Experimental Stroke-Induced Changes in the Bone Marrow Reveal Complex Regulation of Leukocyte Responses

Abstract

Stroke induces a systemic response that involves rapid activation of inflammatory cascades, followed later by immunodepression. Experimental stroke-induced responses in the bone marrow, which is the primary source of circulating monocytes and granulocytes, have not been investigated previously. We show that cerebral ischaemia induced early (4 hours) release of CXCR2-positive granulocytes from the bone marrow, which was associated with rapid systemic upregulation of CXCL1 (a ligand for CXCR2) and granulocyte-colony-stimulating factor, a key cytokine involved in the mobilisation of bone marrow leukocytes. This process involves rapid activation of nuclear factor-κB and p38 mitogen-activated protein kinase in bone marrow myeloid cells. T-cell numbers in the bone marrow increased after stroke, and bone marrow cells did not show suppressed cytokine response to bacterial endotoxin stimulation in vitro. Stroke-induced laterality observed in the brain stem and in the bone marrow indicates direct involvement of the autonomic nervous system in stroke-induced cell mobilisation. We also show that systemic inflammatory changes and leukocyte responses in the bone marrow are profoundly affected by both anaesthetic and surgical stress. We conclude that stroke influences leukocyte responses in the bone marrow through multiple mechanisms and suggest that preclinical studies should take into consideration the effect of surgical manipulation in experimental models of stroke.

Keywords

acute-phase response autonomic bone marrow cytokine granulocyte stroke

Introduction

It is now accepted that inflammatory processes originating both inside and outside the brain are important aetiological and pathologic factors in stroke. Acute or chronic inflammatory diseases often precede and contribute to the onset and outcome of stroke in humans and experimental animals, in part through immune cells such as neutrophils or monocytes and various soluble inflammatory mediators (Denes et al, 2010; Emsley and Hopkins, 2008; McColl et al, 2009). Both clinical and experimental cerebral ischaemia induces activation of the sympathetic nervous system and lymphoid organs such as the spleen, followed by later immunosuppression and splenic atrophy (Dirnagl et al, 2007; Offner et al, 2006b, 2009; Prass et al, 2003). These data indicate that the generation of the stroke-induced acute-phase response involves both humoural and neural signals to various peripheral organs.

We are aware of no previous studies that have examined the consequences of cerebral ischaemia on bone marrow cell composition, activation, and mobilisation. The bone marrow is the primary source of granulocytes and monocytes, which are recruited to the inflamed brain and reportedly contribute to many brain diseases, including stroke (D’Mello et al, 2009; Kim et al, 2009; McColl et al, 2008). Notably, the signals that trigger leukocyte mobilisation from the bone marrow in acute brain diseases are unknown and may represent potential targets for therapy. We showed previously that the bone marrow receives central autonomic innervation, which involves forebrain areas such as the insular and piriform cortex (Denes et al, 2005), which often become ischaemic after experimental stroke. It is also known that humoural factors, including cytokines and chemokines are rapidly elevated in both the brain and the periphery after stroke (Offner et al, 2006a). In addition, we recently showed that cerebral ischaemia induces systemic upregulation of CXCL1 (KC), a chemokine that acts in concert with granulocyte-colony-stimulating factor (G-CSF) to mobilise neutrophils from the bone marrow in response to inflammatory challenges (Chapman et al, 2009; Wengner et al, 2008). The release of neutrophil granulocytes from the bone marrow is a complex process, which involves an antagonistic interaction between the neutrophil chemokine receptors CXCR2 and CXCR4 (Eash et al, 2010). Activation of CXCR2 by its ligands, CXCL1 and/or CXCL2, is a key step in neutrophil mobilisation and recruitment to inflamed tissues in various diseases, including arthritis, ischaemia–reperfusion injury, or inflammatory demyelination in the central nervous system (Jacobs et al, 2010; Li et al, 2010; Liu et al, 2010; Martin et al, 2003; von Vietinghoff et al, 2010; Wengner et al, 2008). Other chemokine receptors such as CCR5 can also interact with key leukocyte mobilisation responses and can promote the recruitment of monocytes, granulocytes, and T cells to the sites of inflammation (Balistreri et al, 2007; Honczarenko et al, 2002; Kohlmeier et al, 2008). Leukocyte mobilisation responses also involve several acutely synthesised inflammatory mediators, such as matrix metalloproteinase protein-9 (MMP-9), which besides its proposed role in the release of leukocytes from the bone marrow, makes a profound contribution to brain damage after stroke (McColl et al, 2008; Pelus et al, 2004).

These findings implicate the bone marrow in inflammatory responses to brain injury; hence, we tested the hypothesis that experimental stroke leads to the activation and mobilisation of bone marrow leukocytes. We show that middle cerebral artery occlusion (MCAo) in mice induced rapid activation of myeloid cells and mobilisation of CXCR2-positive granulocytes, which was followed by changes in bone marrow lymphocytes. Our results also show effects of anaesthesia and surgical stress, which not only altered leukocyte responses but also had a considerable impact on the systemic levels of inflammatory mediators. These findings reveal a previously unrecognised effect of stroke on bone marrow leukocyte responses and have important implications for both therapeutic and modelling aspects of experimental stroke research.

Materials and methods

Animals

Male C57BL6/J mice (Harlan-Olac, Bicester, UK) aged 12 to 14 weeks (weighing 24 to 30 g) were used for the study. Animals had free access to food and water and were maintained under temperature-, humidity-, and light-controlled conditions. All animal procedures were performed under an appropriate Home Office License and adhered to regulations as specified in the Animals (Scientific Procedures) Act (1986).

Surgical Procedures

Transient MCAo was performed using the intraluminal filament method as described previously (Denes et al, 2007). Anaesthesia was induced with 4%, and maintained with 1.5%, isoflurane (Baxter Healthcare Corporation, Thetford, UK) in a mixture of 30% oxygen and 70% nitrous oxide. During surgery, core body temperature was monitored using a rectal probe and maintained at 37°C±0.5°C, using a homeothermic blanket (Harvard Apparatus, Kent, UK). After surgical exposure of the bifurcation of the external carotid artery and the internal carotid artery on the left side, a silicone-coated nylon monofilament (tip diameter 180 μm) was introduced into the origin of the external carotid artery and advanced through the internal carotid artery to occlude the MCA. Reperfusion was induced 60 minutes later, and animals were allowed to recover. In sham-operated animals, the filament was advanced along the internal carotid artery until the origin of the MCA and was retracted immediately. In the case of both MCAo and sham surgery, mice were kept anaesthetised throughout the whole surgical procedure. To control for the effect of isoflurane on bone marrow cells, a group of mice were anaesthetised for 90 minutes (average duration of the complete surgery), without any surgical intervention.

In a separate set of experiments, mice were allowed to recover during occlusion and reanaesthetised at the end of the occlusion to remove the filament, which reduced anaesthesia duration from ∼90 minutes to 20 to 30 minutes (the time spent during surgery to insert the filament and to suture the surgical territory after the occlusion). Control (sham, isoflurane) groups were adjusted accordingly.

After surgery, mice were subjected to 10 minutes to 72 hours reperfusion. Neurologic status was assessed immediately after recovery to confirm successful occlusion, according to a neurologic grading score of increasing severity of deficit (Bederson et al, 1986): (0) no observable deficit; (1) torso flexion to right; (2) spontaneous circling; (3) leaning/falling to right; and (4) no spontaneous movement. Mice showing no functional deficit (i.e., without clear torso flexion and/or circling) after MCAo were excluded from the study. Mice were subcutaneously injected with 0.5 mL of sterile saline daily and continuously monitored for neurologic symptoms.

Tissue Processing

Serial blood samples were obtained from the tail vein and cardiac blood was collected from the right ventricle before perfusion. The plasma was separated with centrifugation at 1,700 × g, 4°C, and blood cells were lysed in ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 5 mmol/L CaCl₂, 0.02% NaN₃, 1% Triton X). Mice were transcardially perfused with 0.9% saline, and then the brains were removed and snap frozen on dry ice. For cytokine measurement, the bone marrow from femurs and tibia was flushed with lysis buffer. For flow cytometric cell surface labelling and for in vitro stimulation assays, bone marrow cells were isolated in medium (RPMI, supplemented with 10% fetal calf serum and penicillin/streptomycin). Bone marrow cells were flushed with 1% paraformaldehyde in phosphate-buffered saline (PBS) for phospho-flow cytometric labelling. In some experiments, the left and right tibial and femoral bone marrow was isolated separately to assess laterality after MCAo. Some animals were perfused with 4% paraformaldehyde for immunohistochemistry and the brains were sectioned on a sledge microtome (Bright, Huntingdon, UK).

Flow Cytometry

Bone marrow cells were isolated as described above. For cell surface labelling, after Fc receptor blocking, cells were incubated with fluorescently conjugated antibodies against CD4 (GK1.5), CD8 (53-6.7), CD3 (17A2), CD19 (MB19-1), MHCII (M5/114.15.2), Gr-1 (RB6-8C5), CD11b (M1/70), c-kit (2B8), CD49b (DX5) (eBioscience, Hatfield, UK), CXCR2 (242216, R&D Systems, Abingdon, UK), and CCR5 (C34-3448, BD Biosciences, Oxford, UK).

For single-cell phosphorylation-state analysis, fixed cells were first incubated with anti-Gr-1 (eBioscience), then permeabilised with BD Phosflow Perm III buffer (BD Biosciences), and then stained with either phospho-nuclear factor (NF)κB p65 (Ser536, Cell Signaling Technology, Danvers, MA, USA, 1:25) or phospho-p38 mitogen-activated protein kinase (MAPK) (pT180/pY182, BD Biosciences, 1:25). Before intracellular cytokine staining, bone marrow cells isolated from naive animals or from mice exposed to anaesthesia, stroke, or sham surgery were incubated with 3 μmol/L monensin (Golgi Stop; BD Biosciences) for 2 hours. Subsequently, cell surface labelling was performed as described above, before permeabilisation and incubation with goat anti-mouse CXCL1 (R&D Systems) overnight and then with donkey anti-goat Alexa 488 (Invitrogen, Paisley, UK) for 1 hour.

Labelled cells were acquired on FACS Calibur (BD Biosciences) and Cyan ADP (Beckman Coulter, High Wycombe, UK) flow cytometers. Data were analysed using CellQuest pro (BD Biosciences), FACSDiva (BD Biosciences), and Summit 4.3 (Dako Ltd, Glostrup, Denmark) software.

Lateralisation of the bone marrow was evaluated by determining the ratio of Gr-1^high granulocytes or CD19-positive B cells in the left and right femur/tibia by flow cytometry, and was then laterality expressed as the % difference between them.

Western Blotting

For NFκB western blots, cell lysates were separated on 4% to 12% SDS-polyacrylamide gels under reducing conditions. Proteins were transferred to polyvinylidene difluoride membrane (Scientific Laboratory Supplies Ltd, Wilford, UK). Membranes were blocked with 10% nonfat milk in PBS-0.1% Tween 20 for 1 hour at room temperature. Membranes were incubatight with the primary antibody (rabbit anti-NFκB p65, Santa Cruz Biotechnology, Wembley, UK) diluted 1:200 in 1% bovine serum albumin/PBS-0.1% Tween 20 at 4°C. Loading volumes were normalised against anti-β-actin peroxidise (1:1,500 dilution, Sigma-Aldrich, St Louis, MO, USA). Membranes were washed with PBS-0.1%Tween 20 and incubated with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit-horseradish peroxidase, Dako Ltd) diluted 1:1,000 in 5% nonfat milk/PBS-0.1% Tween 20 for 1 to 3 hours at room temperature. Membranes were developed by reaction with ECL chemiluminescent substrate (GE Healthcare, Pittsburgh, PA, USA), onto photographic film (Fischer Scientific, Pittsburgh, PA, USA).

Gel Zymography

Bone marrow lysates equilibrated for total protein concentration (2 mg/mL) (25 μL) were separated on SDS-polyacrylamide gels impregnated with 1.5 mg/mL gelatin under nonreducing, nondenaturing conditions. The SDS was removed to renature enzymes by incubating gels in the wash buffer (50 mmol/L Tris-HCl, pH 7.6, 5 mmol/L CaCl₂, 1 μmol/L ZnCl₂, 2.5% Triton X-100). Gels were then incubated in the reaction buffer (wash buffer without detergent) for 72 hours at 37°C to stimulate gelatinolysis. Gels were stained with Coomassie Brilliant Blue R-250 (0.1%) diluted in 40% methanol and 10% acetic acid for 1 hour, and destained in a solution containing 10% methanol and 1% acetic acid until clear gelatinolytic bands appeared.

In Vitro Stimulation of Bone Marrow Cells

Total bone marrow cells collected 72 hours after sham or stroke surgery were stimulated with 1 μg/mL lipopolysaccharide (E. coli O26:B6), at a density of 7.5 × 10⁶ cells/mL in RPMI medium, supplemented with 10% fetal calf serum and penicillin/streptomycin. After 3 hours incubation at 37°C, cells were pelleted with centrifugation at 400 × g, the supernatant was collected, and cells were lysed in lysis buffer.

Cytokine Measurement

For the measurement of CXCL1 (KC), proMMP-9, and interleukin-6, DuoSet ELISA (enzyme-linked immunosorbent assay) kits were purchased from R&D Systems and used according to the manufacturer's protocol. Granulocyte-colony-stimulating factor was measured using the cytometric bead array (BD Biosciences).

Immunohistochemistry

Free-floating, 20-μm-thick brain sections were blocked in 2% normal goat serum and then incubated with rabbit anti-STAT3 (signal transducer and activator of transcription 3) phosphorylation (Cell Signaling Technology) or rabbit anti-neurokinin receptor-1 (NK1) (Millipore, Watford, UK) antibodies. After incubation with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Peterborough, UK) and ABC solution (Vector Laboratories) for 1 hour, colour was developed with diaminobenzidine-tetrahydrochloride (Sigma-Aldrich) in the presence of nickel-ammonium sulphate.

Statistical Analysis

For comparing two groups, Student's t-test (two tailed) was used. Comparison of three or more groups was carried out by one-way or two-way ANOVA (analysis of variance), followed by Bonferroni's correction. Data are expressed as mean±s.e.m. P<0.05 was considered statistically significant.

Results

CXCR2-Positive Granulocytes are Released from the Bone Marrow in Response to Experimental Stroke

Sixty-minute MCAo resulted in ischaemic brain damage in the left hemisphere affecting 35% to 65% of the hemisphere (lesion volume, 40 to 70 mm³), in the MCA territory, including the striatum, the insular, the somatosensory and the somatomotor cortex. Neurologic scores were two or three in average (data not shown). Activation of myeloid cells was associated with mobilisation of Gr-1-positive cells from the bone marrow within 4 hours after MCAo (Figures 1A and 1B) as identified by flow cytometry. Identical results were obtained using CD11b, another marker of granulocytes–monocytes (Figures 1C and 1D). To investigate which population of myeloid cells responds specifically to stroke, we looked at CXCR2-positive granulocytes, because our recent findings indicated a key role for CXCL1 (a CXCR2 ligand) in the stroke-induced early systemic response (Chapman et al, 2009). We observed a marked reduction in CXCR2-positive cells at 4 hours reperfusion (60%, compared with naive mice) in the granulocyte pool of the bone marrow in response to stroke (Figures 1F and 1G). Sham surgery seemed to cause a slight reduction in this population (not significant at 4 hours reperfusion), whereas stroke resulted in a significant (47%) reduction in CXCR2-positive granulocytes (CD11b+SSC^high cells) in the bone marrow compared with sham surgery (Figure 1H). CCR5-positive cells increased in the bone marrow, indicating that stroke is likely to have a differential effect on different populations of leukocytes (Figure 1I).

Figure 1

Experimental stroke induces a rapid release of CXCR2-positive granulocytes from the bone marrow. Flow cytometric analysis was used to assess changes in myeloid cell populations in the bone marrow after isoflurane anaesthesia, sham, or stroke surgery at various reperfusion times. Release of Gr-1- and CD11b-positve cells was induced predominantly by stroke at 4 hours reperfusion, which was followed by a reduction in surgery-induced increase in cell numbers after stroke at 72 hours reperfusion. Both (A) proportion and (B) total number of Gr-1-positive cells in the bone marrow indicate comparable changes to the (C) proportion or (D) total number of CD11b-positive cells over time. (E) Total cell yield in the bone marrow after various reperfusion times. (F) Stroke-induced granulocyte release from the bone marrow was predominantly caused by a marked reduction in CXCR2-positive (CD11b+SSC^high) cells at 4 hours reperfusion. (G) Representative dot blots showing reduction of CXCR2-expressing CD11b+SSC^high cells in the bone marrow at 4 hours reperfusion. Green: gated on CD11b-positive cells. (H) Reduction of CXCR2+granulocytes in the bone marrow 4 hours after stroke is significantly augmented compared with both naive and sham mice. (I) The proportion of CCR5-positive cells increased in the bone marrow at 4 hours reperfusion. ^∗P<0.05; ^∗∗P<0.01; ^∗∗∗P<0.001. Data shown are representative of five to nine mice in each group corresponding to at least two independent experiments. isof., isoflurane.

Both isoflurane and sham surgery seemed to disturb leukocyte responses. A marked increase in bone marrow cell numbers was observed as a result of surgical stress 72 hours after sham or stroke surgery (Figure 1E), which was caused by an increase in the myeloid cell lineage. Total and myeloid cell numbers were significantly lower (20% to 25%) after stroke compared with sham surgery (Figures 1A to 1E). CXCR2-positive granulocytes were less abundant in the bone marrow at 72 hours reperfusion after stroke compared with sham surgery (Supplementary Figure A).

Experimental Stroke Induces Rapid Activation of Myeloid Cells in the Bone Marrow

We evaluated whether experimental stroke led to the activation of leukocytes in the bone marrow, by assessing NFκB and p38 MAPK, two common signalling pathways in various haematopoietic cells (Bottero et al, 2006; Geest and Coffer, 2009). Western blotting identified rapid induction of NFκB p65 in the bone marrow after stroke at 10 minutes reperfusion (70 minutes onset of ischaemia), which was further augmented by 4 hours (Figure 2A). Single-cell analysis with flow cytometry revealed that phosphorylation of NFκB p65 occurs in myeloid cells after stroke, predominantly in Gr-1-positive SSC^high granulocytes (Figure 2B). Phosphorylation of NFκB p65 was significantly increased in granulocytes at 4 hours reperfusion after stroke compared with sham surgery (Figure 2C). Flow cytometry showed a significant increase in phosphorylated p38 MAPK in Gr-1^int SSC^int cells (a population corresponding to various mature and immature myeloid populations including monocytes) at 4 hours reperfusion after stroke (Figures 2D and 2E). These data indicate that experimental stroke induces rapid activation of bone marrow myeloid cells.

Figure 2

Experimental stroke results in rapid activation of myeloid cell populations in the bone marrow. (A) Stroke-induced NFκB activation between 10 minutes and 4 hours reperfusion as assessed by western blotting, using a specific antibody recognising the NFκB p65 subunit. Isoflurane anaesthesia alone (Isof.) or sham surgery also led to mild p65 activation at 4 hours reperfusion. (B) Phosphorylation of NFκB p65 at residues surrounding Ser536 was detected with flow cytometry in Gr-1-positive SSC^high granulocytes in the bone marrow. Histogram shows stroke-induced NFκB p65 phosphorylation, which led to increased intracellular mean fluorescent intensity (MFI) after permeabilisation and immunostaining. (C) NFκB p65 phosphorylation was significantly increased after stroke in bone marrow granulocytes at 4 hours reperfusion compared with sham surgery. (D) Dot blot showing the gates used to assess the phosphorylation of p38 MAPK in different Gr-1-positive cell populations in the bone marrow by flow cytometry. (E) Stroke resulted in a significant increase in phosphorylated p38 MAPK (p-p38) in Gr-1^int cells after 4 hours reperfusion. ^∗P<0.05. One representative experiment (n=3) out of two independent experiments is shown. MAPK, mitogen-activated protein kinase; NFκB, nuclear factor-κB.

Early Inflammatory Changes in the Bone Marrow Parallel the Systemic Response to Experimental Stroke

Stroke induced a transient (50-fold) elevation of CXCL1 (KC) in the circulation 4 hours after reperfusion, which was significantly higher than the effect (25-fold) of surgical stress alone (Figure 3A). At the same time point, CXCL1 was also induced in the bone marrow, but levels were significantly (50%) lower in stroke animals (Figure 3B), which may indicate the increased release of bone marrow-derived CXCL1 (or CXCL1-containing cells) into the blood.

Figure 3

Rapid systemic induction of CXCL1 and proMMP-9 parallel cellular changes in response to experimental stroke and surgical stress. (A) Plasma CXCL1 was induced 4 hours after MCAo at a significantly higher level than sham surgery alone. (B) CXCL1 was upregulated in the bone marrow at 4 hours reperfusion, which was less pronounced after MCAo than after sham surgery. (C) Changes in proMMP-9 levels were measured by ELISA in the bone marrow (fold increase over naive is shown). (D) Gelatinase activity, measured by gel zymography decreases markedly in the bone marrow by 4 hours reperfusion after both sham and MCAo surgery. (E) Intracellular cytokine staining revealed upregulated CXCL1 in CD11b/Gr-1 double-positive, SSC^high bone marrow cells after sham and stroke surgery after 4 hours reperfusion. Circulating blood cells were lysed and (F) CXCL1 or (G) proMMP-9 were detected with ELISA at various reperfusion times. By 10 minutes reperfusion, plasma (H) CXCL1 was elevated after both sham and MCAo surgeries, whereas (I) plasma G-CSF showed a significant increase only after stroke. ^∗P<0.05; ^∗∗P<0.01; ^∗∗∗P<0.001. Data from two to three independent experiments are shown. ELISA, enzyme-linked immunosorbent assay; G-CSF, granulocyte-colony-stimulating factor; MCAo, middle cerebral artery occlusion; MMP-9, matrix metalloproteinase protein-9; NS, not significant.

Bone marrow proMMP-9 levels reduced markedly (by 70%) at 4 hours reperfusion after MCAo (Figure 3C) compared with naive animals. Gelatinase activity in the bone marrow decreased equally after stroke and surgical stress caused by sham surgery (Figure 3D). These results indicated that surgical stress is sufficient to induce inflammatory changes in the bone marrow. Intracellular staining and flow cytometric analysis confirmed upregulation of CXCL1 in granulocytes (CD11b⁺Gr-1⁺SSC^high cells) in the bone marrow after both sham and stroke surgery (Figure 3E and Supplementary Figure B). Correspondingly, at 4 hours reperfusion, CXCL1 levels in circulating blood cells increased in response to surgery and MCAo (25- to 30-fold; Figure 3F). Moreover, surgical stress alone increased proMMP-9 in circulating blood cells by 10 minutes after reperfusion (Figure 3G). Blood cell-derived CXCL1 and proMMP-9 were reduced after stroke compared with sham surgery at 10 minutes and 4 hours, respectively, which indicate that stroke alters the production and/or release of inflammatory mediators compared with surgical stress alone (Figures 3F and 3G).

We also investigated whether circulating levels of key neutrophil-mobilising cytokines are upregulated early enough to drive the release of CXCR2-positive granulocytes from the bone marrow after stroke. Ten minutes after reperfusion, CXCL1 levels were not different after sham and stroke surgery (Figure 3H); in contrast, G-CSF exhibited a rapid increase after stroke, but not sham surgery (Figure 3I).

Experimental Stroke Causes Increased Bone Marrow T Cells and Natural Killer Cells

Isoflurane alone induced an increase in T and B cells in the bone marrow 4 hours after induction, which correlated with lymphocyte responses in animals that had undergone sham or stroke surgery (Figures 4A to 4F). This was largely normalised by 24 to 72 hours reperfusion in sham animals. In contrast, MCAo increased CD4- and CD8-positive T cells in the bone marrow between 24 and 72 hours reperfusion, compared with sham surgery. Total bone marrow T-cell numbers markedly increased at 72 hours after stroke compared with naive mice (Figures 4A to 4D). B cells were significantly reduced in the bone marrow by 72 hours in response to surgical stress, which corresponded to the increase in proportion and number of granulocytes/monocytes (Figures 1A to 1E). In contrast, the ratio of major histocompatibility complex II (MHCII)-positive B cells showed a transient (4 hours) elevation after stroke (Figure 4G). Natural killer cells (Figures 5A and 5B) showed an increase after stroke compared with sham surgery between 24 and 72 hours reperfusion, whereas mast cells increased by 72 hours reperfusion in response to surgical stress (Figures 5C and 5D). The main leukocyte population changes in the bone marrow are summarised in Table 1.

Table 1

Changes in cells populations in the bone marrow over time after experimental stroke

	Early (4 hours)			Late (24–72 hours)
	Anaesthetic	Surgery	Stroke	Anaesthetic	Surgery	Stroke
Total BM cells					↑↑	↑
Granulocytes (CXCR2+)			↓↓		↑	↓
Myeloid cells (total)			↓		↑↑	↑
CCR5+			↑
Mast cells					↑
CD4+ T cells	↑					↑
CD8+ T cells	↑					↑
B cells	↑				↓
MHCII+			↑
NK cells						↑

The main effects of isoflurane anaesthesia, surgical stress, and stroke were summarized at the time points examined. Arrows indicate cell population changes: ↑, increase; ↓, decrease. It must be noted that trends, or transient changes in cell population ratio without real effect in total cell numbers were not included.

In a separate set of experiments, we investigated whether shortening the duration of anaesthesia would prevent the confounding effects seen on various leukocyte populations (see the ‘Materials and methods’ section). The effect of an average of 20- to 30-minute anaesthesia on leukocyte responses in the bone marrow was largely indistinguishable from what we observed earlier with a longer period of anaesthesia, indicating that isoflurane effects occur relatively early after induction (data not shown).

Figure 4

Differential effects of anaesthetic, surgery, and experimental stroke on lymphocyte populations in the bone marrow. Isoflurane increased T and B cells in the bone marrow at 4 hours reperfusion (A–E) as revealed by flow cytometric analysis. (Panels A and B) CD4-positive and (panels C and D) CD8-positive T cells were more abundant at 72 hours reperfusion after stroke surgery compared with sham animals, which is indicated by (panels A and C) increased percentage and by (panels B and D) cell yield. Surgical stress resulted in reduced B cell ratio (E) and numbers (F) after 72 hours reperfusion, which was associated with an increased proportion of myeloid cells (see Figure 1) and no significant effect of stroke surgery versus sham was seen. (G, H) In contrast, MCAo resulted in upregulated MHCII on B cells at 4 hours reperfusion compared with sham surgery. ^∗P<0.05; ^∗∗P<0.01; ^∗∗∗P<0.001. Data shown are representative of five to nine mice in each group, corresponding to at least two independent experiments. MCAo, middle cerebral artery occlusion.

Figure 5

Flow cytometric analysis of natural killer (NK) cells and mast cells in the bone marrow after experimental stroke. (A, B) Stroke resulted in an increase in NK cells in the bone marrow between 24 and 72 hours reperfusion compared with sham surgery. (C) C-kit+Gr-1^int mast cell proportions and (D) yield at various reperfusion times. ^∗P<0.05; ^∗∗P<0.01; ^∗∗∗P<0.001. Data shown are representative of three to eight mice in each group, corresponding to at least two independent experiments.

Bone Marrow Cells do not Exhibit a Suppressed Response to Endotoxin after Experimental Stroke

As stroke-induced immunodeficiency is a known cause of serious bacterial infections in stroke patients and impairs outcome (Dirnagl et al, 2007; Prass et al, 2003), we investigated whether this response can manifest in the bone marrow. Total bone marrow cells isolated at 72 hours reperfusion expressed and released high levels of interleukin-6 after stimulation with lipopolysaccharide in vitro (Figures 6A and 6B). CXCL1 was also induced, but was not released from the cells during the 3-hour incubation period (Figures 6C and 6D). However, no difference between stroke and sham surgery was observed (Figures 6A to 6D), indicating that bone marrow cells after stroke retain their ability to respond to stimulation by bacterial cell-wall compounds. The chronic effect of stroke on lymphopaenia in the blood, thymus, and spleen, and impaired cytokine expression by blood cells in response to lipopolysaccharide or mitogens, are apparent 12 to 48 hours after stroke in mice (Prass et al, 2003).

Figure 6

Bone marrow cell response to endotoxin stimulation after experimental stroke. Seventy-two hours after stroke or sham surgery, total bone marrow cells were isolated and stimulated with LPS in vitro. (A) IL-6 and (C) CXCL1 became upregulated in cell lysates after 3 hours stimulation, but no difference was observed between cells isolated from sham or stroke animals. (B) High levels of IL-6 and (D) low levels of CXCL1 were released into the medium in response to LPS, but no difference in cytokine levels was observed between sham and stroke surgery. ^∗P<0.05; ^∗∗∗P<0.001. Data are representative of two independent experiments. IL-6, interleukin-6; LPS, lipopolysaccharide; NS, not significant.

Lateralisation of the Bone Marrow in Response to Experimental Stroke

Preautonomic neural efferents in the brain stem control the bone marrow (Denes et al, 2005), and two-thirds of the projections originating from the ventrolateral medulla are considered ipsilateral (Moon et al, 2002). Hence, we investigated whether stroke could induce laterality in the brain or in the bone marrow. In the nucleus ambiguus and pre-Bötzinger complex, the adrenal medulla and in the dorsal vagal nucleus (Figures 7A and 7B), obvious ipsilateral–contralateral differences were observed in STAT3 phosphorylation, which was present in most animals up to 48 to 72 hours reperfusion. These medullary neurones are involved in the autonomic regulation of various organs, including respiratory and cardiovascular processes (Doi and Ramirez, 2010; Elenkov et al, 2000). Consequently, 30% to 40% of mice after stroke, but not sham surgery, exhibited marked laterality when comparing the left and right femoral/tibial bone marrow (Figure 7C). The average difference in the ratio of Gr-1^high granulocytes between the left and right bones after stroke was significantly greater than that of sham animals (18% versus 5%, respectively, P<0.05, Figure 7D), whereas B-cell proportions were not different (Figure 7E). Laterality in naive animals did not exceed 2% on average for all cell populations examined (data not shown). No obvious ipsilateral or contralateral dominance was observed in any leukocyte populations in the bone marrow after stroke, indicating that although autonomic projections are likely involved in the regulation of the bone marrow, their lateralisation may be more variable at the individual level than in the case of somatomotor projections.

Figure 7

Experimental stroke resulted in lateralisation in the autonomic brainstem nuclei and in the bone marrow. (A) Marked differences were observed in STAT3 phosphorylation (pSTAT3) in the adrenal medulla, the nucleus ambiguus, and pre-Bötzinger complex between the ipsilateral and contralateral medulla (bottom inserts, arrowhead) at 48 hours reperfusion. Neurokinin receptor-1 (NK1) expression is shown on the corresponding area of parallel sections (top inserts). (B) Laterality was seen in the nucleus tractus solitarius/dorsal motor nucleus of the vagus (inserts, arrows). (C) Overall, 30% of animals after MCAo exhibited marked lateralisation in the bone marrow when comparing left and right femurs/tibia. Gr-1^high granulocytes and CD19-positive B cells are shown. (D) The average difference in Gr-1^high granulocyte proportions between the left and right bones was significantly greater after stroke than sham surgery (P<0.05, two-way ANOVA), (E) whereas laterality was not different for B cells. Scale bars: panel A, 250 μm; panel B, 100 μm. ANOVA, analysis of variance; MCAo, middle cerebral artery occlusion; STAT3, signal transducer and activator of transcription 3.

Discussion

We show in this study that the bone marrow responds rapidly to experimental stroke, which involves changes in various leukocyte populations and inflammatory mediators in concert with the development of the stroke-induced systemic response. These findings have implications for at least three different, but largely overlapping research areas. First, the role of systemic inflammatory changes in stroke is increasingly recognised; our data identify key targets for intervention and show that inflammatory leukocyte responses after stroke are likely to be affected directly by changes in the bone marrow microenvironment. Second, our results may serve as important indications for clinical and experimental stem-cell therapy approaches, which largely rely on the transplantation of bone marrow-derived cells after ischaemia, without fully understanding how the bone marrow itself is affected by the ischaemic challenge. Third, the influence of anaesthetic and surgical stress on the bone marrow indicates that experimental stroke studies need to consider these effects in a translational context.

Myeloid cells in the bone marrow responded rapidly (10 minutes to 4 hours after reperfusion) to experimental stroke. We observed p38 MAPK phosphorylation in Gr-1^int SSC^int cells, which represent a diverse population of immature and mature myeloid cells, including a subset of monocytes. p38 MAPKs are involved in the regulation of various haematopoietic processes, including granulocyte differentiation and respond to a wide range extracellular cues particularly cellular stressors such as ultraviolet radiation, osmotic shock, hypoxia, proinflammatory cytokines, and less often growth factors (Geest and Coffer, 2009; Raman et al, 2007). Activation of the NFκB pathway in neutrophils occurs in response to various stimuli and is involved in the production of inflammatory cytokines (McDonald et al, 1997; McDonald and Cassatella, 1997; Vancurova et al, 2001). Nuclear factor-κB signalling has a key role in granulopoiesis and granulocyte release; mice with I-kappa-B (IκB) deficiency (which leads to constitutive NFκB activation) exhibit granulocytosis and blood neutrophilia (Beg et al, 1995). CXCL1 (KC) production and NFκB activation are involved in neutrophil recruitment to the ischaemic tissue in various organs (Chandrasekar et al, 2001; Ishibashi et al, 1999; Souza et al, 2005). CXCR2 is not only a receptor for CXCL1 but also has a crucial role in neutrophil release from the bone marrow. Defective regulation of CXCR2 facilitates neutrophil release from the bone marrow, whereas CXCR2^−/− neutrophils are retained in the bone marrow (Eash et al, 2010; von Vietinghoff et al, 2010). Our data indicate that Gr-1-positive cells become rapidly activated in response to stroke in the bone marrow, which causes the release of the CXCR2-positive myeloid population within 4 hours reperfusion. It has been shown that CXCL1 and G-CSF exert coordinated actions on neutrophil mobilisation from the bone marrow (Wengner et al, 2008). Although in our study CXCL1 was upregulated in bone marrow-derived neutrophils/monocytes after both sham and stroke surgery, our results indicate a more pronounced release of CXCL1 (at 4 hours reperfusion) and G-CSF (at 10 minutes reperfusion) into the circulation in response to stroke, which correlates with increased stroke-induced cell mobilisation from the bone marrow. Granulocyte-colony-stimulating factor can induce the expression of CXCL2 (another CXCR2 agonist) in bone marrow endothelial cells; therefore, CXCR2-positive cells may be activated synergistically by CXCL1 and CXCL2 (Eash et al, 2010). In contrast to the reduction of CXCR2-positive cells in the bone marrow, we found a transient increase in CCR5 after 4 hours reperfusion. Among several cell types, CCR5 is expressed on murine neutrophils and monocytes, and the absence of CCR5 results in deficient neutrophil migration responses after ischaemia (Reichel et al, 2006). Therefore, our data indicate that experimental stroke may not only exert differential effects on bone marrow myeloid/lymphoid cell populations but is also likely to have differential effects on specific chemokine receptors, which determine leukocyte mobilisation and recruitment.

Neutrophil-derived MMP-9 is involved in the mobilisation of bone marrow cells by G-CSF and CXCR2 ligands in mice (Pelus et al, 2004). Production of MMP-9 by activated human monocytes also involves NFκB activation (Lu and Wahl, 2005). We did not investigate whether proMMP-9 is directly involved in stroke-induced cell mobilisation, but our results indicate that changes in proMMP-9 after MCAo in the bone marrow largely correspond to the release of source cells into the circulation. It is noteworthy that sham surgery alone induced CXCL1 and proMMP-9 in neutrophils between 10 minutes and 4 hours reperfusion. Furthermore, surgical stress and anaesthesia exerted profound effects on both early (4 hours) and late (24 to 72 hours) cellular responses in the bone marrow. Lymphocytes seemed to respond to isoflurane with early (within 4 hours) recruitment into the bone marrow, whereas surgical stress caused a marked increase in bone marrow myeloid cells 72 hours after surgery. Volatile anaesthetics reportedly affect leukocyte responses; e.g., they alter neutrophil adherence to blood vessels (Heindl et al, 1999), induce apoptosis of lymphocytes (Matsuoka et al, 2001), or affect the resolution of inflammation (Chiang et al, 2008). They also contribute to ischaemic brain injury (Lee et al, 2007; Li and Zuo, 2009), but our results are the first to show that both surgery and anaesthesia interact with experimental stroke to cause cellular and cytokine changes directly in the bone marrow. Therefore, these data may have important implications for experimental stroke modelling.

The importance of stroke-induced increases in T cells and natural killer cells in the bone marrow between 24 to 72 hours reperfusion is unclear. Bone marrow T cells have been recently shown to maintain and modulate haematopoiesis. T cell-deficient mice have impaired haematopoietic activity and reduced granulocyte numbers, which can be reversed by intravenous injection of T cells (Monteiro et al, 2005). Further studies are required to determine whether T cells influence granulocyte responses in the bone marrow after stroke.

Brain damage leads to suppression of host immune responses in experimental animals and humans, which then enables opportunistic pathogens to develop serious posttraumatic infections (Dirnagl et al, 2007; Meisel et al, 2004; Prass et al, 2003). We found no evidence of suppressed bone marrow cell responses to bacterial endotoxin, which indicates that myeloid cells in the bone marrow likely retain their ability to respond to infectious stimuli. This is also supported by the fact that although bone marrow cells were less abundant in animals that had undergone stroke surgery compared with sham surgery alone, cellular responses in the bone marrow were largely determined by surgical stress at 72 hours reperfusion as compared with naive animals. Furthermore, T-cell numbers were found to increase in the bone marrow after stroke, and not decrease, as occurs in the case of the spleen after cerebral ischaemia (Offner et al, 2006b).

Lateralisation of the autonomic nervous system in the brain is one of the proposed causes of stroke-induced immunodepression (Dirnagl et al, 2007). Laterality, seen after MCAo in our experiments, indicates that direct neuronal mechanisms are likely to be involved in the activation and/or mobilisation of leukocytes from the bone marrow. This is supported by published findings. Substance P, a neuropeptide released from sensory nerves in the bone marrow controlling haematopoietic cell maturation and release (Broome and Miyan, 2000; Rameshwar et al, 1993) induces NFκB-dependent chemotaxis of neutrophils by upregulation of CXCR2 (Sun et al, 2007). The key role of the autonomic nervous system and bone marrow nerves in haematopoiesis and cell mobilisation has also been confirmed in elegant studies (Busik et al, 2009; Mendez-Ferrer et al, 2008). Further studies are required to assess the role of the autonomic nervous system in bone marrow cell mobilisation after stroke. However, these studies need to consider the difficulty in undertaking specific interventions targeting the bone marrow without affecting the ischaemic brain injury. Our data indicate that neuronal processes are likely to be involved in bone marrow cell mobilisation after experimental stroke, but their impact on immunosuppression may be less than that in other lymphoid organs, such as the spleen.

In conclusion, our results reveal previously unrecognised responses of the bone marrow to experimental stroke, which are likely to affect outcome. Our data also indicate the need to reconsider the relevance of experimental studies on stroke, given the major impacts of anaesthesia and sham surgery.

Footnotes

NJR is a nonexecutive director of AstraZenca, but this has no relation to the current research.

Notes

Supplementary Information accompanies the paper on the Journal of Cerebral Blood Flow & Metabolism website ()

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