoptive transfer of EAE when co-cultured with PLP-encephalitogenic spleen cells (Young et al., 2000). Matsushita et al. (2008) recently showed that cytokine production by B cells affects EAE disease progression. The data presented here indicate that GA administration favors the production by B cells of cytokines associated with a more regulatory profile. We also observed that transfer of GA-sensitized B cells interferes with EAE disease presentation as measured by clinical and histological criteria as well as cytokine levels in recipient animals (Begum-Haque et al., manuscript submitted). IL-13 has been associated with a robust anti-inflammatory response. B cells produce IL-13 in response to EAE, and GA administration significantly enhanced IL-13 production by B cells (Fig. 2). IL-13 was recognized for its effects on B cells and monocytes, where it up-regulated

52

S. Begum-Haque et al. / Journal of Neuroimmunology 219 (2010) 47–53

class II expression, promoted IgE class switching and inhibited inflammatory cytokine production. IL-13 induces its effects through a multisubunit receptor that includes the alpha chain of the IL-4 receptor (IL-4R) (Young et al., 2000; Wynn, 2003). IL-13 production by regulatory T cells protects against EAE independent of antigen sensitization (OchoaReparaz et al., 2008). Thus, a subset of both B and T cells produce this cytokine, which is neuroprotective against EAE. Moreover, most of the biological effects of IL-13, like those of IL-4, are linked to a single transcription factor, STAT6, and the functions of IL-13 overlap considerably with those of IL-4, especially with regard to changes induced in hematopoietic cells (Wynn, 2003). We demonstrate here that GA administration interferes with the expression of B cell survival factors, BAFF and APRIL, and also the BAFFR. GA interfered with BAFF mRNA but not APRIL mRNA expression in brain (Fig. 4), while APRIL mRNA expression was sensitive to GA in spleen (Fig. 5). BAFF and APRIL are not functionally interchangeable, since BAFF-deficient mice have almost no follicular or marginal zone B cells and B cells do not progress across the transitional T2 phase, while APRIL deficient mice have normal immune system development (Schiemann et al., 2001; Rahman et al., 2003; Varfolomeev et al., 2004). One possible differential is that APRIL expression is restricted to B1 cells within the spleen, whereas BAFF is expressed in spleen B cells at all stages of development (Chu et al., 2007). Beside B cells, APRIL may be expressed by other brain cell types, such as astrocytes. Further studies are needed to uncover the molecular basis for the resistance of APRIL to GA in the brain. The fact that the expression of both BAFF and APRIL expression is reduced by GA treatment contrasts with the up-regulation of BAFF expression by interferon beta (Gandhi et al., 2008; Krumbholz et al., 2008). Both these ligands have been implicated in autoimmunity, although BAFF's involvement is more fully understood than that of APRIL, (for reviews see Mackay et al., 2005; Ng et al., 2005). The expression of BAFF and APRIL is associated with EAE and MS advancement (Krumbholz et al., 2005; Magliozzi et al., 2004; Thangarajh et al., 2007; Thangarajh et al., 2005). BAFF and APRIL costimulate T cells when T cell receptor stimulation is sub-optimal and enhances T cell proliferation and cytokine production (Ng et al., 2005; Yang et al., 2005). Indeed, Sutherland et al. (2005) reported that systemic over-expression of BAFF in BAFF transgenic mice exacerbates Th-1 mediated delayed-type hypersensitivity, which co-relates with levels of BAFF in serum. Administration of a BAFF/APRIL antagonist (soluble human B cell maturation antigen fused to the constant region of IgG1) inhibits CNS inflammation and demyelination in an EAE model with an increase in TGF beta levels and a reduction in Th1 cytokine levels (Huntington et al., 2006). Potentially, BAFF, APRIL, and BAFF-R down-regulation in B cells from EAE mice that have been treated with GA may lead to apoptosis in autoimmune B cells (Kern et al., 2004). The modulation of the expression of the BAFF-R and its ligands by GA most likely contributes to the beneficial effects of this therapeutic in CNS inflammatory disease progression. Our data further confirm that B cells are activated during disease and are affected by treatment with GA. Thus, GA can now be associated with a potentially important effect on B cells, which are an increasingly important targets for disease amelioration in patients with MS.

Acknowledgments We thank Dr. Jacqueline Channon-Smith (Dartmouth Medical School, Hanover, NH) for helpful discussions, and Kathy Smith (Dartmouth Medical School, Hanover, NH) for Luminex assays, Dervla Mellerick, PhD (Science Word Doctor, LLC, Ann Arbor MI) and Pippa Loupe, PhD, (Teva Neuroscience, Kansas City MO) for manuscript assistance. This work was supported by a grant from Teva Pharmaceuticals, LTD, Petah Tiqva, Israel, R01 AI061938 — NIAID and CA1027A1/3 — National Multiple Sclerosis Society.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jneuroim.2009.11.016. References Aharoni, R., Teitelbaum, D., Arnon, R., Sela, M., 1999. Copolymer 1 acts against the immunodominant epitope 82–100 of myelin basic protein by T cell receptor antagonism in addition to major histocompatibility complex blocking. Proc. Natl. Acad. Sci. U. S. A. 96, 634–639. Arnon, R., Aharoni, R., 2007. Neurogenesis and neuroprotection in the CNS— fundamental elements in the effect of glatiramer acetate on treatment of autoimmune neurological disorders. Mol. Neurobiol. 36, 245–253. Arnon, R., Sela, M., 2003. Immunomodulation by the copolymer glatiramer acetate. J. Mol. Recognit. 16, 412–421. Begum-Haque, S., Sharma, A., Kasper, I.R., Foureau, D.M., Mielcarz, D.W., Haque, A., Kasper, L.H., 2008. Downregulation of IL-17 and IL-6 in the central nervous system by glatiramer acetate in experimental autoimmune encephalomyelitis. J. Neuroimmunol. 204, 58–65. Bettelli, E., Baeten, D., Jager, A., Sobel, R.A., Kuchroo, V.K., 2006. Myelin oligodendrocyte glycoprotein-specific T and B cells cooperate to induce a Devic-like disease in mice. J. Clin. Invest. 116, 2393–2402. Bouaziz, J.D., Yanaba, K., Venturi, G.M., Wang, Y., Tisch, R.M., Poe, J.C., Tedder, T.F., 2007. Therapeutic B cell depletion impairs adaptive and autoreactive CD4+ T cell activation in mice. Proc. Natl. Acad. Sci. U. S. A. 104, 20878–20883. Chan, O.T., Shlomchik, M.J., 2000. Cutting edge: B cells promote CD8+ T cell activation in MRL-Fas(lpr) mice independently of MHC class I antigen presentation. J. Immunol. 164, 1658–1662. Chan, O.T., Hannum, L.G., Haberman, A.M., Madaio, M.P., Shlomchik, M.J., 1999a. A novel mouse with B cells but lacking serum antibody reveals an antibody-independent role for B cells in murine lupus. J. Exp. Med. 189, 1639–1648. Chan, O.T., Madaio, M.P., Shlomchik, M.J., 1999b. The central and multiple roles of B cells in lupus pathogenesis. Immunol. Rev. 169, 107–121. Chu, V.T., Enghard, P., Riemekasten, G., Berek, C., 2007. In vitro and in vivo activation induces BAFF and APRIL expression in B cells. J. Immunol. 179, 5947–5957. Crawford, A., Macleod, M., Schumacher, T., Corlett, L., Gray, D., 2006. Primary T cell expansion and differentiation in vivo requires antigen presentation by B cells. J. Immunol. 176, 3498–3506. Dalakas, M.C., 2008. Invited article: inhibition of B cell functions: implications for neurology. Neurology 70, 2252–2260. Fillatreau, S., Sweenie, C.H., McGeachy, M.J., Gray, D., Anderton, S.M., 2002. B cells regulate autoimmunity by provision of IL-10. Nat. Immunol. 3, 944–950. Gandhi, K.S., McKay, F.C., Schibeci, S.D., Arthur, J.W., Heard, R.N., Stewart, G.J., Booth, D.R., 2008. BAFF is a biological response marker to IFN-beta treatment in multiple sclerosis. J. Interferon. Cytokine. Res. 28, 529–539. Huntington, N.D., Tomioka, R., Clavarino, C., Chow, A.M., Linares, D., Mana, P., Rossjohn, J., Cachero, T.G., Qian, F., Kalled, S.L., Bernard, C.C., Reid, H.H., 2006. A BAFF antagonist suppresses experimental autoimmune encephalomyelitis by targeting cell-mediated and humoral immune responses. Int. Immunol. 18, 1473–1485. Jee, Y., Piao, W.H., Liu, R., Bai, X.F., Rhodes, S., Rodebaugh, R., Campagnolo, D.I., Shi, F.D., Vollmer, T.L., 2007. CD4(+)CD25(+) regulatory T cells contribute to the therapeutic effects of glatiramer acetate in experimental autoimmune encephalomyelitis. Clin. Immunol. 125, 34–42. Kasper, L.H., Haque, A., Haque, S., 2007. Regulatory mechanisms of the immune system in multiple sclerosis. T regulatory cells: turned on to turn off. J. Neurol. 245S, 10–14. Kern, C., Cornuel, J.F., Billard, C., Tang, R., Rouillard, D., Stenou, V., Defrance, T., AjchenbaumCymbalista, F., Simonin, P.Y., Feldblum, S., Kolb, J.P., 2004. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway. Blood 103, 679–688. Krishnamoorthy, G., Lassmann, H., Wekerle, H., Holz, A., 2006. Spontaneous opticospinal encephalomyelitis in a double-transgenic mouse model of autoimmune T cell/B cell cooperation. J. Clin. Invest. 116, 2385–2392. Krumbholz, M., Theil, D., Derfuss, T., Rosenwald, A., Schrader, F., Monoranu, C.M., Kalled, S.L., Hess, D.M., Serafini, B., Aloisi, F., Wekerle, H., Hohlfeld, R., Meinl, E., 2005. BAFF is produced by astrocytes and up-regulated in multiple sclerosis lesions and primary central nervous system lymphoma. J. Exp. Med. 201, 195–200. Krumbholz, M., Faber, H., Steinmeyer, F., Hoffmann, L.A., Kumpfel, T., Pellkofer, H., Derfuss, T., Ionescu, C., Starck, M., Hafner, C., Hohlfeld, R., Meinl, E., 2008. Interferon-beta increases BAFF levels in multiple sclerosis: implications for B cell autoimmunity. Brain 131, 1455–1463. Kuchroo, V.K., Martin, C.A., Greer, J.M., Ju, S.T., Sobel, R.A., Dorf, M.E., 1993. Cytokines and adhesion molecules contribute to the ability of myelin proteolipid protein-specific T cell clones to mediate experimental allergic encephalomyelitis. J. Immunol. 151, 4371–4382. Mackay, F., Sierro, F., Grey, S.T., Gordon, T.P., 2005. The BAFF/APRIL system: an important player in systemic rheumatic diseases. Curr. Dir. Autoimmun. 8, 243–265. Magliozzi, R., Columba-Cabezas, S., Serafini, B., Aloisi, F., 2004. Intracerebral expression of CXCL13 and BAFF is accompanied by formation of lymphoid follicle-like structures in the meninges of mice with relapsing experimental autoimmune encephalomyelitis. J. Neuroimmunol. 148, 11–23. Matsushita, T., Fujimoto, M., Hasegawa, M., Komura, K., Takehara, K., Tedder, T.F., Sato, S., 2006. Inhibitory role of CD19 in the progression of experimental autoimmune encephalomyelitis by regulating cytokine response. Am. J. Pathol. 168, 812–821.

S. Begum-Haque et al. / Journal of Neuroimmunology 219 (2010) 47–53 Matsushita, T., Yanaba, K., Bouaziz, J.D., Fujimoto, M., Tedder, T.F., 2008. Regulatory B cells inhibit EAE initiation in mice while other B cells promote disease progression. J. Clin. Invest. 118, 3420–3430. Meinl, E., Krumbholz, M., Derfuss, T., Junker, A., Hohlfeld, R., 2008. Compartmentalization of inflammation in the CNS: a major mechanism driving progressive multiple sclerosis. J. Neurol. Sci. 274, 42–44. Menard, L.C., Minns, L.A., Darche, S., Mielcarz, D.W., Foureau, D.M., Roos, D., Dzierszinski, F., Kasper, L.H., Buzoni-Gatel, D., 2007. B cells amplify IFN-gamma production by T cells via a TNF-alpha-mediated mechanism. J. Immunol. 179, 4857–4866. Minns, L.A., Menard, L.C., Foureau, D.M., Darche, S., Ronet, C., Mielcarz, D.W., Buzoni-Gatel, D., Kasper, L.H., 2006. TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii. J. Immunol. 176, 7589–7597. Ng, L.G., Mackay, C.R., Mackay, F., 2005. The BAFF/APRIL system: life beyond B lymphocytes. Mol. Immunol. 42, 763–772. Ochoa-Reparaz, J., Rynda, A., Ascon, M.A., Yang, X., Kochetkova, I., Riccardi, C., Callis, G., Trunkle, T., Pascual, D.W., 2008. IL-13 production by regulatory T cells protects against experimental autoimmune encephalomyelitis independently of autoantigen. J. Immunol. 181, 954–968. Olsson, T., 1995. Critical influences of the cytokine orchestration on the outcome of myelin antigen-specific T-cell autoimmunity in experimental autoimmune encephalomyelitis and multiple sclerosis. Immunol. Rev. 144, 245–268. O'Neill, S.K., Shlomchik, M.J., Glant, T.T., Cao, Y., Doodes, P.D., Finnegan, A., 2005. Antigen-specific B cells are required as APCs and autoantibody-producing cells for induction of severe autoimmune arthritis. J. Immunol. 174, 3781–3788. Park, H., Li, Z., Yang, X.O., Chang, S.H., Nurieva, R., Wang, Y.H., Wang, Y., Hood, L., Zhu, Z., Tian, Q., Dong, C., 2005. A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6, 1133–1141. Patke, A., Mecklenbrauker, I., Tarakhovsky, A., 2004. Survival signaling in resting B cells. Curr. Opin. Immunol. 16, 251–255. Rahman, Z.S., Rao, S.P., Kalled, S.L., Manser, T., 2003. Normal induction but attenuated progression of germinal center responses in BAFF and BAFF-R signaling-deficient mice. J. Exp. Med. 198, 1157–1169. Raine, C.S., 1994. The Dale E. McFarlin Memorial Lecture: the immunology of the multiple sclerosis lesion. Ann. Neurol. 36, S61–72 Suppl. Schiemann, B., Gommerman, J.L., Vora, K., Cachero, T.G., Shulga-Morskaya, S., Dobles, M., Frew, E., Scott, M.L., 2001. An essential role for BAFF in the normal development of B cells through a BCMA-independent pathway. Science 293, 2111–2114.

53

Schrempf, W., Ziemssen, T., 2007. Glatiramer acetate: mechanisms of action in multiple sclerosis. Autoimmun. Rev. 6, 469–475. Sutherland, A.P., Ng, L.G., Fletcher, C.A., Shum, B., Newton, R.A., Grey, S.T., Rolph, M.S., Mackay, F., Mackay, C.R., 2005. BAFF augments certain Th1-associated inflammatory responses. J. Immunol. 174, 5537–5544. Thangarajh, M., Masterman, T., Rot, U., Duvefelt, K., Brynedal, B., Karrenbauer, V.D., Hillert, J., 2005. Increased levels of APRIL (a proliferation-inducing ligand) mRNA in multiple sclerosis. J. Neuroimmunol. 167, 210–214. Thangarajh, M., Masterman, T., Hillert, J., Moerk, S., Jonsson, R., 2007. A proliferationinducing ligand (APRIL) is expressed by astrocytes and is increased in multiple sclerosis. Scand. J. Immunol. 65, 92–98. Varfolomeev, E., Kischkel, F., Martin, F., Seshasayee, D., Wang, H., Lawrence, D., Olsson, C., Tom, L., Erickson, S., French, D., Schow, P., Grewal, I.S., Ashkenazi, A., 2004. APRIL-deficient mice have normal immune system development. Mol. Cell. Biol. 24, 997–1006. Weber, M.S., Prod'homme, T., Youssef, S., Dunn, S.E., Rundle, C.D., Lee, L., Patarroyo, J.C., Stuve, O., Sobel, R.A., Steinman, L., Zamvil, S.S., 2007. Type II monocytes modulate T cell-mediated central nervous system autoimmune disease. Nat. Med. 13, 935–943. Wiesemann, E., Klatt, J., Sonmez, D., Blasczyk, R., Heidenreich, F., Windhagen, A., 2001. Glatiramer acetate (GA) induces IL-13/IL-5 secretion in naive T cells. J. Neuroimmunol. 119, 137–144. Wolf, S.D., Dittel, B.N., Hardardottir, F., Janeway Jr., C.A., 1996. Experimental autoimmune encephalomyelitis induction in genetically B cell-deficient mice. J. Exp. Med. 184, 2271–2278. Wynn, T.A., 2003. IL-13 effector functions. Annu. Rev. Immunol. 21, 425–456. Yanaba, K., Bouaziz, J.D., Haas, K.M., Poe, J.C., Fujimoto, M., Tedder, T.F., 2008. A regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T celldependent inflammatory responses. Immunity 28, 639–650. Yang, M., Hase, H., Legarda-Addison, D., Varughese, L., Seed, B., Ting, A.T., 2005. B cell maturation antigen, the receptor for a proliferation-inducing ligand and B cellactivating factor of the TNF family, induces antigen presentation in B cells. J. Immunol. 175, 2814–2824. Young, D.A., Lowe, L.D., Booth, S.S., Whitters, M.J., Nicholson, L., Kuchroo, V.K., Collins, M., 2000. IL-4, IL-10, IL-13, and TGF-beta from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis. J. Immunol. 164, 3563–3572.