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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci. Author manuscript; available in PMC 2012 June 8.
Published in final edited form as:
PMCID: PMC3111929
NIHMSID: NIHMS291837

Regulatory B-cells limit CNS inflammation and neurologic deficits in murine experimental stroke

Abstract

Evaluation of infarct volumes and infiltrating immune cell populations in mice after middle cerebral artery occlusion (MCAO) strongly implicates a mixture of both pathogenic and regulatory immune cell subsets in stroke pathogenesis and recovery. Our goal was to evaluate the contribution of B-cells to the development of MCAO by comparing infarct volumes and functional outcomes in WT versus B-cell deficient μMT−/− mice. The results clearly demonstrate larger infarct volumes, higher mortality, more severe functional deficits and increased numbers of activated T-cells, macrophages, microglial cells and neutrophils in the affected brain hemisphere of MCAO-treated μMT−/− vs. WT mice. These MCAO-induced changes were completely prevented in B-cell restored μMT−/− mice after transfer of highly purified WT GFP+ B-cells that were detected in the periphery, but not the CNS. In contrast, transfer of B-cells from IL-10−/− mice had no effect on infarct volume when transferred into μMT−/− mice. These findings strongly support a previously unrecognized activity of IL-10-secreting WT B-cells to limit infarct volume, mortality rate, recruitment of inflammatory cells and functional neurological deficits 48h after MCAO. Our novel observations are the first to implicate IL-10-secreting B-cells as a major regulatory cell type in stroke and suggest that enhancement of regulatory B-cells might have application as a novel therapy for this devastating neurologic condition.

INTRODUCTION

Animal data clearly support a biphasic effect of stroke on the peripheral immune system. The initial phase is characterized by early signaling from the ischemic brain to spleen, resulting in a massive production of inflammatory factors, transmigration of splenocytes to the circulation and infiltration of stroke-damaged areas of the brain by inflammatory PMN, macrophages, T-cells and B-cells (Offner et al., 2006a). This early activation phase is followed by compensatory systemic immunosuppression that is manifested within days of focal stroke by a profound (90%) loss of immune T- and B-cells in the spleen and thymus and reduced T-cell activation (Offner et al., 2006b; Offner et al., 2009). These changes were accompanied by an increase in splenocytes that were overtly apoptotic or committed to the apoptotic pathway (i.e. that were TUNEL positive, Annexin V+, or PI+).

To evaluate the contribution of lymphocytes to stroke severity, we performed MCAO in SCID mice that are genetically deficient in T- and B-lymphocytes (Hurn et al., 2007). Cortical and total infarction volumes were strikingly smaller in the SCID mice with MCAO compared to wild type (WT) C57BL/6 control mice (p<0.01). The smaller infarct size in SCID versus WT mice indicated that ~40% of the stroke damage observed within the first 22h of MCAO involves the inflammatory T- and/or B-cells that have migrated from the periphery into the evolving infarct. However, the initial inflammatory ischemic insult may also induce regulatory responses that limit CNS damage.

A promising regulatory candidate was the CD4+CD25+FoxP3+ T-cell population (Tregs), which increased significantly 96h after MCAO (Offner et al., 2006b). However, depletion of Tregs using Foxp3DTR mice (Kim et al., 2007) did not affect infarct volume or behavioral evaluations in mice (Ren et al., 2011). Our failure to implicate Tregs in limiting brain lesion volume after MCAO is of general importance to the field because of the increased interest in utilizing CD4+Foxp3+ Tregs as a possible therapeutic approach in stroke. Our results differed from a recent report (Liesz et al., 2009) in which depletion of the CD25+ population with anti-CD25 mAb significantly increased brain infarct volume and worsened functional outcome. Although these effects cannot be attributed solely to CD4+CD25+Foxp3+ Treg cells due to a wider expression of CD25, this study did implicate activated T- and/or B-cell populations in a regulatory role.

Recent studies have described powerful regulatory effects of B-lymphocytes on inflammatory responses (LeBien and Tedder, 2008; Lund, 2008). Depletion of B-cells worsened disease severity in models of multiple sclerosis, and transfer of WT B-cells provided protection against disease induction (Matsushita et al., 2010). The ability of the CD1dhighCD5+CD19+ regulatory B-cell subset to limit CNS injury is likely associated with the well-recognized anti-inflammatory effects of IL-10 in the CNS (Fillatreau et al., 2002; Mann et al., 2007). We thus evaluated effects of regulatory B-cells in stroke. Our data demonstrate the profound impact of endogenous regulatory B-cells on limiting the infarct volume and neurological deficits after ischemic stroke, and further identify the cellular targets of this highly protective B-cell population.

MATERIALS AND METHODS

Animals

B-cell deficient μMT−/− mice on the C57BL/6 background were bred at the VA Animal Resource Facility. Age-matched 8 – 12 week old C57BL/6 mice (WT, the Jackson Laboratory, USA) were used as control mice for MCAO induction. Green fluorescent protein mice (GFP+) mice (bred at the VA animal facility), WT and IL-10−/− mice (the Jackson Laboratory, USA) on the C57BL/6 background were used as B-cell donors to restore B-cell function in B-cell deficient mice. Animals were bred and cared for according to institutional guidelines in the Animal Resource Facility at the Veterans Affairs Medical Center, Portland, OR. All experiments were performed under approved institutional protocols from the VA and Oregon Health & Science University.

MCAO model

The mice were subjected to MCAO as previously published (Offner et al., 2006b) by reversible right MCA occlusion (60min) under isoflurane anesthesia, followed by 48h of reperfusion. Body and head temperatures were controlled at 37±0.5°C. Occlusion and reperfusion were verified in each animal by laser Doppler flowmetry (Moor Instruments, Oxford, UK).

Quantification of Infarct

As previously published, brains were collected at 48h for standard 2,3,5-triphenyltetrazolium chloride (TTC) histology and digital image analysis of infarct volume. To control for edema, corrected infarct volume is expressed as a percentage of the contralateral structure, i.e. cortex, striatum or total hemisphere.

Neurological Deficit Score

Neurological function was evaluated using a 0–5 point-scale neurological score: 0 = no neurological dysfunction; 1 = failure to extend left forelimb fully when lifted by tail: 2 = circling to the contralateral side; 3 = falling to the left; 4 = no spontaneous walk or in a comatose state; 5 = death. The scores were assessed in a blinded fashion.

Cell isolation

Peripheral blood mononuclear cells were prepared by using red cell lysis buffer (eBioscience, San Diego, CA) following manufacturer’s Instructions. Single-cell suspensions from lymph nodes (superficial cervical, mandibular, axillary, lateral axillary, superficial inguinal and mesenteric) and spleens were prepared by mechanical disruption. For preparation of inflammatory cells in brain infarction, each mouse was transcardially perfused with 30ml saline to exclude blood cells. The forebrain was dissected from the cerebellum and suspended in RPMI-1640 medium. The suspension was digested with type IV collagenase (1 mg/ml, Sigma-Aldrich, St. Louis, MO) and DNase I (50 μg/ml, Roche Diagnostics, Indianapolis, IN) at 37°C for 45 min in a shaker at 180 times per min. Inflammatory cells were isolated by 37–70% Percoll (GE Healthcare, Piscataway, NJ) density gradient centrifugation according to a method described elsewhere (Campanella et al., 2002). Inflammatory cells were removed from the interface for further analysis. The cells were then washed twice with RPMI 1640, counted, and resuspended in stimulation medium containing 10% FBS for phenotyping.

Analysis of cell populations by FACS

Anti-mouse antibodies used for this study included: CD19 (1D3, BD PharMingen, San Diego, CA), CD45 (30-F11, Invitrogen, Carlsbad, CA), CD11b (M1/70, eBioscience), MHCII (2G9, BD PharMingen), Gr1 (IA8, BD Horizon), CD3 (17A2, eBioscience), IFN-γ (XMG1.2, eBioscience), TNF-α (MP6-XT22, BD PharMingen) and IL-10 (JES5-16E3, eBioscience). Single-cell suspensions were washed with staining medium (PBS containing 0.1% NaN3 and 2% FCS). After incubation with appropriate mAb and washing, cells were acquired with LSRII Fluorescence Activated Cell Sorter (BD Biosciences). For each experiment, cells were stained with appropriate isotype control antibodies to establish background staining and to set quadrants before calculating the percentage of positive cells. Data were analyzed using Flowjo software (TreeStar, Ashland, OR).

CD19+ B cell sorting and transfer

B-cells were isolated for transfer experiments by negative magnetic cell sorting (Miltenyi Biotec, Auburn, CA), with GFP+, WT and IL-10−/− mice as donors. μMT−/− mice were injected i.p. with a total of 5×107 CD19+ B cells for all cell transfer experiments. The purity of B cells was >99%. The distribution of GFP+CD19+ B-cells was evaluated 48h after transfer in recipient μMT−/− mice.

IL-10 staining

Intracellular IL-10 expression was visualized by modification of a previously published immunofluorescence staining protocol (Yanaba et al., 2008). Briefly, isolated leukocytes or purified cells were resuspended (2×106 cells/ml) in complete medium (RPMI 1640 media containing 10% FCS, 1 mM pyruvate, 200 μg/ml penicillin, 200 U/ml streptomycin, 4 mM L-Glutamine, and 5×10−5 M 2-beta-ME with LPS (10 μg/ml), PMA (50 ng/ml), ionomycin (500 ng/ml), and Brefeldin A (10 μg/ml) (all reagents are from Sigma-Aldrich) for 5h. For IL-10 detection, Fc receptors were blocked with mouse Fc receptor-specific mAb (2.3G2; BD PharMingen) before cell-surface staining and then fixed and permeabilized with the Fixation/Permeabilization buffer (eBioscience), according to the manufacturer’s instructions. Permeabilized cells were washed with 1×Permeabilization Buffer (eBioscience) and stained with APC-conjugated anti-IL-10 mAb (JES5-16E3; eBioscience). Isotype matched mAb served as negative controls to demonstrate specificity and to establish background IL-10-staining levels.

Immunohistochemistry

Brains, lymph nodes and spleens were collected from perfused μMT−/− mice that received adoptively transferred GFP+ B-cells after 60min MCAO and 48 hours reperfusion. Tissues were fixed with 4% buffered formalin, paraffin embedded, and sectioned. Sections were incubated with anti-GFP (Cell signaling), followed by incubation with secondary biotinylated antibody (goat anti-rabbit, Cell signaling) and staining with VECTASTAIN ABC Peroxidase Kit (Vector) and 3′, 3-diaminobenzidine (Sigma-Aldrich). Nuclear staining was carried out with hematoxylin (Sigma-Aldrich).

Statistical Analysis

Data were reported as means ± SEM. Statistical analyses were performed using the appropriate test indicated in the Figure Legends as follows: Student’s t test for infarct volume; Mann-Whitney-U-test for neuroscores; and Fisher’s exact test for comparison of mortality rates incurred from surgery and induction of MCAO. P-values ≤0.05 were considered statistically significant.

RESULTS

B cell deficiency exacerbates stroke outcomes and alters cerebral inflammatory cell invasion

B-cell deficient μMT−/− mice sustained significantly larger total hemisphere infarcts (P < 0.05) relative to WT mice (Fig. 1A). Representative histologic staining of injured brain is shown in Figure 1B. These data clearly implicate the role of B-cells in limiting histological damage after MCAO. Mortality was also higher in B-cell deficient vs. WT mice (11/35 vs. 3/35, respectively, P = 0.017). Neurological scores were similar among all animals as assessed during MCAO immediately prior to reperfusion (Figure 1C, P = 0.36). However, B-cell deficient mice exhibited worse functional outcomes at both 24h (Figure 1C, P = 0.02) and 48h of reperfusion (Figure 1C, P = 0.002) compared to WT mice. To confirm that the ischemic insult was equivalent among all animals, relevant physiological parameters were assessed before and during MCAO. As is shown in Table 1, rectal temperature, mean arterial blood pressure, arterial blood gases and pH were comparable between groups. Similarly, intra-ischemic cortical blood flow, as estimated by laser Doppler flowmetry, was not different between B-cell deficient and WT mice.

Figure 1
Deficiency of B-cells exacerbates ischemic infarct volume and behavioral outcome after MCAO
Table 1
Physiological parameters at baseline, mid-MCAO and end-MCAO in wild-type (WT) and B-cell deficient (μMT−/−) mice.

Leukocytes are major effectors of inflammatory damage after experimental brain ischemia (Gee et al., 2007; Wang et al., 2007). To determine if the lack of B-cells altered leukocyte composition in brain after MCAO, numbers of infiltrating Gr1+ neutrophils, CD3+ T-cells, CD11b+CD45low microglia and CD11b+CD45high macrophages were evaluated by flow-cytometry. After 48h reperfusion, accumulation of all of these leukocyte subtypes was significantly greater in the affected hemisphere of MCAO-treated μMT−/− mice as compared to MCAO-treated WT mice (Figure 2A–D). Lack of B-cells in μMT−/− mice further permitted significant increases in the absolute number of IFN-γ- and TNF-α-secreting CD3+ T cells and MHC class II+ and TNF-α-secreting microglia and macrophages in the ipsilateral hemisphere of MCAO mice at 48h reperfusion (Figure 3A–F). In addition to the cell types mentioned above, we detected 6,576 ± 829 CD19+ B-cells per hemisphere in sham-treated brains (n=5), not different from 7,092 ± 637 “resident” B-cells in naïve brains (n=5). In MCAO mice, there were modest but significant increases to 9,766 ± 1,832 B-cells in the non-ischemic hemisphere and 12,282 ± 824 B-cells in the ischemic hemisphere (n=5).

Figure 2
B-cells reduce the infiltration of inflammatory cells into the ischemic brain after MCAO
Figure 3
B-cells reduce the activation of infiltrating inflammatory cells in the ischemic brain after MCAO

Adoptive transfer of B-cells to μMT−/− mice reduces ischemic infarct size and improves neurological deficits

To specifically implicate B-cells as the protective cell type, highly enriched populations of B-cells were transferred from WT donors to B-cell deficient recipient mice prior to MCAO. As illustrated in Figure 4A, CD19+ B-cells were obtained and enriched to 99% purity by negative selection from splenocytes of transgenic green fluorescent (GFP+) mice, and 50 million GFP+CD19+ B-cells were injected i.p. into μMT−/− mice one day prior to MCAO. The B-cell deficient animals that received adoptively transferred GFP+CD19+ B-cells had reduced infarct volumes (P > 0.05) after MCAO compared to no cell transfer (PBS) controls (Figs. 5A and 5B), as well as a lower mortality rate (P = 0.05). Consistent with smaller infarction size, neurological outcome scores were also improved in B-cell restored μMT−/− mice with stroke after 48h reperfusion compared to no cell (PBS) transferred control mice (Fig. 5C). We also confirmed that the adoptive transfer of CD19+ B-cells from C57BL/6 WT donors (GFP) limited stroke infarct size and functional outcome (data not shown). These findings clearly demonstrate that WT CD19+ B-cells can restore improved ischemic outcomes in B-cell deficient μMT−/− mice. However, after 48h reperfusion, transferred GFP+ B-cells could be detected in blood, lymph nodes (LN), spleen and peritoneal cavity, but not in ischemic (left) or non-ischemic (right) brain of recipient μMT−/− mice by FACS (Fig. 4B). Immunohistochemical staining showed GFP+ cells distributed in lymph nodes and spleens, but not in non-ischemic (L) or ischemic (R) hemispheres (Fig. 4C).

Figure 4
Distribution of GFP+CD19+ B-cells in μMT−/− mice at 48h reperfusion after 60min MCAO
Figure 5
Transfer of GFP+CD19+ B-cells reduces infarct volume and improves behavioral outcome of μMT−/− B-cell deficient mice

B cells, but not T cells, are the major producer of IL-10 in MCAO mice

Because of the significant B-cell dependent activity in limiting stroke infarct size and functional outcome demonstrated above, we hypothesized that the protective actions of CD19+ B-cells might be linked to IL-10 production, a major regulatory cytokine known to be produced by both B-cells and T-cells. Thus, intracellular staining of IL-10 was carried out in CD19+ B-cells and CD3+ T-cells harvested from immune organs after MCAO and stimulated ex vivo with LPS, PMA and ionomycin. As is shown in Figure 6, an increased percentage of IL-10-secreting CD3-negative cells was observed in MCAO WT mice but not μMT−/− mice after 48hr reperfusion in blood, but not spleen or lymph nodes. These IL-10- secreting cells in blood were identified as CD19+ B-lymphocytes (Figure 7). These data demonstrate enhanced availability of B-cells with potential to limit ischemic and neurological outcomes after MCAO through secretion of IL-10.

Figure 6
IL-10-secreting non-T-cells, but not T-cells are increased after activation ex vivo in WT but not B-cell deficient mice with MCAO
Figure 7
IL-10-secreting B-cells are increased after activation ex vivo in the blood of WT mice with MCAO

Adoptive transfer of IL-10−/− B cells to μMT−/− mice does not reduce ischemic infarct size or improve neurological deficits

To specifically address the mechanism of B-cells as the protective cell type producing IL-10, highly enriched populations of B-cells were transferred from IL-10−/− donors to B-cell deficient recipient mice prior to MCAO. As shown in Figure 8A&B, the B-cell deficient animals that received adoptively transferred IL-10−/− B-cells did not exhibit significantly reduced infarct volumes after 60min MCAO followed by 48h reperfusion compared to no cell transfer (PBS) controls. The mortality rate of the PBS and IL-10−/− B cell transfer groups was 7/16 and 6/14 respectively. Moreover, there were no differences of neurological outcome scores after 24h and 48h reperfusion between PBS and IL-10−/− B cell transfer groups. Taken together, these data clearly demonstrate that WT CD19+ B-cells can restore improved ischemic outcomes that were shown to be lacking in B-cell deficient μMT−/− mice through the secretion of IL-10.

Figure 8
Transfer of IL-10−/− CD19+ B-cells does not alter infarct volume or improve behavioral outcome of μMT−/− mice

Regulatory B-cells inhibit inflammatory responses in the periphery of MCAO mice

To further evaluate possible regulatory effects of B-cells on T-cell cytokine production during MCAO, inflammatory factors were quantified in blood and spleens after 60min MCAO and 48h reperfusion in WT mice, WT B-cell restored μMT−/− mice and IL-10−/− B-cell restored μMT−/− mice. As is shown in Figure 9, the percentages of both IFN-γ and TNF-α secreting CD3+ T-cells were significantly increased in blood and spleen from B-cell deficient vs. WT mice, with a reduction to WT levels of these peripheral T-cells in μMT−/− mice after restoration with WT B-cells, but not with IL-10−/− B-cells. Thus, WT B-cells with the potential for IL-10 secretion limited both inflammatory cytokine production of peripheral T-cells (Fig. 9) and infiltration of inflammatory T-cells (Figure 3A&B), into the MCAO-affected hemisphere during MCAO.

Figure 9
Increased cytokine production by T-cells after MCAO in peripheral blood and spleen of B-cell deficient μMT−/− mice is normalized after B-cell restoration

DISCUSSION

Much has been learned about factors that worsen or modulate stroke severity in animal models such as middle cerebral artery occlusion (MCAO). Of importance are the effects on and contribution of the immune system in MCAO. The occlusion triggers early signaling from the ischemic brain to spleen, resulting in a massive production of inflammatory factors and transmigration of splenocytes to the circulation and brain. Whereas inflammatory cells from the periphery have now been shown to contribute to CNS damage and cell death, other regulatory immune cells can reduce inflammation and limit damage within the brain. A major conundrum in the immunology of stroke is how to enhance the early immunoregulation that limits CNS inflammation while preventing excessive systemic suppression. To do this in a strategic manner requires a full understanding of the involved inflammatory and regulatory immune pathways. To this end, we evaluated regulatory B-cells from the peripheral immune system that can diminish stroke lesion size and protect from neurological damage.

The results presented above demonstrate the previously unrecognized activity of WT B-cells to limit infarct volume and functional neurological deficits as well as to inhibit activation and recruitment of inflammatory T-cells, macrophages and microglia into the growing CNS infarct after experimental stroke in mice. These regulatory activities were not only significantly decreased in MCAO-treated B-cell deficient μMT−/− mice, but also were fully restored after passive transfer of WT B-cells, thus implicating unequivocally the protective activity of regulatory B-cells. These regulatory functions were associated with increased percentages of IL-10-secreting CD19+ B-cells in blood, but not IL-10-secreting T-cells, including Treg cells that have received much previous attention as possible immune regulators in stroke (Liesz et al., 2009). Our novel observations are the first to implicate B-cells as a major regulatory cell type in stroke.

We demonstrated previously that the loss of B- and T-cells in SCID mice resulted in smaller infarct volumes (Hurn et al., 2007). Consistent with this concept, lymphocyte-deficient RAG-1−/− mice sustained smaller infarct volumes and improved neurological deficit after MCAO (Yilmaz et al., 2006), and splenectomized rats given permanent MCAO exhibited reduced neurodegeneration and numbers of activated microglia, macrophages and neutrophils in brain tissue (Ajmo et al., 2008). In the Yilmaz study, CD4+ and CD8+ T-cells contributed largely to post-ischemic intravascular inflammatory and pro-thrombotic responses in cerebral venules, and thus apparently could not account for the observed reduction in infarct volumes at 24h post-injury (Yilmaz et al., 2006). However, unlike our current results, their study did not detect improvement in MCAO outcomes in B-cell deficient mice, perhaps due to differences in the animal model or experimental paradigm (Yilmaz et al., 2006). Another recent study (Kleinschnitz et al., 2010) failed to observe a protective effect of adoptively transferred B-cells in RAG-1−/− mice treated with MCAO, a result possibly explained by the use of only 10 million transferred B-cells (compared to 50 million cells used in our study) as well as the genetically induced lack of potentially neurotoxic T-cells that would need to be present to observe protective B-cell effects. It is noteworthy that we observed significant changes in neurological scores after MCAO, further strengthening our observations of worsened tissue injury size in B-cell deficient mice (Fig. 1). Both the previous studies and our present data were obtained in animals at fairly early recovery time points, when full maturation of the infarct has not yet occurred. This early window of observation is a limitation for all of these studies. We chose a 48h recovery time in order to accommodate a potentially high mortality rate in the B-cell deficient μMT−/− mice treated with a standard MCAO and to minimize survivorship effects that are present in an animal that lacks all B-cell functions.

As for regulatory T-cells, there remains substantial discord. The recent report by Liesz and colleagues (Liesz et al., 2009) found that depletion of the CD25+ population with anti-CD25 mAb significantly increased brain infarct volume and worsened functional outcome. These effects were attributed to CD4+CD25+Foxp3+ Treg cells, even though the anti-CD25 mAb only depleted ~50% of this Treg phenotype. On the other hand, our recent study using conditional Treg-deficient mice failed to implicate CD4+CD25+FoxP3+ Treg cells as having protective activity against MCAO (Ren et al., 2011). It should be noted that CD25, the IL-2 receptor chain-α (IL-2Rα), has a broad expression on early progenitors of the T- and B-cell lineages, as well as on activated mature T-cells and B-cells. Thus, although our study did not demonstrate larger infarct volumes in Treg-deficient mice, it did not exclude the contribution of other regulatory cells. From this perspective, the data from these previous reports do not preclude our current identification of regulatory B-cells in MCAO.

A key function of B-cells is their secretion of IL-10 (Carter et al., 2011), an anti-inflammatory cytokine that has been studied extensively in stroke (Planas et al., 2006). IL-10 deficient mice develop larger infarcts after permanent focal ischemia (Grilli et al., 2000), whereas administration of IL-10 to the lateral ventricle (Spera et al., 1998) or intraperitoneally in combination with hypothermia (Dietrich et al., 1999), by adenoviral vectors (Ooboshi et al., 2005), after induction of mucosal tolerance by IL-10-producing MOG-reactive T cells (Frenkel et al., 2005) or by transgenic over-expression of IL-10 (de Bilbao et al., 2009) all reduced infarct volume. Moreover, IL-10 was found to prevent neuronal damage induced by excitotoxicity in vitro (Grilli et al., 2000). In the clinic, early worsening of stroke was associated with lower IL-10 plasma levels in patients with subcortical infarcts or lacunar stroke, but not in patients with cortical lesions (Vila et al., 2003). Conversely, excessive levels of IL-10 may predispose to increased infections (Chamorro et al., 2006). Taken together, these findings suggest that local secretion of IL-10 may be preferable to systemic delivery. Our study demonstrated that B-cells are the major producer of IL-10 in WT mice and are enriched in the blood after MCAO (Figures 6 and and7),7), thus providing locally secreted IL-10 that would be missing in B-cell deficient mice.

Expression of CD19 is almost completely restricted to B-cells but may also be expressed in follicular dendritic cells, with some shared phenotypes with plasmacytoid dendritic cells (Bao et al., 2011). A minor population of splenic dendritic cells expresses CD19 and has been shown to mediate aspects of T-cell suppression through IFN signaling (Mellor et al., 2005). In our study, we employed a negative B-cell sorting strategy, which depleted mature T-cells, NK cells and myeloid lineage cells by anti-CD43 antibody. Thus, the significant protective effect in stroke provided by highly purified B-cells would not likely be affected by the few remaining dendritic cells or other cell types. Although we cannot rule out that other protective mediators from Bregs, such as TGF-β (Tian et al., 2001) or direct cellular interactions, could contribute to B-cell mediated protection, we concentrated on IL-10 as a universal protective mechanism used by regulatory B-cells (Breg). IL-10 potently reduced infarct size in normal mice in a previous study, and our present report strongly supports the contention that B-cells do not protect against ischemic injury (Fig. 8) or peripheral inflammation (Fig. 9) in the absence of IL-10.

In the adoptive transfer assay, we detected a small percentage of engrafted B-cells in peripheral blood and immune organs after stroke (Fig. 4B&C), even though some B-cells were retained in the peritoneal cavity three days after adoptive transfer (Fig. 4B). Higher engraftment has been reported previously in μMT−/− using 1.5×107 transferred B cells (Roth and Mamula, 1997), compared to the 5×107 CD19+ B cells used in the current study. Some of the transferred B cells in our study likely did not survive due to ischemia-induced B-cell depletion (Offner et al. 2006), although it has been reported that resting B-cells may survive for at least 2 months after transfer into recipient mice (Gray, 1988). In the current study, transferred GFP+ B-cells could not be detected in the brain (Fig. 4), which suggested that the regulatory effects of Breg cells in this model could occur entirely in the periphery (Fig. 9). However, the stronger inflammatory response in CNS (Figs. 2&3) of μMT−/− mice could be from infiltrating inflammatory cells from the periphery. Moreover, CNS microglia might be directly or indirectly activated (Fig. 3) by inflammatory components.

Recent studies have identified a subpopulation of CD1dhighCD5+CD19+ “regulatory B cells”, and we also investigated changes in these CD1dhighCD5+ Breg cells in stroke. To our surprise, the IL-10 secreting population was not restricted to either the CD1dhigh or the CD5+ population post stroke (data not shown). We thus conclude that IL-10 is more specific than the CD1dhighCD5+19+ B-cell subset markers for identification of Breg cells in stroke. In conclusion, our study provides new insights into the endogenous inflammatory response after acute brain ischemia. Specifically, we have described a previously unknown role for B-cells as cerebroprotective immunomodulators after stroke, a function that affects diverse cytokine-dependent and cellular inflammatory targets through the anti-inflammatory effects of IL-10.

Acknowledgments

The authors wish to thank, Dr. Heng Hu, Dr. Sushmita Sinha, Dr. Sheetal Bodhankar and Ms. Sandhya Subramanian for helpful discussions, Dr. Takeru Shimizu for performing part of animal surgeries, and Ms. Eva Niehaus for assistance in preparing the manuscript. This work was supported by NIH grants NR03521, NS49210. This material is based upon work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

Footnotes

Conflict of Interest: The authors affirm that they have no conflict of interest to declare.

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