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. Author manuscript; available in PMC: 2008 Dec 2.
Published in final edited form as: J Cereb Blood Flow Metab. 2007 Mar 28;27(11):1798–1805. doi: 10.1038/sj.jcbfm.9600482

T- and B-cell-deficient mice with experimental stroke have reduced lesion size and inflammation

Patricia D Hurn 1, Sandhya Subramanian 2, Susan M Parker 1, Michael E Afentoulis 2, Laurie J Kaler 2, Arthur A Vandenbark 2,3,4, Halina Offner 1,2,3
PMCID: PMC2592689  NIHMSID: NIHMS41815  PMID: 17392692

Abstract

Stroke induction in immunologically competent mice not only produces local ischemia and brain damage, but also induces early inflammatory changes in brain and peripheral immune responses. Although immune elements clearly are activated after brain vascular occlusion, the relative contribution of T and B lymphocytes to the developing lesion has not been quantified. We evaluated effects 22 h after middle cerebral artery occlusion (90 mins) on histologic injury and peripheral immune activation in severe combined immunodeficient (SCID) mice lacking T and B cells. Cortical and total infarct volumes were strikingly reduced in male SCID mice (n = 14, 33±4% of contralateral cortex, n = 10, 52±3% of contralateral hemisphere) versus immunologically intact C57BL/6 mice (wild type, n=9, 57±5% of contralateral cortex, 57±4% of contralateral hemisphere) (P < 0.01). Striatal infarction was not altered (77±7% of contralateral striatum in SCID, 84±7% in wild type), suggesting that the core of the evolving ischemic lesion was not impacted by lack of T and B cells. As expected, inflammatory factors from immune cells in ischemic SCID brains were essentially absent, with the exception of interleukin-1β increase in both SCID and wild type tissue. Spleen cell numbers were low in SCID mice, but were further reduced 22 h after stroke, with substantial reduction in most inflammatory factors except for increased expression of interferon-γ and macrophage inflammatory protein (MIP)-2. These data quantify the damaging effect of T and B lymphocytes on early, evolving ischemic brain injury, and further implicate interleukin-1β in brain and interferon-γ and MIP-2 in spleen as inflammatory factors produced by cells other than T and B cells.

Keywords: cerebral ischemia, chemokines, cytokines, spleen, T/B-deficient mice

Introduction

It is increasingly clear that human stroke is not solely a brain lesion, but a multiorgan systemic disease. Stroke-induced systemic inflammation and immunodeficiency have been described in humans and modeled in animals (Prass et al, 2003; Audebert et al, 2004; for review see Meisel et al, 2005). In patients, C-reactive protein, white blood cell counts, and plasma interleukin-6 (IL-6) levels are increased on admission for stroke and persisted for > 7 days (Emsley et al, 2003). In mice, focal cerebral ischemia leads to reduced numbers of immune cells in peripheral lymphoid organs and decreased secretion of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) that contributed to spontaneous bacterial infections, a leading cause of mortality in stroke patients (Prass et al, 2003). These results suggest that there may be systemic repercussions in lymphoid organs that occur in response to postischemic injury in the brain.

Using a murine middle cerebral artery occlusion (MCAO) model, we have characterized the systemic immunopathology that evolves in tandem with the maturing central cerebral infarct. Our previous work indicates that cerebral ischemic injury has strong deleterious consequences for the immune system (Offner et al, 2006, 2006a). One consequence is a rapid intra-splenic activation of T lymphocytes and enormous local cytokine elaboration within 6 h of reperfusion after MCAO. The importance of early peripheral immune activation of T lymphocytes to the evolving brain injury is not clear. It seems likely that these immunocytes are released from an activated ‘poststroke’ spleen, migrate across the blood–brain barrier, and are primed to contribute to well-studied local inflammatory processes arising from infiltrating polymorphonuclear neutrophils and resident microglia. T cells have been identified by immunohistochemistry in postischemic brain as early as 24 h after reperfusion, and appear localized to infarction boundary zones, typically close to blood vessels (Schroeter et al, 1994; Jander et al, 1995). Previous work suggests that antibodies against rat α4 integrin that prevents lymphocyte infiltration into postischemic brain can reduce damage after transient MCAO (Becker et al, 2001; Relton et al, 2001). However, the importance of T lymphocytes in the quantity and diversity of inflammatory mediators expressed in injured brain or to the evolving infarct remains to be quantified. Less is known about a role for B lymphocytes in the brain inflammatory process, for example if they present antigens to T cells or produce antibodies in the penumbra. Accordingly, we hypothesized that inducing stroke in severe combined immunodeficient (SCID) mice that lack T and B cells might result in improved outcome for the brain relative to immunologically intact mice. Furthermore, we utilized this novel ischemic model to partition out residual splenic and brain immune activation that occurs through inflammatory cells other than T and B cells. We show here for the first time that SCID mice are robustly protected from ischemic injury, measured early in the evolving infarction process, relative to their background C57BL/6 wild-type (WT) strain. These data emphasize that T and B lymphocytes are highly injurious players in early ischemic brain injury.

Materials and methods

Animals

The study was conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, and all protocols were approved by the Institutional Animal Care and Use Committee of Oregon Health and Sciences University and the Portland Veterans Affairs Medical Center. Severe combined immunodeficient male mice that had been backcrossed at least 10 generations on the C57BL/6 background were purchased from The Jackson Laboratories (Bar Harbor, ME, USA) at 8 weeks of age and compared with male C57BL/6 WT mice.

Ischemic Model

Focal cerebral ischemia was induced by 90 mins of reversible MCAO under halothane anesthesia, as described previously (McCullough et al, 2003; Sawada et al, 2000). In brief, mice were anesthetized with 1.5% to 2.0% halothane in O2-enriched air. The common carotid artery was exposed and the external carotid artery was ligated and cauterized. Unilateral MCA occlusion was performed by inserting a 6-0 nylon monofilament surgical suture with heat-blunted tip into the internal carotid artery via the external carotid artery stump. The tip was positioned at a distance of 6 mm beyond the internal carotid/ pterygopalatine artery bifurcation, and occlusion was confirmed by a laser-Doppler flow (Moor Instruments Ltd, Oxford, England) probe positioned over the ipsilateral hemisphere at the mid ear-to-eye distance. The suture was then secured in place, and the animal was awakened and assessed for intraischemic neurological deficit, that is presence or absence of forelimb weakness; torso turning to the ipsilateral side when held by tail; circling to affected side; inability to bear weight on affected side; or spontaneous locomotor activity or barrel rolling. Any animal without a visible deficit was excluded from the study. At end-ischemia (90 mins), the animal was briefly reanaesthetized and reperfusion was initiated by filament withdrawal. Sham-operated mice were treated identically with the exception of insertion of the filament to produce occlusion.

Terminal Histopathology

The brains were harvested after 22 h of reperfusion and sliced into five 2-mm-thick coronal sections for staining with 1.2% triphenyltetrazolium chloride (Sigma, St. Louis, MO, USA) in saline as described previously (Offner et al, 2006). Infarction volume was measured using digital imaging and image analysis software (Sigma Scan Pro, Jandel, San Rafeal, CA, USA). The area of infarct was measured on the rostral and caudal surfaces of each slice and numerically integrated across the thickness of the slice to obtain an estimate of infarct volume in each slice. Spleens were harvested and fixed in 10% formalin. Histologic sections were stained with hematoxylin and eosin and examined by light microcopy.

Isolation of Mononuclear Cells from Spleen and Blood

Spleen was isolated from naïve, sham, and post-MCAO (22 h reperfusion) SCID and WT mice and a single-cell suspension was prepared by passing the tissue through a 100 µm nylon mesh screen. The cells were washed using RPMI, and the red cells were lysed using red cell lysis buffer (8.3 g NH4Cl in 0.01 mol/L Tris–HCl, pH 7.4). The cells were then washed twice with RPMI, counted, and resuspended in staining medium (1 × phosphate-buffered saline, 2% fetal bovine serum, and 0.02% sodium azide) for fluorescence activated cell sorter (FACS) staining. For reverse transcription polymerase chain reaction (PCR), splenocytes were pelleted, snap-frozen and stored at −80°C until tested. Cardiac blood was collected in 1 ml of 3 mg/ml EDTA. Cells were then pelleted, red cells lysed using red cells lysis buffer, and incubated at room temperature for 5 mins Cells were washed twice using RPMI, counted, and resuspended in staining medium for FACS staining.

Analysis of Cell Populations by FACS

Two-color (fluorescein isothiocyanate, phycoerythrin) fluorescence flow cytometry analysis was performed to evaluate the phenotype of splenic macrophages. Spleens were harvested, and single-cell suspensions were obtained by mechanical disruption. Cells were washed with staining medium (phosphate-buffered saline containing 0.1% NaN3 and 2% fetal calf serum) and stained with a combination of monoclonal antibodies specific for CD11b (Clone M1/70, PharMingen Cat. #553310, FITC) and VLA-4 (Clone 9C10, PharMingen Cat. #557420, PE) for 20 mins, on ice. After incubation with monoclonal antibody, cells were analyzed with a FACSCalibur (BD Biosciences, San Diego, CA, USA). Forward and side-scatter parameters were chosen to identify mononuclear cells. Dead cells were gated out using propidium iodide discrimination. Data were analyzed using CellQuest software (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.

RNA Isolation and Reverse transcription-Polymerase Chain Reaction

Cerebral hemispheres were sliced coronally, discarding slices 1 and 5 that are most anterior and posterior, then subdissecting the central 3mm that contain the target tissue in the MCAO mouse model into contralateral and ipsilateral sides. Each cube of tissue was flash-frozen and then processed for RNA. Total RNA was isolated from the cerebral hemispheres and splenocytes using the RNeasy mini kit protocol (Qiagen, Valencia, CA, USA) and converted into cDNA using oligo-dT, random hexamers, and Superscript RT II (Invitrogen, Grand Island, NY, USA). Reverse transcription-PCR was performed using Quantitect SYBR Green PCR master mix (Applied Biosystems, Foster City, CA, USA) and primers. Reactions were conducted on the ABI Prism 7000 Sequence Detection System (Applied Biosystems) to detect mRNA quantified as relative units compared with the ribosomal reference gene, L32. The following genes were detected:

L32: (F: GGA AAC CCA GAG GCA TTG AC; R: TCA GGA TCT GGC CCT TGA AC); IFN-γ: (F: TGC TGA TGG GAG GAG ATG TCT; R: TGC TGT CTG GCC TGC TGT TA); TNF-α: (F: CAG CCG ATG GGT TGT ACC TT; R: GGC AGC CTT GTC CCT TGA); IL-1β: (F: TTG ACG GAC CCC AAA AGA TG; R: TGG ACA GCC CAG GTC AAA G); TGF-β1: (F: ACC ATG CCA ACT TCT GTC TG; R: CGG GTT GTG TTG GTT GTA GA); IL-6: (F: CCA CGG CCT TCC CTA CTT C; R: TGG GAG TGG TAT CCT CTG TGA A); IL-10: (F: GAT GCC CCA GGC AGA GAA; R: CAC CCA GGG AAT TCA AAT GC); IL-13: (F: ACT GCT CAG CTA CAC AAA GCA ACT; R: TGA GAT GCC CAG GGA TGG T); RANTES: (F: CCT CAC CAT CAT CCT CAC TGC A; R: TCT TCT CTG GGT TGG CAC ACA C); macrophage inflammatory protein (MIP)-2: (F: TGG GCT GCT GTC CCT CAA; R: CCC GGG TGC TGT TTG TTT T); interferon-inducible protein-10 (IP-10): (F: CGA TGA CGG GCC AGT GA; R: CGC AGG GAT GAT TTC AAG CT); CCR1: (F: GGG CCC TAG CCA TCT TAG CT; R: TCC CAC TGG GCC TTA AAA AA); CCR2: (F: GTG TAC ATA GCA ACA AGC CTC AAA G; R: CCC CCA CAT AGG GAT CAT GA); CCR3: (F: GGG CAC CAC CCT GTG AAA; R: TGG AGG CAG GAG CCA TGA); CCR5: (F: CAA TTT TCC AGC AAG ACA ATC CT; R: TCT CCT GTG GAT CGG GTA TAG AC); CCR6: (F: AAG ATG CCT GGC TTC CTC TGT; R: GGT CTG CCT GGA GAT GTA GCT T); CCR7: (F: CCA GGC ACG CAA CTT TGA G; R: ACT ACC ACC ACG GCA ATG ATC); CCR8: (F: CCA GCG ATC TTC CCA TTC TTC; R: GCC CTG CAC ACT CCC CTT A).

Statistical Analysis

Values are expressed as means ± SEM. Differences among naïve, sham, and MCAO groups in inflammatory genes were analyzed by Student’s t-test. Infarction volume was analyzed by one-way analysis of variance with post hoc Neuman–Keuls test, whereas laser-Doppler flow and physiologic parameters were analyzed by two-way analysis of variance with Neuman–Keuls. P-values ≤ 0.05 were considered significant.

Results

A total of 24 SCID mice and 22 WT mice were used in this study. There was no difference in poststroke mortality rates between groups (1/24 in SCID and 1/22 WT). In SCID mice, three animals were excluded because of subarachnoid or intracerebral hemorrhage and two animals were excluded because of lack of visible intra-ischemic neurological deficit. In WT mice, two were excluded because of hemorrhage, and three were excluded for lack of neurologic deficit.

Stroke-Induction in Male Severe Combined Immunodeficient Mice

To evaluate the contribution of T and B cells to the development of stroke, we compared infarction at 22 h after MCAO in SCID versus WT C57BL/6 mice. There were no differences in poststroke mortality rates between groups. Body weight was similar in both groups (WT, 23±1 g; SCID, 22±2), as was body temperature (WT, 37.0±0.5°C; SCID, 37.3±0.4) and intraocclusion laser-Doppler flow (WT, 10±1% of baseline signal; SCID, 17±1%). As shown in Figure 1, both cortical and total infarct volumes were strikingly reduced by ~ 40% in SCID (n = 14) versus control (n = 10) mice (P < 0.01). Striatal infarction was not altered in the SCID mice (not shown), suggesting that the core of the evolving infarction was not protected by the lack of T and B cells.

Figure 1.

Figure 1

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Infarction volume (percentage of contralateral structure) is reduced in SCID (n = 14) versus immunologically intact C57BL/6 mice (n = 10, P < 0.01). Data are presented as mean ± SEM.

Middle Cerebral Artery Occlusion-Induced Changes in Cytokines and Chemokines in Brain

In a previous report, we found striking increases in many inflammatory cytokines, chemokines, and chemokine receptor levels in WT postischemic brain after 22 h as compared with sham-operated mice (Offner et al, 2006). In Figure 2 (lower panel, filled bars), increased message levels in WT mice are presented for TNF-α, IL-1β, IL-6, IL-10, IP-10, CCR1, CCR2, CCR3, and CCR5 as a relative expression of MCAO versus sham WT brain. To identify which of these inflammatory factors in the lesioned brain were due to infiltrating T and B cells, we also assessed changes mRNA expression in the ischemic hemisphere of MCAO versus sham-operated SCID mice. As expected for SCID mice, expression levels for most lymphocyte-derived factors and chemokines and chemokine receptors were greatly reduced in the ischemic hemisphere, particularly IL-10, CCR2, and CCR3 (Figure 2, lower panel, open bars). Only mRNA expression for IL-1β was significantly elevated by ischemia in SCID brain (MCAO versus sham SCID). Elevated IL-1β production after stroke thus appeared to be produced by resident or infiltrating macrophages or central nervous system parenchymal cells rather than T or B cells.

Figure 2.

Figure 2

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Effects of MCAO on expression of cytokines and chemokines/receptors in spleen and brain of SCID and C57BL/6 WT mice. Spleen, ipsilateral, and contralateral cerebral hemispheres were collected from three MCAO and two sham-operated mice from each treatment group (WT and SCID) at 22 h of reperfusion. mRNA was prepared from each sample, then analyzed by reverse transcription–PCR in triplicate for relative expression (RE) of cytokines, chemokines and their receptors. RE in each sample is compared with expression of ribosomal reference gene L32. These initial REs for SCID or WT stroke groups were normalized to the relevant companion sham groups. Therefore, data are shown as the fold increase (or decrease) in MCAO tissue relative to matched sham-operated controls: WT (filled bars) and SCID (open bars).

Middle Cerebral Artery Occlusion-Induced Changes in the Spleens of Severe Combined Immunodeficient Mice

We previously reported that focal cerebral ischemia in immunologically intact C57BL/6 mice resulted in a marked reduction in spleen cell numbers when evaluated at 22 h postischemia (16±3 million in MCAO versus 35±5 million in sham versus 91±8 million in naïve mice; Offner et al, 2006). We show here that naïve SCID mice have drastically reduced splenocyte numbers (2.7±1.4 million cells, Table 1) as would be expected in the absence of T and B cells. Sham-treated SCID mice had only 1.8±0.9 million cells per spleen, and MCAO treatment further reduced the cell number to 1.2 + 0.6 million per spleen (P < 0.05, Table 1). In contrast, there was no difference in the number of blood mononuclear cells in MCAO versus sham or naïve SCID mice (Table 1), although the total cell numbers (0.5 to 0.8 million/ml) were reduced relative to immunologically normal mice (~ 1 million mononuclear cells per ml).

Table 1.

Cell counts from spleen and blood of SCID mice after 22 h MCAO

Spleen Naïve (× 106) Sham (× 106) MCAO (× 106)
Exp. 1 2.6 2.6 1.6
1.4 1.0 0.8
2.0 1.8
2.8
0.8
Exp. 2 2.0 1.8 1.8
3.4 2.4 1.0
1.2 1.2
Exp. 3 3.2 1.8 0.6
2.0 3.4 2.2
Exp. 4 2.5 0.9
2.5 0.8
1.0
Exp. 5 1.5 0.7 1.3
0.7 0.9
5.8 0.4 0.8
2.2 2.0 0.8
SCID average 2.7±1.4 (n = 9) 1.8±0.9 (n = 14) 1.2±0.6a,b (n = 17)
WT average (Offner et al, 2006) 91±8 (n = 4) 35±5a (n = 15) 16±3a,b (n = 16)
Blood cells (per ml)
  Exp. 1 1.00 0.46 1.20
0.32 0.06 0.34
0.24 0.04
0.02
0.80
  Exp. 2 0.57 0.35 1.10
0.13 0.40 0.50
1.106 0.40
  Exp. 3 0.97 1.30 2.30
0.10 0.87 1.00
  Exp. 4 ND ND ND
  Exp. 5 0.30 0.30
0.20 1.20
  Exp. 6 0.20 0.30 1.10
0.70 0.50 0.60
  SCID average 0.5±0.4 (n = 8) 0.5±0.4 (n = 12) 0.8±0.6 (n = 14)
WT average
(Offner et al, 2006) ~1 1.3±0.7 0.7±0.3c
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MCAO, middle cerebral artery occlusion; ND, not determined; SCID, severe combined immunodeficient; WT, wild type.

a

P-value < 0.05 compared with naïve mice.

b

P-value < 0.05 compared with sham mice.

c

P<0.05 compared with sham mice.

Middle cerebral artery occlusion induced marked increases in secretion of inflammatory cytokines in spleens from intact WT mice at 6 and 22 h after occlusion (Offner et al, 2006). As is shown in Figure 2 (upper panel, filled bars), these changes at 22 h of MCAO were reflected by increased expression of mRNA for IFN-γ, TNF-α, IL-6, MIP-2, IP-10, CCR1, and CCR2. In contrast, the only large changes observed in spleens from MCAO versus sham-MCAO SCID mice were increases in IFN-γ and MIP-2 messages and decreases in IP-10 and CCR5 messages (Figure 2, upper panel, open bars). The changes in IFN-γ and MIP-2 indicate a stroke-dependent effect on non-T and -B cells, including residual splenocytes that are mainly natural killer cells, monocytes, macrophages, and dendritic cells.

Another important change that occurred in the periphery after stroke in WT mice was the marked emergence in the spleen of CD11b + VLA-4− macrophages (Offner et al, 2006). We evaluated this subpopulation in SCID mice 22 h after occlusion and again found a striking increase in the percentage of CD11b + VLA-4− macrophages in the spleen of MCAO-treated versus Sham or naïve mice (Table 2).

Table 2.

Percentage of surface markers in spleens of SCID and WT mice after 22 h MCAO

SCID CD11b+VLa-4−
  Naïve 4.1±1.2
  Sham 7.2±3.8
  MCAO 24.2±8.5*
WT CD11b+VLa-4−
  Naïve 0.4±0.1
  Sham 0.7±0.1
  MCAO 3.1±1.4*
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MCAO, middle cerebral artery occlusion; SCID, severe combined immuno-deficient;WT, wild type.

Spleen cells from two mice in each group were pooled and analyzed by FACS. The data indicate mean ± SD for three replicate experiments for both SCID and WT mice.

*

indicates a significant increase (P ≤ 0.05) in MCAO mice vs. naïve or sham-treated mice as determined by Student–Newman–Keuls multiple comparison test. Data from WT mice were obtained from experiments reported previously in Offner et al (2006).

Discussion

This study demonstrates three important findings. First, loss of T and B lymphocytes by a genetic mutation in SCID mice results in significant improvement in early ischemic histologic damage. The target region is cortex, presumably in penumbral areas rather than in the core of the infarct. Second, when T and B cells are absent, postischemic induction of inflammatory mediators in brain is largely suppressed within the window of our observations. Only IL-1β was elevated in ischemic SCID brain relative to sham-operated mice. Third, poststroke loss of splenocytes is blunted but not completely ablated in SCID mice. Further, post-stroke expression of intrasplenic cytokines/chemokines is also blocked, with the exception of IFN-γ and MIP-2. The source of residual splenic inflammatory factor production is likely VLA-4– macrophages. These data are the first to quantify the relative contribution of T and B lymphocytes to production of inflammatory mediators in the context of a developing infarct and emphasize that spleen-derived immunocytes are a potential target for therapeutic intervention.

A number of cellular elements are responsible for postinjury inflammation in brain, with largely cytotoxic outcomes. Such cells may arise from central origins (astrocytes and microglia) or are recruited from the peripheral circulation as activated immune cells. Peripherally derived players such as polymorphonuclear neutrophils and mononuclear phagocytes have been exhaustively studied in experimental stroke (for review see del Zoppo et al, 2001; Stoll 2002; Meisel et al, 2005). In contrast, the role of T or B lymphocytes in ischemic lesions remains unclear. Lymphocytes are ordinarily excluded from the central nervous system but can be visualized in postischemic brain by immunohisto-chemistry (for review, see Stoll et al, 1998). The precise time frame and mechanism behind their recruitment into brain, and their functionality once in residence are unclear. The early appearance of T-cell infiltration (24 h; Schroeter et al, 1994; Jander et al, 1995) may indicate that recruitment of activated cells is antigen-nonspecific, perhaps generated by sympathetic signaling from brain to the periphery (Offner et al, 2006). Alternatively, leakage of brain antigens across a compromised blood–brain barrier could initiate a peripheral immune response. Activated T cells have the capacity to infiltrate brain, and could contribute to expansion of the ischemic penumbra, an area that already contains infiltrating neutrophils. The functionality of this response has received much interest because of reports that tolerance to brain antigens can be induced with beneficial effects on stroke severity (Becker et al, 1997). The context of lymphocyte activation is likely important, as proinflammatory CD4+ CD28− lymphocyte subsets in blood are well recognized in clinical ischemic stroke, and rising CD4 + CD28− counts are associated with increased risk of stroke recurrence and death (Nadareishvili et al, 2004).

We hypothesized that activated T and B lymphocytes of splenic origin would home to injured brain and alter the trajectory of the evolving infarct. To test this directly, we employed C.B-17scid/scid mice that lack T and B lymphocytes with a consequent loss of all immune functions that require these cells. The immunodeficiency in SCID mice results from a mutation on chromosome 16, which causes deficient recombinase activity required for immunoglobulin and T-cell receptor gene rearrangement (Jones et al, 1993). Despite the lack of T and B cells, SCID mice have normal functioning macrophages, dendritic cells, and natural killer cells (but not NK-T cells that have a rearranged T-cell receptor, as well as neutrophils. We compared early histologic outcome in this strain as compared with its back-ground WT strain, C57BL/6, as a means of determining the contribution of T and B cells to focal ischemia. Intraischemic physiologic parameters, anesthesia requirement, and residual cortical perfusion as assessed by laser-Doppler flow were not different in SCID versus WT mice, suggesting that the insult was comparable. The present data suggest that lymphocytes are strongly involved in the size of the evolving infarct, and are a significant source of selected inflammatory mediators in brain. These data are consistent with a recent report that lymphocyte-deficient Rag1−/− mice sustain smaller infarct volumes and improved neurologic deficit after MCAO (Yilmaz et al, 2006). Importantly, CD4 + and CD8 + T lymphocytes contributed largely to postischemic intravascular inflammatory and prothrombotic responses in cerebral venules. In this report, mice deficient in B lymphocytes did not experience improved ischemic outcome; however, the treatment group size was small and possibly underpowered. Accordingly, further work is needed to evaluate the role of B lymphocytes in cerebral ischemia and their interaction with T-cell subpopulations.

In the present study, tissue outcomes were region-specific, in that the cortex was largely spared in SCID mice and striatal infarction was similar to that of the WT brain. One explanation may be that vascular collateralization is more pronounced in cortex, offering an effective route for T-cell entry into brain during reperfusion. It must also be emphasized that these data provide a window only into early brain inflammation, and that in this model of ischemia and reperfusion injury, final tissue damage may be strongly altered by cellular mechanisms not fully engaged at 22 h. Nevertheless, we chose to measure infarction at 22 h because longer survival times would increase the potential vulnerability of SCID mice to postoperative immune challenges that would confound stroke outcome. Lastly, the present experimental protocol does not prove that infarction matures at equivalent rates in SCID and WT mice. Further experiments are necessary to track the rate of lesion formation when T and B lymphocytes are absent versus the immunologically intact brain.

Extensive previous work has characterized cytokine and chemokine levels and their time course of appearance or persistence in postischemic brain (Barone and Feuerstein, 1999). Nevertheless, it has proved a challenge to localize inflammatory factors to cell source because of the orchestra of inflammatory and immune cells that are present after dense focal injury. The present data offer an opportunity to dissect residual levels of these mediators when T and B lymphocytes are removed from the orchestra. We utilized a fairly comprehensive PCR-based evaluation of RNA transcripts, but recognize that one limitation of this study is that protein translation was not confirmed in postischemic brain or spleen. Surprisingly, although hemispheric levels of proinflammatory TNF-α, IL-1β, and IL-6 are elevated in postischemic WT brain, the expression of TNF-α and IL-6 was strongly depressed in SCID brain to below the levels observed in companion SCID shams. TNF-α and IL-1β are well documented to be cytotoxic in the setting of cerebral ischemia, whereas the toxicity of proinflammatory IL-6 in experimental or human stroke is less clear. In our experiments, IL-1β, although reduced, persisted at detectable levels in SCID mice with stroke, indicating a non-T or -B cell source of production. IL-1β and TNF-α are thought to be largely produced by resident-activated microglial cells, but may also be secreted by other brain cells, including astrocytes, endothelial cells, and neurons, and later by infiltrating mononuclear cells from blood (Liu et al, 1993, 1994; Wang et al, 1994, 1995; del Zoppo et al, 2001). The present results suggest that expression of TNF-α and IL-6, and to a lesser degree, IL-1β, is closely linked to the presence of T and B lymphocytes, at least within the window of our observations. The only mediator that persisted in postischemic SCID brain to levels greater than companion shams was proinflammatory cytokine, IL-1β. Probably non-T and -B cell sources of production for IL-1β are known and include resident microglia, infiltrating macrophages, and neutrophils (Liu et al, 1993; Wang et al, 1997; Stevens et al, 2002). We also evaluated selected chemokines that cause neuronal death either directly through neuronal receptors or indirectly via microglial activation and killing. Previous work indicates these factors are induced after MCAO, for example IP-10, CXCR3 (that binds IP-10), and CCR5 (that binds MIP-1α/β, RANTES, and MCP-2) (Wang et al, 1998; Spleiss et al, 1998). We also observed induction of these factors in WT, but not SCID, ischemic brain, again emphasizing that these mediators are linked to T/B lymphocytes of lymphoid origin.

Although the spleen is comprised mainly of T and B cells that are drastically reduced in WT MCAO mice, it was of importance to detect a further reduction in the non-T and -B cell fraction of spleens in SCID MCAO mice that was not apparent in blood. The stroke-induced increase in CD11b + VLA-4− splenocytes apparent in WT mice was accentuated even more in SCID mice in the absence of T and B cells. The net result in the spleens of SCID mice with stroke was the absence of most inflammatory factors, but persistence of message for IFN-γ and MIP-2, both likely secreted by the enriched residual population of CD11b + VLA-4− macrophages.

In summary, by comparing stroke in WT versus T-and B-cell-deficient SCID mice, we here show for the first time a significant contribution of inflammatory T and B cells to the early penumbral infarct that predominantly affects cortex but not the core striatal insult in which IL-1β was the only remaining cytokine. Additionally, there were substantial reductions in inflammatory factors in the spleen of stroke-induced SCID mice, with the exception of IFN-γ and MIP-2 that appeared to be the product of residual CD11b + VLA-4− macrophages that were relatively enriched in the absence of T and B cells. Future studies using B-cell-deficient mice will clearly distinguish the role of T versus B cells in stroke development.

Acknowledgements

The authors thank Ms Eva Niehaus for assistance in preparing and submitting this paper.

This work was supported by US Public Health Service NIH grants NS33668, NR03521, NS49210, RR00163, and the Biomedical Laboratory R&D Service, Department of Veterans Affairs.

References

  1. Audebert HJ, Rott MM, Eck T, Haberl RL. Systemic inflammatory response depends on initial stroke severity but is attenuated by successful thrombolysis. Stroke. 2004;35:2128–2133. doi: 10.1161/01.STR.0000137607.61697.77. [DOI] [PubMed] [Google Scholar]
  2. Barone FC, Feuerstein GZ. Inflammatory mediators and stroke: New opportunities for novel therapeutics. J Cereb Blood Flow Metab. 1999;19:819–834. doi: 10.1097/00004647-199908000-00001. [DOI] [PubMed] [Google Scholar]
  3. Becker KJ, Kindrick D, Relton J, Harlan J, Winn R. Antibody to the α4 integrin decreases infarct size in transient focal cerebral ischemia in rats. Stroke. 2001;32:206–211. doi: 10.1161/01.str.32.1.206. [DOI] [PubMed] [Google Scholar]
  4. Becker KJ, McCaron RM, Ruetzler C, Laban O, Sternberg E, Flanders KC, Hallenbeck JM. Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia. Proc Natl Acad Sci. 1997;94:10873–10878. doi: 10.1073/pnas.94.20.10873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. del Zoppo GJ, Becker KJ, Hallenbeck JM. Inflammation after stroke: is it harmful? Arch Neurol. 2001;58:669–672. doi: 10.1001/archneur.58.4.669. [DOI] [PubMed] [Google Scholar]
  6. Emsley HCA, Smith CJ, Gavin CM, Georgiou RF, Vail A, Barberan EM, Hallenbeck JM, del Zoppo GJ, Rothwell NJ, Tyrrell PJ, Hopkins SJ. An early and sustained peripheral inflammatory response in acute ischemic stroke: relationships with infection and atherosclerosis. J Neuroimmunol. 2003;139:93–101. doi: 10.1016/s0165-5728(03)00134-6. [DOI] [PubMed] [Google Scholar]
  7. Jander S, Karemer M, Schroeter M, Witte OW, Stoll G. Lymphocytic infiltration and expression of intercellular adhesion molecule-1 in photochemically induced ischemia of the rat cortex. J Cereb Blood Flow Metab. 1995;15:42–51. doi: 10.1038/jcbfm.1995.5. [DOI] [PubMed] [Google Scholar]
  8. Jones RE, Bourdette DN, Whitham RH, Offner H, Vandenbark AA. Induction of experimental autoimmune encephalomyelitis in severe combined immunodeficient mice reconstituted with allogeneic or xenogeneic hematopoietic cells. J Immunol. 1993;150:4620–4629. [PubMed] [Google Scholar]
  9. Liu T, Clark RK, McDonnell PC, Young PR, White RF, Barone FC, Feuerstein GF. Tumour necrosis factor a expression in ischemic neurons. Stroke. 1994;25:1481–1488. doi: 10.1161/01.str.25.7.1481. [DOI] [PubMed] [Google Scholar]
  10. Liu T, McDonnell PC, White RF, Barone FC, Feuerstein GZ. Interleukin-1B mRNA expression in ischemic rat cortex. Stroke. 1993;24:1746–1751. doi: 10.1161/01.str.24.11.1746. [DOI] [PubMed] [Google Scholar]
  11. McCullough LD, Blizzard K, Simpson ER, Oz OK, Hurn PD. Aromatase cytochrome P450 and extragonadal estrogen play a role in ischemic neuroprotection. J Neurosci. 2003;23:8701–8705. doi: 10.1523/JNEUROSCI.23-25-08701.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Meisel C, Schwab JM, Prass K, Meisel A, Dirnagl U. Central nervous system injury-induced immune deficiency syndrome. Nat Rev. 2005;6:775–786. doi: 10.1038/nrn1765. [DOI] [PubMed] [Google Scholar]
  13. Nadareishvili ZG, Li H, Wright V, Maric D, Warach S, Hallenbeck JM, Dambrosia J, Barker JL, Baird AE. Elevated pro-inflammatory CD4+CD28+-lymphocytes and stroke recurrence and death. Neurology. 2004;63:1446–1451. doi: 10.1212/01.wnl.0000142260.61443.7c. [DOI] [PubMed] [Google Scholar]
  14. Offner H, Subramanian S, Parker SM, Afentoulis ME, Vandenbark AA, Hurn PD. Experimental stroke induces massive, rapid activation of the peripheral immune system. J Cereb Blood Flow Metab. 2006;26:654–665. doi: 10.1038/sj.jcbfm.9600217. [DOI] [PubMed] [Google Scholar]
  15. Offner H, Subramanian S, Parker SM, Wang C, Afentoulis ME, Lewis A, Vandenbark AA, Hurn PD. Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages. J Immunol. 2006a;176:6523–6531. doi: 10.4049/jimmunol.176.11.6523. [DOI] [PubMed] [Google Scholar]
  16. Prass K, Meisel C, Hoflich C, Braun J, Halle E, Wolf T, Ruscher K, Victorov IV, Priller J, Dirnagl U, Volk H-D, Meisel A. Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation reversal by post-stroke T helper cell type 1-like immunostimulation. J Exp Med. 2003;198:725–736. doi: 10.1084/jem.20021098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Relton JK, Sloan KE, Frew EM, Whalley ET, Adams SP, Lobb RR. Inhibition of α4 integrin protects against transient focal cerebral ischemia in normotensive and hpertensive rats. Stroke. 2001;32:199–205. doi: 10.1161/01.str.32.1.199. [DOI] [PubMed] [Google Scholar]
  18. Sawada M, Alkayed NJ, Got S, Crain BJ, Traystman RJ, Shaivitz A, Nelson RJ, Hurn PD. Estrogen receptor antagonist ICI182,780 exacerbates ischemic injury in female mouse. J Cereb Blood Flow Metab. 2000;20:112–118. doi: 10.1097/00004647-200001000-00015. [DOI] [PubMed] [Google Scholar]
  19. Schroeter M, Jander S, Witte OW, Stoll G. Local immune responses in the rat middle cerebral artery occlusion. J Neuroimmunol. 1994;55:195–203. doi: 10.1016/0165-5728(94)90010-8. [DOI] [PubMed] [Google Scholar]
  20. Spleiss O, Gourmala N, Boddeke HW, Sauter A, Fiebich BL, Berger M, Gebicke-Haerter PJ. Cloning of rat HIV-1-chemokine co-receptor CKR5 from microglia and upregulation of its mRNA in ischemic and endotoxinemic rat brain. J Neurosci Res. 1998;53:16–28. doi: 10.1002/(SICI)1097-4547(19980701)53:1<16::AID-JNR3>3.0.CO;2-M. [DOI] [PubMed] [Google Scholar]
  21. Stevens SL, Bao J, Hollis J, Lessov NS, Clark WM, Stenzel-Poore MP. The use of flow cytometry to evaluate temporal changes in inflammatory cells following focal cerebral ischemia in mice. Brain Res. 2002;932:110–119. doi: 10.1016/s0006-8993(02)02292-8. [DOI] [PubMed] [Google Scholar]
  22. Stoll G. Inflammatory cytokines in the nervous system: multifunctional mediators in autoimmunity and cerebral ischemia. Rev Neurol. 2002;158:887–891. [PubMed] [Google Scholar]
  23. Stoll G, Jander S, Schroeter M. Inflammation and glial responses in ischemic brain lesions. Prog Neurobiol. 1998;56:149–171. doi: 10.1016/s0301-0082(98)00034-3. [DOI] [PubMed] [Google Scholar]
  24. Wang X-K, Barone FC, Aiyar NV, Feuerstein GF. Increased interleukin-1 receptor and interleukin-1 receptor antagonist gene expression after focal stroke. Stroke. 1997;28:155–162. doi: 10.1161/01.str.28.1.155. [DOI] [PubMed] [Google Scholar]
  25. Wang X-K, Ellison JA, Siren A-L, Lysko PG, Yue T-L, Barone FC, Shatzman A, Feuerstein GF. Prolonged expression of interferon inducible protein-10 in ischemic cortex after permanent occlusion of the middle cerebral artery in the rat. J Neurochem. 1998;71:1194–1204. doi: 10.1046/j.1471-4159.1998.71031194.x. [DOI] [PubMed] [Google Scholar]
  26. Wang XK, Yue TL, Barone FC, Feuerstein GF. Monocyte chemoattractant protein-1 (MCP-1) mRNA expression in rat ischemic cortex. Stroke. 1995;26:661–666. doi: 10.1161/01.str.26.4.661. [DOI] [PubMed] [Google Scholar]
  27. Wang XK, Yue T-L, Barone FC, White R, Young PR, McDonnell PC, Feuerstein GZ. Concomitant cortical expression of TNFα and IL-1β mRNA following transient focal ischemia. Mol Chem Neuropathol. 1994;23:103–114. doi: 10.1007/BF02815404. [DOI] [PubMed] [Google Scholar]
  28. Yilmaz G, Arumugam TV, Stokes KY, Granger DN. Role of T lymphocytes and interferon-gamma in ischemic stroke. Circulation. 2006;113:2105–2112. doi: 10.1161/CIRCULATIONAHA.105.593046. [DOI] [PubMed] [Google Scholar]

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