Gr1+ Macrophages and Dendritic Cells Dominate the Inflammatory Infiltrate 12 Hours After Experimental Intracerebral Hemorrhage

Abstract

Intracerebral hemorrhage (ICH) is a devastating disease lacking an effective treatment. While the initial injury occurs within minutes, an inflammatory response contributes to ongoing tissue damage over hours to days. Relatively little is known about leukocyte trafficking into the brain in the hours after ICH onset. Understanding these events may lead to identification of new therapeutic targets. Using the blood injection mouse model of ICH, the numbers of leukocytes in the ipsilateral and contralateral brain were quantified by flow cytometry 12 hours after surgery. Perihematomal inflammation was confirmed by histology and chemokines and cytokines in the brain quantified by multiplex ELISA. Few neutrophils were detected in the brain 12 hours after ICH. The majority of leukocytes consisted of inflammatory macrophages (CD45.1(hi)CD3(-)Ly6G(-)CD11c(-)CD11b(+)Gr1(+) cells) and inflammatory dendritic cells (CD45.1(hi)CD3(-)Ly6G(-)CD11c(int)CD11b(+)Gr1(+) cells). Microglia numbers did not differ between the hemispheres. These results indicate that blood-derived monocyte populations traffic into brain early after ICH and outnumber neutrophils at 12 hours.

Fig. 1

Microglia and blood-derived leukocytes can be distinguished from injected leukocytes. a Representative plots from ipsilateral brain samples showing few donor CD45.2⁺ cells at 12 hours. The CD45.1^int population (black gate, center) is 99.7% CD11b⁺ and are classified as microglia. The blue gate (lower right corner) represents the host-derived CD45.1^hi leukocyte population. b Total microglia numbers are not different between hemispheres. n=5

Fig. 2

Representative plots showing leukocyte populations present after ICH. a Inflammatory macrophages (I.Mϕs) and Gr1⁻ macrophages (Gr1⁻ Mϕs) are both present in brain, but the inflammatory macrophages are much more numerous. This plot is gated on CD45.1^hiCD3⁻Ly6G⁻CD11c⁻ cells. b Plot showing CD11c^int (blue gate, center) and CD11c^hi dendritic cell populations (DCs, black gate, right) is gated on CD45.1^hiCD3⁻Ly6G⁻ cells. The majority of CD11c^int cells express Gr1 (inset) and are termed inflammatory dendritic cells. c Few neutrophils (PMNs) are found in brain at 12 hours. The plot is gated on CD45.1^hiCD3⁻. d T cells are found in the ipsilateral hemisphere by 12 hours. The plot is gated on CD45.1^hi cells. e Pie chart showing the distribution of leukocyte populations expressed as the mean percentage of infiltrating cells from 5 mice. “Other” indicates CD45.1^hi cells that do not fall into any of the aforementioned gating strategies

Fig. 3

Immunofluorescence staining demonstrates local inflammation. Representative perihematomal sections from 12 hours after ICH or sham surgery are shown in a–d. a–b Staining for CD11b, a marker for myeloid cells, demonstrates most CD11b+ cells after sham surgery (a) have branched cytoplasmic processes consistent with resting microglia. b Arrows indicate larger, spherical CD11b+ cells consistent with macrophages at the border of the ICH that were not found after sham surgery. Astrocytic processes, identified by GFAP+ staining, were difficult to identify after sham surgery but became more evident after ICH. c–d Neutrophils, identified by staining with Ly6G, could not be found after sham surgery and were found in small numbers around the ICH at 12 hours (arrowhead). Scale bar indicates 10 μm

Fig. 4

ICH induces elevations of several chemokines and cytokines within 12 hours. The chemokines CCL2, CXCL1, and CXCL10 are significantly higher in ipsilateral ICH (n=4) brain versus ipsilateral sham (n=3) brain. The pro-inflammatory cytokine IL-6 is also elevated, but TNF is lower in ipsilateral ICH brain. All values are shown in pg/g brain protein. * p<0.05 by t-test after Holm-Bonferroni correction for multiple hypotheses