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Functional differences between microglia and monocytes after ischemic stroke.

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD - J Neuroinflammation (2015)

Bottom Line: The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA. rritzel@uchc.edu.

ABSTRACT

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.

Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.

Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

No MeSH data available.


Related in: MedlinePlus

Measurement of cytokine production by microglia and monocytes at 24 and 72 h and 7 days following stroke. A representative histogram showing microglia (red) have relatively higher reactive oxygen species levels than monocytes (blue) at 72 h after stroke as measured with dihydrorhodamine (DHR) 123 (a). The mean fluorescence intensity of DHR123 was quantified (b). Representative dot plots showing microglia and monocyte production of TNF (c) and IL-1β (e) at 72 h reveals differential expression patterns after stroke. The respective percentages of cells positive for these cytokines are quantified at 0, 24, and 72 hrs and 7 days (d, f). For all experiments, N = 5–7/group. Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, MFI mean fluorescence intensity. **p < 0.01; ***p < 0.001
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Fig3: Measurement of cytokine production by microglia and monocytes at 24 and 72 h and 7 days following stroke. A representative histogram showing microglia (red) have relatively higher reactive oxygen species levels than monocytes (blue) at 72 h after stroke as measured with dihydrorhodamine (DHR) 123 (a). The mean fluorescence intensity of DHR123 was quantified (b). Representative dot plots showing microglia and monocyte production of TNF (c) and IL-1β (e) at 72 h reveals differential expression patterns after stroke. The respective percentages of cells positive for these cytokines are quantified at 0, 24, and 72 hrs and 7 days (d, f). For all experiments, N = 5–7/group. Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, MFI mean fluorescence intensity. **p < 0.01; ***p < 0.001

Mentions: Reactive oxygen species production drives the oxidative stress response to stroke and is critical to injury progression [18]. We examined free radical formation in microglia and monocytes after stroke. Resident microglia expressed significantly more ROS relative to the infiltrating monocytes (p < 0.01), as evidenced by increased DHR123 staining intensity (Fig. 3a, b). Because oxidative stress precipitates cytokine production, we next evaluated pro-inflammatory cytokine levels in these cells. Newly recruited monocytes expressed higher levels of TNF at 24 h; however, microglia expressed significantly greater levels by 72 h (Fig. 3c, d). Infiltrating monocytes expressed relatively higher levels of IL-1β at all time points (p < 0.001; Fig. 3e, f). The number of microglia expressing TNF remained elevated above sham levels at 7 days, whereas those expressing IL-1β did not differ. Neither microglia nor brain-recruited monocytes expressed detectable levels of the anti-inflammatory cytokines IL-4 and IL-10 after stroke (data not shown). These results suggest that microglia play a critical role in oxidative injury and that these two cell types may mediate cell death via distinct signaling pathways.Fig. 3


Functional differences between microglia and monocytes after ischemic stroke.

Ritzel RM, Patel AR, Grenier JM, Crapser J, Verma R, Jellison ER, McCullough LD - J Neuroinflammation (2015)

Measurement of cytokine production by microglia and monocytes at 24 and 72 h and 7 days following stroke. A representative histogram showing microglia (red) have relatively higher reactive oxygen species levels than monocytes (blue) at 72 h after stroke as measured with dihydrorhodamine (DHR) 123 (a). The mean fluorescence intensity of DHR123 was quantified (b). Representative dot plots showing microglia and monocyte production of TNF (c) and IL-1β (e) at 72 h reveals differential expression patterns after stroke. The respective percentages of cells positive for these cytokines are quantified at 0, 24, and 72 hrs and 7 days (d, f). For all experiments, N = 5–7/group. Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, MFI mean fluorescence intensity. **p < 0.01; ***p < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4465481&req=5

Fig3: Measurement of cytokine production by microglia and monocytes at 24 and 72 h and 7 days following stroke. A representative histogram showing microglia (red) have relatively higher reactive oxygen species levels than monocytes (blue) at 72 h after stroke as measured with dihydrorhodamine (DHR) 123 (a). The mean fluorescence intensity of DHR123 was quantified (b). Representative dot plots showing microglia and monocyte production of TNF (c) and IL-1β (e) at 72 h reveals differential expression patterns after stroke. The respective percentages of cells positive for these cytokines are quantified at 0, 24, and 72 hrs and 7 days (d, f). For all experiments, N = 5–7/group. Cell-specific FMO controls were used to determine positive gating. Error bars show mean SEM. Abbreviation: SEM standard error of the mean, SH sham, ST stroke, MFI mean fluorescence intensity. **p < 0.01; ***p < 0.001
Mentions: Reactive oxygen species production drives the oxidative stress response to stroke and is critical to injury progression [18]. We examined free radical formation in microglia and monocytes after stroke. Resident microglia expressed significantly more ROS relative to the infiltrating monocytes (p < 0.01), as evidenced by increased DHR123 staining intensity (Fig. 3a, b). Because oxidative stress precipitates cytokine production, we next evaluated pro-inflammatory cytokine levels in these cells. Newly recruited monocytes expressed higher levels of TNF at 24 h; however, microglia expressed significantly greater levels by 72 h (Fig. 3c, d). Infiltrating monocytes expressed relatively higher levels of IL-1β at all time points (p < 0.001; Fig. 3e, f). The number of microglia expressing TNF remained elevated above sham levels at 7 days, whereas those expressing IL-1β did not differ. Neither microglia nor brain-recruited monocytes expressed detectable levels of the anti-inflammatory cytokines IL-4 and IL-10 after stroke (data not shown). These results suggest that microglia play a critical role in oxidative injury and that these two cell types may mediate cell death via distinct signaling pathways.Fig. 3

Bottom Line: The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations.We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham.In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA. rritzel@uchc.edu.

ABSTRACT

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models.

Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days.

Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke.

Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells.

No MeSH data available.


Related in: MedlinePlus