Interleukin-1beta and tumor necrosis factor-alpha are expressed by different subsets of microglia and macrophages after ischemic stroke in mice
- Bettina H Clausen†1,
- Kate L Lambertsen†1Email author,
- Alicia A Babcock1,
- Thomas H Holm1,
- Frederik Dagnaes-Hansen2 and
- Bente Finsen1
© Clausen et al; licensee BioMed Central Ltd. 2008
Received: 17 May 2008
Accepted: 23 October 2008
Published: 23 October 2008
Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) are expressed by microglia and infiltrating macrophages following ischemic stroke. Whereas IL-1β is primarily neurotoxic in ischemic stroke, TNF-α may have neurotoxic and/or neuroprotective effects. We investigated whether IL-1β and TNF-α are synthesized by overlapping or segregated populations of cells after ischemic stroke in mice.
We used flow cytometry and immunohistochemistry to examine cellular co-expression of IL-1β and TNF-α at 6, 12 and 24 hours after permanent middle cerebral artery occlusion in mice, validating the results by the use of bone marrow chimeric mice.
We found that IL-1β and TNF-α were expressed in largely segregated populations of CD11b+CD45dim microglia and CD11b+CD45high macrophages, with cells expressing both cytokines only rarely. The number of Gr1+ granulocytes producing IL-1β or TNF-α was very low, and we observed no IL-1β- or TNF-α-expressing T cells or astrocytes.
Taken together, the results show that IL-1β and TNF-α are produced by largely segregated populations of microglia and macrophages after ischemic stroke in mice. Our findings provide evidence of a functional diversity among different subsets of microglia and macrophages that is potentially relevant to future design of anti-inflammatory therapies in stroke.
The proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) play key roles in the pathogenesis of ischemic stroke [1–3]. IL-1β exerts neurotoxic effects in ischemic stroke and blocking its action has been shown to reduce ischemic brain damage [4, 5]. In comparison, there is evidence that TNF-α has both neurotoxic [6, 7] and neuroprotective [8–10] roles after ischemic stroke in rats and in mice. Increasing evidence implicates both cytokines in the early inflammatory response that precedes and accompanies ischemia-induced neuronal damage [6, 11]. However, detailed knowledge about the contribution of different cell types to the production of IL-1β and TNF-α is still not available.
The relative physiological outcome of increased IL-1β and TNF-α signaling in ischemic stroke may depend on the kinetics and location of cytokine producing cells. There is compelling evidence that IL-1β and TNF-α are primarily synthesized by activated microglia and infiltrating macrophages [12–14], although granulocytes and astrocytes have also been suggested to produce both IL-1β [15–17] and TNF-α [18, 19]. Precise identification of cell source has, however, been compromised by the lack of microglial and macrophage specific markers, which prevents discrimination of these cell types at the histological level [12, 14]. Furthermore, it is presently unknown whether IL-1β and TNF-α are expressed to the same extent by the same or different subsets of microglia and macrophages following ischemic stroke.
We have previously shown that IL-1β mRNA and TNF-α mRNA, and TNF-α protein are produced by CD11b+ microglia and by CD11b+ macrophages at the edge of and within areas of infarction, and that this production reaches maximum levels of expression between 12 and 24 hours after permanent middle cerebral artery occlusion (pMCAO) in mice [12, 14, 20]. The objective of the present study was to provide additional insight into the cell types and cell subpopulations that produce IL-1β and TNF-α within the first 24 hours following ischemic stroke in mice . To distinguish microglia from infiltrating macrophages after pMCAO, we used flow cytometry with CD45 and CD11b as myeloid-lineage specific markers, we used a radiated, bone marrow (BM) chimeric mouse model; and we used intracellular cytokine-staining, and double immunofluorescence staining. In addition, since CD11b is expressed by both macrophages and granulocytes [21, 22], we also analyzed cytokine production by granulocytes using the granulocyte specific marker Gr1. Our results show that IL-1β and TNF-α are produced by largely segregated subsets of microglia and macrophages, and that very few cells express both cytokines.
Breeding pairs of CD45.1+ (B6.SJL-Ptprca Pepcb/BoyJ) mice and CD45.2+ C57BL/6-Tg(UBC-GFP)30Scha/J (GFP-Tg)  mice were purchased from the Jackson Laboratory (Bar Harbour, Maine, USA) and transferred to the Department of Medical Microbiology and Immunology, University of Aarhus, where they were maintained as a colony. GFP-Tg mice, which express the CD45.2+ allotype, were used as BM donors and congenic BoyJ male mice, which express the CD45.1+ allotype, were used as BM recipients. This combination of mice was chosen so that infiltrating cells could be identified by two different markers (GFP+ and CD45.2+), however the GFP signal alone proved to be sufficiently strong and reliable  to identify infiltrating cells. Peritoneal macrophages were obtained from C57BL/6 mice, which were purchased from Taconic (Ry, Denmark). Mice were housed under diurnal lightning conditions with free access to food and water. The experiments were approved by the Danish Animal Inspectorate (J. no. 2005/561–1068).
Generation of bone marrow chimeras
BM cells from GFP-Tg mice were grafted into lethally irradiated mice as described by Wirenfeldt et al. . For donor BM recovery, the proximal and distal ends of tibia and femur were removed and BM was flushed from the medullary channel into sterile 50 mL polypropylene tubes using cold RPMI 1640 medium (Gibco, Paisley, UK). The BM cells were rinsed in RPMI medium and centrifuged at 1,000 rpm for 10 min, then filtered through a sterile cotton sieve. After 3 rounds of rinsing and filtering, cells were kept on ice until use. Recipient mice (6–8 weeks of age) were lethally irradiated with a single dose of 9.5 Gy from a 137Cs source (Risø National Laboratory, Roskilde, Denmark). Within 2 hours of irradiation, BM cells were injected into the tail veins of recipient mice (approximately 107 cells/mouse). For 3 days after grafting, mice received oxytetracycline (2 g/L Terramycin vet. 20%; Pfizer, Amoise, France) in autoclaved drinking water. Thereafter they were supplied with acidified water (pH 3). The mice were fed irradiation-sterilized mouse chow and maintained under pathogen-free conditions for 6 weeks until they were subjected to surgery.
Permanent middle cerebral artery occlusion
Under anaesthesia, mice were subjected to focal cerebral ischemia by permanent occlusion of the distal part of the left middle cerebral artery (pMCAO), as previously described . During surgery, mice were placed on a 37°C ± 0.5°C warm heating pad. An incision was made from eye to ear, the parotid gland and the upper part of the temporal muscle were split, and a small hole was drilled over the distal part of the MCA. The MCA was occluded by electrocoagulation and the incision was stitched with a 4.0 nylon-suture. After surgery, mice were injected with 1 ml of isotonic saline and their eyes were coated with ointment. Mice were kept at 28°C until sacrificed, 24 hours post-surgery. For post-surgical analgesia mice were treated with Temgesic (0.001 mg/20 g buprenorphinum, Reckitt & Colman, Hull, UK) three times at 8-hour intervals starting immediately after surgery .
Mice were anesthetized i.p. with an overdose of pentobarbital and perfused through the left ventricle using 10 ml of Soerensen's phosphate buffer (SB) (0.03 M KH2PO4 and 0.12 M Na2HPO4, pH 7.4) followed by 20 ml of cold 4% paraformaldehyde (PFA) in 0.15 M SB, pH 7.4. The brains were post-fixed in 4% PFA for 1 hour followed by immersion in 20% sucrose in 0.15 M SB overnight. Next, the brains were frozen and cut into 16-μm frontal sections. For verification of chimerism in BM-chimeric mice, samples of spleen, heart, kidney and liver were frozen and processed to obtain 16-μm cryostat sections.
Cell culture preparation
Thioglycolate-elicited peritoneal macrophages were obtained after i.p. injection of 1 ml 3% thioglycolate (Sigma) into C57BL/6 mice. Three days after injection, the mice were killed by cervical dislocation and 10 ml Dulbecco's modified Eagle Medium (Invitrogen) was injected into the peritoneum. Subsequently, the peritoneal fluid was withdrawn. After two washes in phosphate buffered saline (PBS, pH 7.4) the cells were counted and plated in T25 flasks in DMEM Medium containing 5% FBS, 100 units/ml of penicillin, and 100 mg/ml streptomycin (Sigma). The macrophages were kept in a humidified 37°C incubator with 5% CO2 for 2 days and then stimulated for 24 hours with 100 ng/ml LPS (Sigma).
Mice were perfused through the left ventricle using 10 ml PBS, whereafter the ipsi- and contralateral cortices were isolated. Infarcts were macroscopically visible at 6, 12 and 24 hours post-occlusion, verifying successful occlusion of the MCA. Blood samples were collected in heparinized Eppendorf tubes and Hanks' balanced salt solution (HBSS: 0.14 M NaCl, 5.4 mM KCl, 0.4 mM MgSO4•7H2O, 0.4 mM Na2HPO4(anhydrate), 1.3 mM CaCl22H2O, 4.2 mM NaHCO3, 0.4 mM KH2PO4, 0.5 mM MgCl26H2O, and 5 mM glucose) for validation of BM reconstitution. Lymph nodes were removed and collected as control tissue.
Immunohistochemistry and cell counting
Immunohistochemistry for CD11b, CD45, IL-1β and TNF-α
Antibodies applied for flow cytometry, immunohistochemistry and fluorescence stainings.
Species of origin
553174, BD Biosciences*
553126, BD Biosciences
TNF producing cells
559503, BD Biosciences
IL-1β producing cells
556923, BD Biosciences
554711, BD Biosciences
558141, BC Biosciences
TNF producing cells
TNF producing cells
IL-1β producing cells
CD45.1 Biotin-conjugated (1:80)
553774, BD Biosciences
CD45.2 Biotin-conjugated (1:80)
553771, BD Biosciences
Ly-6G and Ly-6C(Gr1) (1:200)
01211A, BD Biosciences
von Willebrandt's Factor (1:200)
557355, BD Biosciences
sc-6170, Santa Cruz
553455, BD Biosciences
Alexa Fluor594-conjugated (1:200)
Alexa Fluor546-conjugated (1:200)
Alexa Fluor488-conjugated (1:200)
Alexa Fluor488-conjugated (1:200)
Alexa Fluor568-conjugated (1:200)
Alexa Fluor594-conjugated (1:200)
Alexa Fluor488-conjugated (1:200)
HRP-conjugated Streptavidin (1:200)
Immunohistochemistry for GFP+cells
To detect infiltrating GFP+ cells, sections were rinsed 3 × 15 min in 0.05 M Tris-buffered saline (TBS, pH 7.4), and endogenous peroxidase activity was blocked by rinsing the sections in 30% H2O2, methanol and TBS (1:1:8) for 30 min at room temperature (RT). After rinsing 2 × 10 min in TBS + 0.5% Triton, the sections were incubated with 10% fetal calf serum (FCS) in TBS for 30 min at RT to reduce non-specific staining. Sections were then incubated with rabbit anti-GFP (Table 1, Abcam) diluted 1:1,000 in TBS containing 10% FCS overnight at 4°C. Next, sections were rinsed 3 × 15 min in TBS + 0.5% Triton and incubated with peroxidase-labeled "ready-to-use" EnVision+ polymer (DakoCytomation) overnight at 4°C. Sections were then rinsed 3 × 15 min in TBS and developed for 7 min in TBS containing 0.05% diaminobenzidine and 0.033% H2O2. Finally, sections were rinsed in TBS, dehydrated in a graded series of alcohol, cleared in xylene and mounted in Depex. Antibody specificity was assessed by negative controls using substitution of the primary antibody with rabbit IgG (Table 1, DakoCytomation) or omission of the primary antibody. These sections were devoid of signal.
Double immunofluorescence staining for cell specific markers and IL-1β and/or TNF-α
Sections were air dried, rinsed in TBS for 10 min, and blocked with 10% FCS in TBS + 0.5% Triton for 30 min at RT. Next, the sections were incubated with primary antibody (Table 1) overnight at 4°C, followed by rinsing for 10 min in TBS and incubation with a species-specific secondary fluorescent antibody (Table 1) diluted 1:200 for 1–2 hours the next day. From this point forward, the sections were protected from light. After 10 min in TBS, sections were incubated for 2 hours with the second primary antibody diluted in TBS + 0.5% Triton containing 10% FCS, rinsed in TBS for 10 min at RT, and then incubated with the secondary antibody diluted 1:200 in TBS + 0.5% Triton containing 10% FCS for 1–2 hours at room temperature (RT). Finally, sections were rinsed 2 × 10 min in TBS, followed by 2 × 10 min in distilled H2O, and mounted in ProLong® Gold antifade reagent with DAPI (Invitrogen, Taastrup, Denmark). Negative controls, where primary antibodies were replaced by isotype controls (Table 1) or omitted from the protocol, were devoid of signal.
Estimation of numbers of infiltrating GFP+cells
The number of GFP+ cells in the ipsilateral cortex was estimated using the cell counting method described in Lambertsen et al. . GFP+ cells with an identifiable nucleus were counted in approximately 16 sections from each animal, 384 μm apart. This was done using a 100× objective and a 25% frame area, stepping 300 μm/300 μm in the x-y position using the CAST Grid System from Olympus. The total number (N) of GFP+ cells in each animal was estimated using the formula: Estimate of N = ΣQ × (1/ssf) × (1/asf) × (1/tsf), where Q was the number of cells counted, 1/ssf the sampling section fraction (1/ssf = 24), and 1/asf the area sampling fraction (1/asf = 36.4) . The thickness sampling fraction (1/tsf) was set to 1, since cells were counted in the entire height of the sections .
Preparation of cell suspensions
Tissues from individual mice were processed separately. Single-cell suspensions of ipsi- or contralateral cortices or lymph nodes were obtained by homogenisation using 70-μm nylon cell strainers (BD Falcon, Franklin Lakes, NJ, USA) [24, 27], in RPMI containing 10% FCS and 1 μl/ml BD GolgiPlug™ with Brefeldin A (BD Biosciences). Cells were transferred to sterile petri dishes and placed in a 37°C incubator with 5% CO2 for 4 1/2 hours. Cell suspensions were transferred to polystyrene tubes and pelleted by centrifugation (1,400 rpm for 6 min at 4°C).
Cells were incubated for 30 min in staining-buffer (staining-buffer: Hank's Buffered Salt Solution with 2% FCS and 0.1% sodium azide), containing 50 μg/ml Syrian hamster Ig (Jackson Immunoresearch, West Grove, PA, USA) and 1 μg/ml anti-FcγIII/II receptor (BD FcBlock™, BD Biosciences), to block non-specific staining. After rinsing, cells were stained for surface antigens (or their isotype controls) (Table 1) for 30 min at RT, and then rinsed in staining-buffer [24, 27]. Following staining of surface markers (CD11b, CD45, Gr1, TCRβ), the cells were fixed and permeabilized in Cytofix/Cytoperm™ (BD Biosciences) for 20 min at 4°C, washed in 1 ml 1× PermWash™ buffer (BD Biosciences), and incubated with antibodies recognizing intracellular antigens (IL-1β, TNF-α or isotype controls) (Table 1) for 30 min at RT. Cells stained with unconjugated antibodies were rinsed, then incubated with fluorescently labelled goat anti-rabbit secondary antibodies for 30 min at RT. After the final rinse, cells were resuspended in staining-buffer.
For blood samples, cells were stained using antibodies recognizing CD11b, CD45 and TCRβ (Table 1), as described for brain homogenates. Red blood cells were then lysed using Serotec Erythrolyse Red Blood Cell buffer (Serotec) diluted 1:10 in distilled water for 7 min at RT. Finally, cells were rinsed three times and resuspended in staining-buffer.
Flow cytometric analysis
Staining was analyzed using a FACSCalibur flow cytometer and CellQuest Pro Software (BD Biosciences). Estimation of numbers of cells was done as previously described [24, 27]. Data are presented as an average of these numbers. Preparation and staining of cortical homogenates inevitably resulted in cell loss, and the cell numbers and proportions presented are based on cells remaining in suspension. For analysis of microglia and macrophages/granulocytes, cells were gated on side scatter (SSC) versus CD11b, followed by forward scatter (FSC) versus CD11b, and FSC versus CD45. Microglia were separated from macrophages/granulocytes based on CD45 expression, with intermediate levels of CD45 (CD45dim) identifying CD11b+ parenchymal microglia, and high levels of CD45 (CD45high) identifying CD11b+ macrophage/granulocytes . GFP+, Gr1+, IL-1β+ and TNF-α+ cells were identified on additional colour channels. GFP+ BM-derived cells were separated from GFP- resident cells based on GFP expression. For quantification of IL-1β/TNF-α co-expression, resident microglia (GFP-CD45dim) and infiltrating leukocytes (GFP+CD45high) were gated on SSC versus CD45 followed by FSC versus CD45. For analysis of expression of IL-1β and TNF-α in TCRβ+ T cells, cells were gated on SSC versus CD45 followed by FSC versus CD45. IL-1β+, TNF-α+ and TCRβ+ cells were identified on additional colour channels. Positive staining was determined based on fluorescence levels of isotype controls or autofluorescence controls (for GFP).
Histology results were documented using an Olympus DP70 digital camera mounted on an Olympus BX51 microscope connected to a PC with Olympus DP-software. Figures were organized using Adobe Photoshop CS. The data were evaluated using Kruskal-Wallis test (non-parametric ANOVA) with Dunn's multiple comparison test as follow-up test. Comparisons of mean values between two groups of mice were done using the non-parametric Mann-Whitney test using the GraphPad Prism 4.0b software for Macintosh, GraphPad Software, San Diego, California, USA. Data are presented as means ± SD. Statistically significant differences were established at P < 0.05.
Regional distribution of cells producing IL-1β and TNF-α
Blood-borne, bone marrow-derived cells infiltrate after pMCAO
The distribution of infiltrating GFP+ cells was visualized using immunohistochemistry. Twenty-four hours after pMCAO, GFP+ cells were distributed throughout the infarct, such that the border of the infarct was easily delineated (Figure 2E). The GFP+ cells occurred both as single cells (Figure 2F), and as vessel-associated aggregates (Figure 2G). Microscopic analysis of the perivascular vessel-associated aggregates showed two morphologically different cell types; round cells, which appeared to locate to the juxtavascular space and to infiltrate the neuropil (Figure 2G), and elongated cells lining the microvascular wall (insert in Figure 2G). In order to determine the extent of cellular recruitment, we estimated the total number of GFP+ cells within the ischemic cortex using both morphometry and flow cytometry. By morphometry, the total number of single GFP+ cells was estimated to be 130,000 ± 24,000 (mean ± SD) cells (Figure 2H). The number of vessel-associated aggregates of GFP+ cells were quantified separately (8,000 ± 2,200) (Figure 2H). Only a few GFP+ single cells were present in the cortex of the contralateral hemisphere (Figure 2H), or in brains from unmanipulated BM-chimeric mice. GFP+ cells were identified in sections from all organs from BM-chimeric mice (data not shown). Using flow cytometry, we estimated the total number of GFP+ cells within the isolated cortex to be 42,655 ± 23,465 (mean ± SD) cells (Figure 2C), which corresponded to 33% of the number of GFP+ cells estimated by morphometry (Figure 2H).
Kinetics of the microglial and macrophage/granulocyte response
Finally, since microglia become activated by stroke [14, 20], and evidence shows that CD45 is inducible in microglia , we investigated whether cellular levels of CD45 expression were affected by the ischemic insult. For this, we analysed mean fluorescence intensity (MFI) of the CD45 signal on CD11b+CD45dim microglia and CD11b+CD45high macrophages/granulocytes. MFI values were obtained for unmanipulated controls, in mice with 6-, 12-, and 24-hour survivals, and in sham-operated mice at 24 hours. We observed a significant upregulation of CD45 on microglia by 12 hours, which reached peak levels 24 hours after pMCAO compared to unmanipulated controls and sham-operated mice (Figure 4G). Similarly, CD45 levels were increased on infiltrating macrophages/granulocytes by 24 hours compared to unmanipulated control and sham-operated mice (Figure 4H).
IL-1β and TNF-α are expressed by largely non-overlapping subsets of cells
The time profile for TNF-α-expressing CD11b+CD45dim microglia showed a significant increase in numbers of TNF-α+ cells at 12 hours, which peaked 24 hours after pMCAO compared to unmanipulated control mice. Similarly there was a injury-induced increase in numbers of CD11b+CD45high macrophages/granulocytes that expressed TNF-α at 24 hours after pMCAO (Figure 5F). Calculation of the proportion of TNF-α-expressing CD11b+CD45dim microglia and CD11b+CD45high macrophages/granulocytes showed that the proportion of TNF-α+ microglia peaked with 2% of the CD11b+CD45high population at 12–24 hours after pMCAO, while the proportion of TNF-α+ macrophages/granulocytes was 17% of the CD11b+CD45high population in unmanipulated controls and 4% at 24 hours after pMCAO. This most of all reflected that a relatively large proportion of the very few CD11b+CD45high cells present in the unmanipulated mouse cortex expressed TNF-α (Figure 5G). Generation of MFI values for TNF-α showed a significant increase in the amount of TNF-α expressed by microglia by 12 and 24 hours compared to unmanipulated control mice. In contrast, the level of TNF-α expression by CD11b+CD45high macrophages/granulocytes seemed unaffected by ischemic injury (Figure 5H).
We additionally identified a subpopulation of CD11b+CD45dim microglia and CD11b+CD45high macrophages/granulocytes that co-expressed IL-1β and TNF-α (Figure 5I). The proportion of IL-1β/TNF-α co-expressing microglia increased from 0.05% in unmanipulated controls to 1.2% after 24 hours of pMCAO, whereas co-expressing macrophages doubled from 2% in unmanipulated controls to 4% after 12 hours of pMCAO, before declining to 2% after 24 hours (Figure 5J).
Interestingly, the proportions of IL-1β-, TNF-α-, and IL-1β/TNF-α-expressing macrophages/granulocytes were relatively high in sham-operated mice (Figure 5D, G, J). However, since the number of CD11b+CD45dim microglia and CD11b+CD45high macrophages/granulocytes did not differ between unmanipulated controls and sham-operated mice (Figure 4E), the overall number of cytokine expressing cells was much lower in sham-operated versus pMCAO operated mice (Figure 5C, F, I).
Taken together, these results clearly show that largely non-overlapping subsets of microglia and macrophages/granulocytes produce IL-1β and TNF-α after pMCAO in mice.
Finally, since both macrophages and granulocytes express CD11b and high levels of CD45 , we used Gr1 to determine which cell population was the predominant CD45high source of IL-1β and TNF-α. We found a small but significant increase in both IL-1β- and TNF-α expressing CD45highGr1+ granulocytes at 24 hours after pMCAO compared to unmanipulated control mice. However, the CD45highGr1- macrophages were clearly the major producers of both IL-1β and TNF-α (Figure 5K). A small number of CD45high cells identified as T cells (TCRβ+CD45high) was also observed. The number of T cells in ischemic cortex by 6, 12 and 24 hours did not differ from the number in cortex of unmanipulated or sham-operated mice (< 200), and none of the TCRβ+CD45+ T cells were observed to express either IL-1β or TNF-α (data not shown).
Comparison of cytokine expression in LPS-stimulated and ischemia-activated macrophages
Expression of IL-1β and TNF-α by Gr1-CD11b+microglia and macrophages
We have shown for the first time that IL-1β and TNF-α are produced by largely non-overlapping subsets of microglia and macrophages after induction of ischemic stroke in mice. Using flow cytometry and histology, validated by the use of BM-chimeric mice, we showed that microglia and macrophages are the major producers of IL-1β and TNF-α after pMCAO, and that at maximum 1.2% of microglia and 4.5% of infiltrating macrophages co-express IL-1β and TNF-α within the first 24 hours after pMCAO. Granulocytes, which accounted for 50% of the CD11b+CD45high cell population and which are known to exacerbate ischemic brain damage , accounted for only 2.4% of IL-1β and 0.8% of TNF-α expression by CD11b+CD45high cells after 24 hours of pMCAO, indicating that the contribution of these cells to the production of IL-1β and TNF-α after pMCAO in mice is negligible. A scenario therefore emerges wherein different subsets of microglia and macrophages may have different roles in ischemic stroke, and may thus either improve or reduce the chance of survival of ischemic neurons.
Our observation of a peak in the total number of IL-1β- and TNF-α-expressing cells 24 hours after pMCAO is in line with previous demonstrations of a time-dependent peak in the number of IL-1β mRNA-, TNF-α mRNA-, and TNF-α protein-expressing cells in SJL and C57BL/6 mice, 12–24 hours after pMCAO [12, 14, 25]. By taking advantage of the ability of flow cytometry to distinguish between CD11b+CD45dim microglia and infiltrating CD11b+CD45high macrophages, we showed that the number of IL-1β- and TNF-α-expressing microglia by far exceeded the number of cytokine-expressing macrophages 12 hours after pMCAO. This result was expected based on the known steady increase in the number of infiltrating macrophages over the first 24 hours of pMCAO, but this result has not been previously demonstrated.
It was striking to observe that a relatively large proportion of the small number of macrophages present in the cortex from unmanipulated mice express TNF-α, and that the MFI levels of TNF-α expression in these cells are comparable to mean cellular TNF-α expression levels in pMCAO and sham-operated mice. This indicates that TNF-α expression by macrophages is relatively constant no matter whether the cells have infiltrated the cortex prior to or after pMCAO. Interestingly, the proportion of cytokine-expressing macrophages in sham-operated mice was at least as high as that in mice subjected to pMCAO. This likely reflects that sham surgery in itself induces a focal lesion in the cortex [12, 14]. It is important to note, however, that overall numbers of cytokine-expressing macrophages are not increased versus control mice, since there was no significant recruitment of CD11b+CD45high cells to the cortex of sham-operated mice.
Permanent MCAO results in formation of a pan-necrotic infarct, with loss of all cell types including microglial cells. Although neurons and microglia can still be clearly detected after 6 hours of pMCAO [12, 14], the developing infarct is usually characterised by severe cell loss 12 hours after pMCAO [12, 14, 20] We were therefore surprised not to observe a reduction in the number of CD11b+CD45dim microglia 12 and 24 hours after pMCAO. We wondered whether determination of upregulation of CD45, which is widely used to detect both resting and activated microglia in flow cytometry [24, 27–29, 31] and histology [32, 33], might lead to increased detection. Indeed, MFI analysis showed that CD45 levels are upregulated in microglia 12–24 hours after pMCAO. However, microglial expression of CD45 was far below that expressed by macrophages/granulocytes, confirming results by others . Using a bone marrow chimeric approach, we observed that approximately 7% of the CD45dim microglia were GFP+ after 24 hours of pMCAO, suggesting that BM-derived microglial precursors could also contribute to the expansion of the microglial population after ischemic stroke in non-chimeric mice. Infiltration of GFP+ cells was specific to the infarcted cortex, since no increase was observed in the contralateral hemisphere of BM-chimeric mice or in unmanipulated BM-chimeric mice. Other factors contributing to the expansion of the microglial population 12 and 24 hours after pMCAO might be immigration of microglia from regions of the brain not included in the preparation used for flow cytometry, or microglial proliferation. However, microglial proliferation is not prominent at 12 and 24 hours, but is first evident at 48 and 72 hours after induction of focal cerebral ischemia , a finding that is similar to observations in other models of acute neural injury [32, 36, 37].
The use of a GFP BM-chimeric approach in our study of pMCAO served two purposes: 1) direct visualization of infiltrating GFP+ cells in tissue sections and 2) validation of the BM origin of the CD11b+CD45high macrophages/granulocytes identified by flow cytometry in our model. In addition to the massive infiltration of GFP+ macrophages/granulocytes 24 hours after pMCAO, we found that a small proportion of microglia in the infarcted cortex could be classified as GFP+ CD11b+CD45dim microglial cells. These observations confirm findings by others [38–42], showing a lesion-induced recruitment of microglial progenitors of bone marrow origin into the infarcted cortex. Recently it has been suggested that these microglial progenitors would not enter into the bloodstream or cross the blood-brain barrier under normal physiological conditions [43, 44]. However, BM-derived cells have been reported to infiltrate non-irradiated normal brain [45–47], and CD45high leukocytes are routinely detected in unmanipulated, perfused brains by flow cytometry [24, 27]. Although focal cerebral ischemia disrupts the blood brain barrier [33, 39], and irradiation in itself preconditions the brain for cells to infiltrate the neuropil [43, 44], we in line with earlier findings  observed only sporadic GFP+ cells in contralateral, non-ischemic cortex and in the brains of unmanipulated BM-chimeric mice. Furthermore, numbers of CD11b+CD45high macrophages/granulocytes recruited to ischemic cortex 24 hours after pMCAO were not different between BM-chimeric mice and non-irradiated mice. This suggests that any damage induced by irradiation alone was insufficient to trigger excessive entry of BM-derived cells into the CNS.
Our observation of a significant reduction in microglial population in the cortex of unmanipulated BM-chimeric mice compared to unmanipulated non-chimeric mice indicates that irradiation might impair microglial turnover in normal brain. This is supported by observations by Wirenfeldt et al. , who reported that microglial numbers were reduced by approximately 30% in unmanipulated contralateral hippocampi of perforant pathway-lesioned BM-chimeric mice. That study also reported a lesion-induced impairment of the mitotic capacity of microglia in BM-chimeric mice . Taken together, we ascribe the reduced microglial numbers in BM-chimeric mice to irradiation damage.
The quantification of BM-derived GFP+ cells was done by use of an approximated stereological approach, and by use of flow cytometry. The results show that approximately 2/3 of GFP+ cells are lost during tissue processing procedures prior to flow cytometric analysis. Since GFP+ cells located in perivascular aggregates were not included in the approximated stereological analysis, cell loss using flow cytometry might be larger than 2/3. However, the consistency in the data shows that flow cytometry is a reliable and robust tool with which to obtain quantitative data on microglia and infiltrating macrophages/granulocytes in brain pathology, as also has been shown in previous studies [24, 27]. Observations made by others using BM-chimeric mice have shown that BM cells have the capacity to differentiate into microglia and perivascular cells [24, 39, 48], as well as non-myeloid cell types such as endothelial cells, pericytes, astrocytes and neurons [38, 42, 47, 49]. However, in our study of mice with 24-hour survival after pMCAO, we observed no evidence of co-expression of GFP with the astroglial marker GFAP, confirming earlier studies [38, 49]. Similarly, we find no clear evidence of co-expression of GFP and vWF/CD31, although such co-expression has been previously reported at later times of observation (3 days – 1 month) after induction of ischemia [38, 50].
We show that IL-1β and TNF-α are produced by largely non-overlapping subsets of microglia and macrophages after pMCAO in mice. This observation is indicative of activation of distinct signalling pathways in different subpopulations of microglia and macrophages after ischemic stroke. Resolution of these pathways may further the development of cytokine-based therapies in stroke.
List of abbreviations
fetal calf serum
glial fibrillary acidic protein
green fluorescent protein
Hank's balanced salt solution
middle cerebral artery
phosphate buffered saline
platelet endothelial cell adhesion molecule-1
permanent middle cerebral artery occlusion
Soerensens phosphate buffer
tris buffered saline
tumor necrosis factor
von Willebrand factor
This study received financial support from Hørslev Fonden, Legat til yngre kvindelige forskere på Sundhedsvidenskab, Fonden til Lægevidenskabens Fremme, Familien Hede Nielsens Fond, Kong Christian d. 10 Fond, Frimodt-Heineke Fonden, Direktør Kurt Bønnelycke og hustru Grethe Bønnelyckes Fond, Mogens Svarre Mogensens Fond, Carla Cornelia Storch Møllers legat, Else Poulsens Mindelegat (B. Clausen), Augustinusfonden, Beckett Fonden, Aase og Ejnar Danielsens Fond, Overlægerådets Legatudvalg, Fhv. Dir. Leo Nielsen og Hustru Karen Magrethe Nielsens Legat for Lægevidenskabelig Grundforskning, Fonden til Lægevidenskabens Fremme, Tømrermester Alfred Andersen og Hustru's Fond, Harboefonden and the Danish MRC (K.L. Lambertsen). The technical assistance provided by Lene Jørgensen, Inger-Margrethe Rasmussen, Sussanne Petersen, Inger Nissen and Inger Kathrine Andersen, University of Southern Denmark is greatly appreciated. The assistance with animal care by Aarhus University and the Laboratory of Biomedicine, University of Southern Denmark is also greatly appreciated
- Boutin H, LeFeuvre RA, Horai R, Asano M, Iwakura Y, Rothwell NJ: Role of IL-1α and IL-1β in ischemic brain damage. J Neurosci. 2001, 21: 5528-5534.PubMedGoogle Scholar
- Hallenbeck JM: The many faces of tumor necrosis factor in stroke. Nat Med. 2002, 8: 1363-1368. 10.1038/nm1202-1363.View ArticlePubMedGoogle Scholar
- Schielke GP, Yang GY, Shivers BD, Betz AL: Reduced ischemic brain injury in interleukin-1β converting enzyme-deficient mice. J Cereb Blood Flow Metab. 1998, 18: 180-185. 10.1097/00004647-199802000-00009.View ArticlePubMedGoogle Scholar
- Loddick SA, Rothwell NJ: Neuroprotective effects of human recombinant interleukin-1 receptor antagonist in focal cerebral ischaemia in the rat. J Cereb Blood Flow Metab. 1996, 16: 932-940. 10.1097/00004647-199609000-00017.View ArticlePubMedGoogle Scholar
- Mulcahy NJ, Ross J, Rothwell NJ, Loddick SA: Delayed administration of interleukin-1 receptor antagonist protects against transient cerebral ischemia in the rat. Br J Pharmacol. 2003, 140: 471-476. 10.1038/sj.bjp.0705462.PubMed CentralView ArticlePubMedGoogle Scholar
- Barone FC, Arvin B, White RF, Miller A, Webb CL, Willette RN, Lysko RG, Feuerstein GZ: Tumor necrosis factor-alpha. A mediator of focal ischemic brain injury. Stroke. 1997, 28: 1233-1244.View ArticlePubMedGoogle Scholar
- Dawson DA, Martin D, Hallenbeck JM: Inhibition of tumor necrosis factor-alpha reduces focal cerebral ischemic injury in the spontaneously hypertensive rat. Neurosci Lett. 1996, 18: 41-44. 10.1016/0304-3940(96)13116-5.View ArticleGoogle Scholar
- Bruce AJ, Boling W, Kindy MS, Peschon J, Kraemer PJ, Carpenter MK, Holtsberg FW, Mattson MP: Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat Med. 1996, 2: 788-794. 10.1038/nm0796-788.View ArticlePubMedGoogle Scholar
- Gary DS, Bruce-Keller AJ, Kindy MS, Mattson MP: Ischemic and excitotoxic brain injury is enhanced in mice lacking the p55 tumor necrosis factor receptor. J Cereb Blood Flow Metab. 1998, 18: 1283-1287. 10.1097/00004647-199812000-00001.View ArticlePubMedGoogle Scholar
- Nawashiro H, Tasaki K, Ruetzler CA, Hallenbeck JM: TNF-alpha pretreatment induces protective affects against focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 1997, 17: 483-490. 10.1097/00004647-199705000-00001.View ArticlePubMedGoogle Scholar
- Basu A, Lazovic J, Krady JK, Mauger DT, Rothstein RP, Smith MB, Levison SW: Interleukin-1 and the interleukin-1 type 1 receptor are essential for the progressive neurodegeneration that ensues subsequent to a mild hypoxic/ischemic injury. J Cereb Blood Flow Metab. 2005, 25: 17-29. 10.1038/sj.jcbfm.9600002.View ArticlePubMedGoogle Scholar
- Clausen BH, Lambertsen KL, Meldgaard M, Finsen B: A quantitative histological and RT, rt-PCR analysis of microglial-macrophage expression of IL-1β mRNA following permanent middle cerebral artery occlusion in the mouse. Neurosci. 2005, 132: 879-892. 10.1016/j.neuroscience.2005.01.031.View ArticleGoogle Scholar
- Davies CA, Loddick SA, Toulmond S, Stroemer RP, Hunt J, Rothwell NJ: The progression and topographic distibution of interleukin-1beta expression after permanent middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab. 1999, 19: 87-98. 10.1097/00004647-199901000-00010.View ArticlePubMedGoogle Scholar
- Lambertsen KL, Meldgaard M, Ladeby R, Finsen B: A quantitative study of microglial-macrophage synthesis of tumor necrosis factor during acute and late focal cerebral ischemia in mice. J Cereb Blood Flow Metab. 2005, 25: 119-135. 10.1038/sj.jcbfm.9600014.View ArticlePubMedGoogle Scholar
- Acarin L, Gonzales B, Castellano B: Neuronal, astroglial and microglial cytokine expression after an excitotoxic lesion in the immature rat brain. Eur J Neurosci. 2000, 12: 3505-3520. 10.1046/j.1460-9568.2000.00226.x.View ArticlePubMedGoogle Scholar
- Pearson Vl, Rothwell NJ, Toulmond S: Excitotoxic brain damage in the rat induces interleukin-1β protein in microglia and astrocytes. Glia. 1999, 25: 311-323. 10.1002/(SICI)1098-1136(19990215)25:4<311::AID-GLIA1>3.0.CO;2-E.View ArticlePubMedGoogle Scholar
- Rydström A, Wick MJ: Monocyte recruitment, activation, and function in the gut associated lymphoid tissue during oral salmonella infection. J Immunol. 2007, 178: 5789-5801.View ArticlePubMedGoogle Scholar
- Sairanen TR, Lindsberg PJ, Brenner M, Carpen O, Siren A: Differential cellular expression of tumor necrosis factor-alpha and type 1 tumor necrosis factor receptor after transient global forebrain ischemia. J Neurol Sci. 2001, 186: 87-99. 10.1016/S0022-510X(01)00508-1.View ArticlePubMedGoogle Scholar
- Nguyen HX, O'Barr TJ, Anderson AJ: Polymorphonuclear leukocytes promote neurotoxicity through release of matrix metalloproteinases, reactive oxygen species, and TNF-alpha. J Neurochem. 2007, 102: 900-912. 10.1111/j.1471-4159.2007.04643.x.View ArticlePubMedGoogle Scholar
- Gregersen R, Lambertsen KL, Finsen B: Microglia and macrophages are the major source of tumor necrosis factor in permanent middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab. 2000, 20: 53-65. 10.1097/00004647-200001000-00009.View ArticlePubMedGoogle Scholar
- Campanella M, Sciorati CS, Tarozzo G, Beltramo M: Flow cytometric analysis of inflammatory cells in ischemic rat brain. Stroke. 2002, 33: 586-592. 10.1161/hs0202.103399.View ArticlePubMedGoogle Scholar
- Zehntner SP, Brickman C, Bournonniére L, Remington L, Caruso M, Owens T: Neutrophils that infiltrate the central nervous system regulate T cell responses. J Immunol. 2005, 174: 5124-5131.View ArticlePubMedGoogle Scholar
- Schaefer BC, Schaefer ML, Kappler JW, Marrack P, Kedl RM: Observation of antigen-dependent CD8+ T-cell/dendritic cell interactions in vivo. Cell Immunol. 2001, 214: 110-122. 10.1006/cimm.2001.1895.View ArticlePubMedGoogle Scholar
- Wirenfeldt M, Dissing-Olesen L, Anne Babcock A, Nielsen M, Meldgaard M, Zimmer J, Azcoitia I, Leslie RG, Dagnaes-Hansen F, Finsen B: Population control of resident and immigrant microglia by mitosis and apoptosis. Am J Pathol. 2007, 171: 617-631. 10.2353/ajpath.2007.061044.PubMed CentralView ArticlePubMedGoogle Scholar
- Lambertsen KL, Gregersen R, Finsen B: Microglial-macrophage synthesis of tumor necrosis factor following focal cerebral ischemia in mice is strain dependent. J Cereb Blood Flow Metab. 2002, 22: 785-797. 10.1097/00004647-200207000-00004.View ArticlePubMedGoogle Scholar
- West MJ, Ostergaard K, Andreassen OA, Finsen B: Estimation of the number of somatostatin neurons in the striatum: an in situ hybridization study using the optical fractionator method. J Comp Neurol. 1996, 370: 11-22. 10.1002/(SICI)1096-9861(19960617)370:1<11::AID-CNE2>3.0.CO;2-O.View ArticlePubMedGoogle Scholar
- Babcock AA, Wirenfeldt M, Holm T, Nielsen HH, Dissing-Olesen L, Toft-Hansen H, Millward JM, Landmann R, Rivest S, Finsen B, Owens T: Toll-like receptor 2 signaling in response to brain injury: an innate bridge to neuroinflammation. J Neurosci. 2006, 26: 12826-12837. 10.1523/JNEUROSCI.4937-05.2006.View ArticlePubMedGoogle Scholar
- Sedgwick JD, Schwender S, Imrich H, Dörries R, Butcher GW, ter Meulen V: Isolation and direct characterization of resident microglial cells from the normal and inflamed central nervous system. Proc Natl Acad Sci USA. 1991, 88: 7438-7442. 10.1073/pnas.88.16.7438.PubMed CentralView ArticlePubMedGoogle Scholar
- Carson MJ, Reilly CR, Sutcliffe JG, Lo D: Mature microglia resemble immature antigen-presenting cells. Glia. 1998, 22: 72-85. 10.1002/(SICI)1098-1136(199801)22:1<72::AID-GLIA7>3.0.CO;2-A.View ArticlePubMedGoogle Scholar
- McColl BW, Rothwell NJ, Allen SM: Systemic inflammatory stimulus potentates the acute phase and CXC chemokine responses to experimental stroke and exacerbates brain damage via interleukin-1 and neutrophil-dependent mechanisms. J Neurosci. 2007, 27: 4403-4412. 10.1523/JNEUROSCI.5376-06.2007.View ArticlePubMedGoogle Scholar
- Ford AL, Goodsall AL, Hickey WF, Sedgewick JD: Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. J Immunol. 1995, 154: 4309-4321.PubMedGoogle Scholar
- Ladeby R, Wirenfeldt M, Dalmau I, Gregersen R, Garcia-Ovejero D, Babcock A, Owens T, Finsen B: Proliferating resident microglia express the stem cell antigen CD34 in response to acute neural injury. Glia. 2005, 50: 121-131. 10.1002/glia.20159.View ArticlePubMedGoogle Scholar
- Lehrmann E, Christensen T, Zimmer J, Diemer NH, Finsen B: Microglial and macrophage reactions mark progressive changes and define the penumbra in the rat neocortex and striatum after transient middle cerebral artery occlusion. J Comp Neurol. 1997, 386: 461-476. 10.1002/(SICI)1096-9861(19970929)386:3<461::AID-CNE9>3.0.CO;2-#.View ArticlePubMedGoogle Scholar
- Renno T, Krakowski M, Piccirillo C, Lin JY, Owens T: TNF-alpha expression by resident microglia and infiltrating leukocytes in the central nervous system of mice with experimental allergic encephalomyelitis. J Immunol. 1995, 154: 944-953.PubMedGoogle Scholar
- Denes A, Vidyasagar R, Feng J, Narvainen J, McColl BW, Kauppinen RA, Allen SM: Proliferating resident microglia after focal cerebral ischaemia in mice. Cereb Blood Flow Metab. 2007, 27: 1941-1953. 10.1038/sj.jcbfm.9600495.View ArticleGoogle Scholar
- Dissing-Olesen L, Nielsen HH, Toft-Hansen H, Dalmau I, Finsen B: Axonal lesion-induced microglial proliferation and microglial cluster formation in the mouse. Neurosci. 2007, 149: 112-122. 10.1016/j.neuroscience.2007.06.037.View ArticleGoogle Scholar
- Hailer NP, Grampp A, Nitsch R: Proliferation of microglia and astrocytes in the dentate gyrus following entorhinal cortex lesion: a quantitative bromodeoxyuridine-labelling study. Eur J Neurosci. 1999, 11: 3359-3364. 10.1046/j.1460-9568.1999.00808.x.View ArticlePubMedGoogle Scholar
- Hess DC, Abe T, Holl WD, Studdard AM, Carothers J, Masuya M, Fleming PA, Drake CJ, Ogawa M: Hematopoietic origin of microglial and perivascular cells in brain. Exp Neurol. 2004, 186: 134-144. 10.1016/j.expneurol.2003.11.005.View ArticlePubMedGoogle Scholar
- Priller J, Flügel A, Wehner T, Boentert M, Haas CA, Prinz M, Fernández-Klett F, Prass K, Bechmann I, De Boer BA, Frotscher M, Kreutzberg GW, Persons DA, Dirnagl U: Targeting gene-modified hematopoietic cells to the central nervous system: Use of green fluorescent protein uncovers microglial engraftment. Nat Med. 2001, 7: 1356-1361. 10.1038/nm1201-1356.View ArticlePubMedGoogle Scholar
- Schilling M, Besselmann M, Leonhard C, Mueller M, Ringelstein EB, Kiefer R: Microglia activation precedes and predominates over macrophage infiltration in transient focal cerebral ischemia: a study in green fluorescent protein transgenic bone marrow chimeric mice. Exp Neurol. 2003, 183: 25-33. 10.1016/S0014-4886(03)00082-7.View ArticlePubMedGoogle Scholar
- Tanaka R, Komine-Kobayashi M, Mochizuki H, Yamada M, Furuya T, Migita M, Shimada T, Mizumo Y, Urabe T: Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia. Neurosci. 2003, 117: 531-539. 10.1016/S0306-4522(02)00954-5.View ArticleGoogle Scholar
- Tran-Dihn A, Kubis N, Tomita Y, Karaszewski B, Calando Y, Oudine K, Petite H, Seylaz J, Pinard E: vivo imaging with cellular resolution of bone marrow cells transplanted into the ischemic brain of a mouse. Neuroimage. 2006, 31: 958-967. 10.1016/j.neuroimage.2006.01.019.View ArticleGoogle Scholar
- Mildner A, Schmidt H, Nitsche M, Merkler D, Hanisch UK, Mack M, Heikenwalder M, Brück W, Priller J, Prinz M: Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions. Nat Neurosci. 2007, 10 (12): 1544-1553. 10.1038/nn2015.View ArticlePubMedGoogle Scholar
- Ajami B, Bennett JL, Krieger C, Tetzlaff W, Rossi FMW: Local self-renewal can sustain CNS microglia maintenance and function throughout adult life. Nat Neurosci. 2007, 10: 1538-1543. 10.1038/nn2014.View ArticlePubMedGoogle Scholar
- Eglitis M, Mezey E: Hematopoietic cells differentiate into both microglia and macroglia in the brains of adult mice. Proc Natl Acad Sci. 1997, 94: 4080-4085. 10.1073/pnas.94.8.4080.PubMed CentralView ArticlePubMedGoogle Scholar
- Massengale M, Wagers AJ, Vogel H, Weismann IL: Hematopoitic cells maintain hematopoeitic fates upon entering the brain. J Exp Med. 2005, 201: 1579-1589. 10.1084/jem.20050030.PubMed CentralView ArticlePubMedGoogle Scholar
- Ono K, Takii T, Onozaki K, Ikawa M, Okabe M, Sawada M: Migration of exogenous immature hematopoietic cells into adult mouse brain parenchyma under GFP-expressing bone marrow chimera. Biochem Biophys Res Commun. 1999, 262: 610-614. 10.1006/bbrc.1999.1223.View ArticlePubMedGoogle Scholar
- Bechmann I, Priller J, Kovac A, Böntert M, Wehner T, Klett FF, Bohsung J, Stuschke M, Dirnagl U, Nitsch R: Immune surveillance of mouse brain perivascular spaces by blood-borne macrophages. Eur J Neurosci. 2001, 14: 1651-1658. 10.1046/j.0953-816x.2001.01793.x.View ArticlePubMedGoogle Scholar
- Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR: Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science. 2000, 290: 1779-1782. 10.1126/science.290.5497.1779.View ArticlePubMedGoogle Scholar
- Zhang ZG, Zhang L, Jianf Q, Chopp M: Bone marrow derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res. 2002, 90: 284-288. 10.1161/hh0302.104460.View ArticlePubMedGoogle Scholar
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