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(Stroke. 2003;34:1809.)
© 2003 American Heart Association, Inc.

Original Contributions

Adoptive Transfer of Myelin Basic Protein–Tolerized Splenocytes to Naive Animals Reduces Infarct Size

A Role for Lymphocytes in Ischemic Brain Injury?

Kyra Becker, MD; Darin Kindrick, BS; Richard McCarron, PhD; John Hallenbeck, MD Robert Winn, PhD

From the Departments of Neurology (K.B., D.K.) and Surgery (R.W.), University of Washington School of Medicine, Harborview Medical Center, Seattle, Wash; Resuscitative Medicine Department, Naval Medical Research Center, Silver Spring, Md (R.M.); and Stroke Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Md (J.H.).

Reprint requests to Kyra J. Becker, MD, Box 359775, Harborview Medical Center, 325 Ninth Ave, Seattle, WA 98104-2499. E-mail kjb{at}u.washington.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Background and Purpose— Breakdown of the blood-brain barrier during stroke allows central nervous system antigens to leak into the systemic circulation and allows circulating leukocytes access to the brain. Encounter of central nervous system antigens by the peripheral immune system can be capitalized on to modulate the postischemic inflammatory response and potentially improve outcome from stroke.

Methods— Male Lewis rats were tolerized to myelin basic protein (MBP) or ovalbumin (OVA) and subjected to 3 hours of middle cerebral artery occlusion (MCAO) or used as splenocyte donors for immunologically naive animals undergoing MCAO. Infarct size was determined at 24 hours by 2,3,5-triphenyltetrazolium chloride staining. In separate studies, mononuclear cells were removed from the brains of animals after MCAO for enzyme-linked immunospot (ELISPOT) assay and flow cytometry.

Results— Median infarct volume in animals tolerized to MBP and those receiving splenocytes from MBP-tolerized donors was less than in animals tolerized to OVA and those receiving splenocytes from OVA-tolerized donors (87.7±54.9 versus 148±61.6 mm³ [P=0.01] and 89.2±77.5 versus 153±77.1 mm³ [P=0.05], respectively). There was an increase in the number of transforming growth factor-ß1–secreting mononuclear cells in MBP-tolerized animals undergoing sham surgery (P=0.001) as well as in ischemic animals 48 hours (P=0.02) and 336 hours (P=0.04) after stroke. A distinct subset of T cells was present in the brains of MBP-tolerized but not control animals after stroke.

Conclusions— Immunologic tolerance and its neuroprotective effects can be transferred to naive animals and appear to be related to antigen-specific induction of transforming growth factor-ß1.

Key Words: cytokines • immune tolerance • inflammation • stroke • T-lymphocytes

	Introduction

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The blood-brain barrier (BBB) is disrupted after stroke, and leukocytes enter the brain. This postischemic inflammatory response contributes to brain injury.¹ Another consequence of BBB disruption is that antigens found either primarily or exclusively in the central nervous system (CNS), such as neuron-specific enolase, glial fibrillary acidic protein, myelin basic protein (MBP), and S-100 protein, "leak" into the peripheral circulation; serum levels of these antigens correlate to infarct size and neurological outcome.^2–5 While healthy individuals may have circulating T cells that react to CNS antigens, the number and affinity of such cells are higher in persons who have had a stroke.^6–8 The functional consequences of this autoimmune response are not known but could potentially contribute to neurological injury, especially in persons suffering recurrent stroke.

Despite evidence showing that the acute inflammatory response contributes to ischemic brain injury,¹ recent data suggest that certain aspects of the inflammatory response, especially the delayed response, may be important for recovery.^9,10 Thus, the effects of inflammation on ischemic brain injury appear to be modulated in a time-dependent fashion. Stimulating immunologic tolerance to brain antigens through mucosal exposure to these antigens might therefore be a useful paradigm for limiting the inflammatory response associated with cerebral ischemic injury since the immunologic tolerance would only be triggered when the BBB is compromised and CNS antigens are exposed to the systemic circulation.

Mucosal tissue is enriched with T cells,¹¹ and mucosal exposure to antigen results in immunologic tolerance to that antigen.¹² The nature of the acquired tolerance depends on the amount and schedule of antigen delivery; active immunologic tolerance results after repetitive low-dose mucosal exposure to antigen.^13–15 On restimulation with the appropriate antigen, T cells in tolerized animals secrete TH2 cytokines such as transforming growth factor-ß1 (TGF-ß1), interleukin-4 (IL-4), and interleukin-10 (IL-10), which suppress cell-mediated or TH1 immune responses.^14,15 In rodents, nasal administration of antigen induces tolerance through induction of regulatory lymphocytes possessing T cell receptors.¹⁶ T cells from tolerized animals are activated in an antigen-specific fashion, but the effects of activation, ie, secretion of immunomodulatory cytokines, have antigen-nonspecific effects. Thus, suppression of cell-mediated immune responses occurs wherever antigen is present, regardless of whether or not the initial inflammatory response was prompted by that antigen. This phenomenon is referred to as bystander suppression.¹⁷

We previously showed a decrease in infarct size after transient focal cerebral ischemia in animals orally tolerized to MBP.¹⁸ In the present study we replicate these findings by exposing the nasal mucosa to MBP and better define the mechanisms of neuroprotection afforded by induction of immunologic tolerance to MBP.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
Experiments were approved by the institutional Animal Care and Use Committee. Male Lewis rats (weight, 250 to 325 g) were used for all studies.

Induction of Tolerance
Animals were tolerized to bovine MBP or ovalbumin (OVA) through nasal installation of the antigen on 5 separate occasions over 2 weeks. Antigens were purchased from Sigma Chemical Company. After brief exposure to anesthesia, 100 µg of antigen suspended in 40 µL of PBS was instilled into each naris (Figure 1a). Previous experiments showed no significant difference in infarct volume or lymphocyte proliferation to MBP between OVA-tolerized and non-tolerized animals¹⁸; OVA-tolerized animals were used as controls in these experiments.

View larger version (19K): [in this window] [in a new window]   Figure 1. Experimental protocols for induction (a) and transfer of tolerance (b). ICC indicates immunocytochemistry.

Delayed-Type Hypersensitivity Responses
Tolerance to either MBP or OVA was induced as described above. Two days after the last exposure to antigen, animals were immunized to MPB by subcutaneous injection of MBP in the hind footpad (50 µg MBP in 50 µL PBS and 50 µL complete Freund’s adjuvant). On day 12 after immunization, animals were rechallenged with MBP by injection of MBP (50 µg in 50 µL PBS) into the ear. Ear thickness was measured with a micrometer 72 hours later.

Adoptive Transfer
Single cell suspensions were prepared from the spleens of animals tolerized to MBP (n=13) or OVA (n=15). Erythrocytes were removed with ACK lysing buffer (BioWhittaker), and splenocytes were suspended at 5x10⁶ cells per milliliter in RPMI-1640 media (supplemented with 10% fetal calf serum, 2-mercaptoethanol, sodium pyruvate, nonessential amino acids, L-glutamine, penicillin-streptomycin, and HEPES). Cell culture reagents were purchased from BioWhittaker. After a 48-hour incubation period with concanavalin A (Sigma; 2 µg/mL), cells were washed and injected (1x10⁸ cells in 1 mL PBS) into naive animals intraperitoneally immediately after middle cerebral artery occlusion (MCAO) (Figure 1b).

Stroke
Anesthesia was induced with 5% halothane and maintained with 1.5% halothane. After a midline neck incision, the right common carotid, internal carotid, and pterygopalatine arteries were ligated. A monofilament suture (4.0) was inserted into the common carotid artery and advanced into the internal carotid artery approximately 20 mm.¹⁹ Animals were maintained at normothermia during the surgery and allowed to spontaneously thermoregulate thereafter. Reperfusion was performed 3 hours after the onset of ischemia. Temperature was monitored 1, 2, 3, 4, 5, 6, and 24 hours after ischemia. Body weight was assessed before and 24 hours after surgery. Neurological outcome was assessed with the use of a modification of a previously published scale.²⁰

Determination of Infarct Volume
At the time of the animals’ death, brains were removed, placed in 4°C PBS for 10 minutes, and then sectioned at 2-mm intervals and incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (Sigma) at 38°C for 15 minutes.²¹ Brain sections (bregma +2.40, +1.00, -0.40, -1.80, -3.20, -4.40) were scanned and digitized; infarct volume, corrected for the presence of edema,²² was determined with the MetaMorph Image System V4.1.1 (Universal Imaging Corporation).

Carboxyfluorescein Diacetate Succinimidyl Ester Labeling
In a separate group of animals tolerized to either OVA (n=5) or MBP (n=4), splenocytes were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) before adoptive transfer. Briefly, cells were suspended at a concentration of 1x10⁷/mL in PBS; CFSE was added to achieve a final concentration of 0.5 µmol/L, and the suspension was incubated at 37°C for 10 minutes. After they were washed extensively, 1x10⁸ cells in 1 mL of PBS were injected intraperitoneally at the time of stroke.

Immunohistochemistry
In animals receiving CFSE-labeled cells, brains were removed, frozen, and stored at -80°C. To detect CFSE-labeled cells, sections (12 µm) were fixed in acetone and methanol at 4°C and stained with anti–fluorescein isothiocyanate (FITC) antibodies (Dako). The primary antibodies were detected with the use of immunoperoxidase (Vector Laboratories); sections were counterstained with cresyl violet. The number of FITC-positive cells within 6 high-power fields (x100) in each of 6 predefined brain regions on a coronal section at the level of the anterior commissure was counted (Figure 3a). The number of TGF-ß1 (R&D Systems) cells was similarly determined.

View larger version (35K): [in this window] [in a new window]   Figure 3. CFSE-labeled and TGF-ß1–secreting cells were counted in the ipsilateral and contralateral hemispheres after stroke (a). Splenocytes from MBP-tolerized donors were more likely to transit into ischemic brain when injected into naive recipients than splenocytes from OVA-tolerized donors (CFSE-labeled cells), and more TGF-ß1–secreting cells were seen in recipients of MBP-tolerized cells (b). At 48 hours after stroke, fewer MNCs were seen in the ischemic hemisphere of MBP-tolerized animals (c) (*P<0.05).

Lymphocyte Isolation
Lymphocytes were isolated from the brain by previously described methods.²³ Briefly, brains were removed and pressed through a 70-µm mesh into a solution containing HBSS (BioWhittaker), 10 µL/mL DNAse (Sigma), and 0.1% collagenase (Boehringer-Mannheim). The cell suspension was layered over Ficoll-Paque Plus (Amersham-Pharmacia Biotech) and centrifuged at 1500 rpm for 30 minutes. Erythrocytes were removed with ACK lysing reagent (BioWhittaker).

Enzyme-Linked Immunospot Assay
Enzyme-linked immunospot (ELISPOT) assay was performed as follows. Mononuclear cells (MNCs) were isolated from the brain of sham-operated and ischemic animals either 48 or 336 hours after stroke. MNCs (1x10⁶/mL) were cultured in 96 well plates (MultiScreen-IP; Millipore) with media alone or MBP (25 µg/mL) for 48 hours. The number of cells secreting TGF-ß1, TNF-, interferon-, and IL-4 was assessed. Antibodies were purchased from R&D Systems. The reaction product was developed with the use of streptavidin, alkaline phosphatase (Calbiochem), and alkaline phosphatase substrate (Vector Laboratories). Experiments were performed in triplicate; the capture antibody was omitted as a negative control. Spots were counted under a dissecting microscope by 2 independent persons blinded to treatment status. Results are expressed as the number of MBP-reactive cells per 1x10⁵ total MNCs.

Flow Cytometry
In some animals, MNCs (1x10⁷) isolated from the brain 24 hours after stroke were stained with the following primary antibodies: mouse anti-rat TCR (clone MCA1146); mouse anti-rat TCR ß (clone MCA453G); and a B cell marker (mouse anti-rat CD45RA:RPE; clone MRC OX-33). Secondary antibodies (goat anti-mouse IgG:FITC) were used as appropriate. Antibodies were purchased from Serotec. Cells were analyzed with the Coulter EPICS XL flow cytometer and WinMDI software.

Statistical Analysis
Nonparametric data were evaluated with the Mann-Whitney U test and are presented as median±SD. Parametric data were evaluated with the t test and are presented as mean±SD. Significance was set at P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of Immunologic Tolerance
Animals were effectively tolerized to MBP. The delayed-type hypersensitivity response to MBP in animals tolerized to MBP (n=3) was less than that of animals tolerized to OVA (n=4) (ear thickness was 0.036±0.002 versus 0.048±0.007 inch, respectively; P=0.03).

Effect of Immunologic Tolerance on Infarct Size
Infarct volume in MBP-tolerized animals (n=12) was significantly less than that in OVA-tolerized animals (n=14) (87.7±54.9 versus 148±61.6 mm³, respectively; P=0.01) (Figure 2a) 24 hours after MCAO. Similarly, naive animals that received MBP-tolerized splenocytes (n=15) at the time of stroke had smaller infarcts than animals that received OVA-tolerized splenocytes (n=16) (89.2±77.5 versus 153±77.1 mm³, respectively; P<0.05) (Figure 2b).

View larger version (17K): [in this window] [in a new window]   Figure 2. Stroke volume was attenuated in animals tolerized to MBP. Infarct volume and temperature of animals directly tolerized to MBP were less than those of animals tolerized to OVA (a and c). In animals receiving splenocytes from MBP-tolerized donors, infarct volume was less than in animals receiving splenocytes from OVA-tolerized donors; temperatures were similar (b and d) (*P<0.05; **P<0.01).

Modulation of Postischemic Inflammatory Response by Induction of Immunologic Tolerance
The neuroprotective benefit of immunologic tolerance was associated with modulation of postischemic fever (Figure 2c). In animals directly tolerized to MBP, temperatures were significantly less than those of OVA-tolerized animals by 5 hours after MCAO (38.8±0.76°C versus 39.4±0.45°C; P=0.02); this difference persisted at 6 (38.8±0.65°C versus 39.4±0.47°C; P=0.01) and 24 hours (37.3±0.62°C versus 38.0±0.46°C; P<0.01). There was also a trend for OVA-tolerized, but not MBP-tolerized, animals to be persistently febrile at 24 hours. Temperatures among animals receiving MPB- and OVA-tolerized splenocytes were similar throughout the course of the experiment (Figure 2d). With the use of a standard measure of neurological dysfunction in rats,²⁰ there was a trend toward better neurological performance at 24 hours in animals receiving cells from MBP-tolerized animals (P=0.07; data not shown).

To determine whether, after adoptive transfer, splenocytes from MBP-tolerized (n=6) and OVA-tolerized (n=7) animals traffic into the ischemic brain of naive animals, splenocytes were labeled with CFSE before injection. At 24 hours after stroke, the number of CFSE-positive cells in the ischemic hemisphere of animals receiving MBP-tolerized splenocytes was greater than that in the ischemic hemisphere of animals receiving OVA-tolerized splenocytes (13.0±7.5 versus 9.00±3.0; P=0.02) (Figure 3b). In recipients of MBP-tolerized splenocytes, CFSE-labeled cells were found largely in the peri-infarct region; they were seen in the vessels as well as in the parenchyma. In animals receiving cells from OVA-tolerized donors, CFSE-labeled cells were also seen in the peri-infarct region but were generally confined to the intravascular space. Immunohistochemistry revealed an increase in the number of TGF-ß1–labeled cells in the peri-infarct region of animals receiving splenocytes from MBP-tolerized animals (Figure 3b).

There was no difference in the total number of MNCs isolated from the brains of MBP-tolerized and OVA-tolerized animals undergoing sham surgery. By 48 hours after stroke, however, there were significantly more MNCs in the ischemic hemisphere of OVA-tolerized animals (n=5) compared with MBP-tolerized animals (n=14) (1.0x10⁷±3.9x10⁶ versus 3.6x10⁶±1.7x10⁶; P=0.01) (Figure 3b). This difference in cell number disappeared by 336 hours after stroke. The phenotype of the MNCs from MBP-tolerized and OVA-tolerized animals differed; more MNCs from MBP-tolerized animals secreted TGF-ß1 in response to MBP; this increase was seen in tolerized animals undergoing sham surgery (MBP, 2.67/1x10⁵±0.98/1x10⁵; n=4; OVA, 0.64/1x10⁵±0.50/1x10⁵; n=2; P=0.001) and in ischemic animals 48 hours (MBP, 1.01/1x10⁵±1.80/1x10⁵; n=13; OVA, 0.12/1x10⁵±0.25/1x10⁵; n=5; P=0.02) and 336 hours (MBP, 2.38/1x10⁵±1.90/1x10⁵; n=4; OVA, 0.67/1x10⁵±1.16/1x10⁵; n=8; P=0.04) after stroke (Figure 4). There was no difference between MBP-tolerized and OVA-tolerized animals with regard to the number of cells secreting TNF-, interferon-, or IL-4 at any of these time points.

View larger version (9K): [in this window] [in a new window]   Figure 4. More MNCs from the brains of tolerized animals secreted TGF-ß1 in response to MBP than from OVA-tolerized animals. Shown are results in sham-operated animals (a), animals sacrificed 48 hours after MCAO (b), and animals sacrificed 336 hours after MCAO (c). Data are expressed as the number of MBP-reactive cells per 1x105 total MNCs (*P<0.05).

At 24 hours after stroke, the population of B cells in brain was more prominent in MBP-tolerized animals (Figure 5a). There were significant numbers of ß T cells in OVA-tolerized animals (Figure 5b); a population of T cells was present in the brains of MBP- but not OVA-tolerized animals (Figure 5c).

View larger version (8K): [in this window] [in a new window]   Figure 5. The CNS inflammatory infiltrate differs in MBP- and OVA-tolerized animals. Flow cytometry shows a more prominent population of B cells in MBP-tolerized animals (a). Most MNCs isolated from the brains of OVA-tolerized animals appear to be ß T cells (b). A small but distinct population of T cells is seen in the brain of MBP-tolerized animals but not OVA-tolerized animals (c). Histograms for MBP-tolerized animals are black, and those for OVA-tolerized animals are gray; dotted lines indicate unlabeled cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study we show that immunologic tolerance to MBP decreases infarct size in an animal model of transient focal cerebral ischemia and that adoptive transfer of splenocytes from MBP-tolerized animals decreases infarct size in immunologically naive animals. The inflammatory response in the brain of ischemic MBP-tolerized animals is less robust than that in control animals, given that fewer MNCs are found within the brains of these animals 48 hours after stroke. More of the MNCs isolated from the brains of MBP-tolerized animals, however, secrete TGF-ß1 in response to MBP, consistent with a deviation of the immune response from a TH1 to TH2 response. Moreover, there is a distinct population of T cells in the ischemic brains of animals receiving MBP-tolerized splenocytes that is not present in animals receiving OVA-tolerized splenocytes. These results prove that manipulation of the immune response attenuates brain injury.

While our model utilizes the CNS antigen MBP to manipulate the immune response, it does not imply that MBP incites a deleterious inflammatory response after stroke. We chose MBP as the antigen with which to initiate the TH2 response in brain since MBP is released into the circulation in stroke and there is extensive literature regarding immunologic tolerance to MBP. In stroke, CNS antigens enter the systemic circulation after reperfusion, and tolerized lymphocytes enter the brain, where they secrete cytokines like TGF-ß1. TGF-ß1 suppresses inflammation^24,25 and may have neuroprotective properties independent of its immunomodulatory properties.^26,27 Exogenous administration of TGF-ß1 decreases infarct size,^27,28 and inhibition of endogenous TGF-ß1 increases infarct size.²⁹

In animals tolerized to CNS antigens, the tolerized lymphocytes should traffic to the site of highest antigen expression (ie, brain). Thus, by inducing mucosal tolerance to CNS antigens, a relatively organ-specific (and lesion-specific) immunomodulatory response is generated at the time of BBB breakdown. Mucosal tolerance to brain antigens could therefore be induced in persons at risk for stroke. While the inflammatory response begins shortly after the onset of ischemia, with neutrophils evident in the cerebral vasculature within minutes,^30,31 inflammation persists for days and evolves into a predominantly MNC-mediated event over time.^32,33 Since changes in leukocyte function and leukocyte trafficking occur within hours after mucosal exposure to an antigen,³⁴ immunologic tolerance could be induced after stroke onset and still improve outcome.

This study has a number of limitations. First, infarct size was assessed 24 hours after MCAO, while cytokine responses to MBP were assessed in sham-operated animals and in animals 48 and 336 hours after stroke. Nevertheless, since the findings are consistent at each of these time points, it seems reasonable to expect similar data at 24 hours. Second, there were significant temperature differences between MPB- and OVA-tolerized animals after stroke (Figure 2c). Temperature is a potent modulator of brain injury,³⁵ and fever after stroke is associated with increased morbidity and mortality.^36,37 It is therefore tempting to speculate that the observed neuroprotection conferred by immunologic tolerance to MBP was related solely to modulation of body temperature. The degree of neuroprotection was similar in animals tolerized directly to MBP and in animals tolerized through adoptive transfer; no temperature differences were seen between MBP- and OVA-tolerized animals in the latter group (Figure 2d), making it unlikely that temperature changes accounted for the benefit of immunologic tolerance. Finally, the inflammatory infiltrate was less pronounced in MBP-tolerized animals, suggesting that there could have been changes in cerebral blood flow since activated leukocytes impair flow through the microvasculature.³⁰ Future studies will need to incorporate measures of cerebral blood flow to determine the effect of immunologic tolerance on blood flow.

Conclusions
Immunologic tolerance to CNS antigens reduces infarct size in a rodent model of transient focal cerebral ischemia. This neuroprotection can be transferred from tolerized animals to immunologically naive animals through splenocytes, showing that modulation of the immune response can improve outcome from ischemic brain injury. Active immunologic tolerance to brain antigens, by virtue of bystander effects, could be exploited for the treatment of stroke and other forms of brain injury.

	Acknowledgments

 
This work was supported by National Institute of Neurological Disorders and Stroke grant KO2 NS02160-01 NST.

Received October 14, 2002; revision received December 11, 2002; accepted February 21, 2003.

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