Chemokines regulate the migration of neural progenitors to sites of neuroinflammation

Abstract

Many studies have shown that transplanted or endogenous neural progenitor cells will migrate toward damaged areas of the brain. However, the mechanism underlying this effect is not clear. Here we report that, using hippocampal slice cultures, grafted neural progenitor cells (NPs) migrate toward areas of neuroinflammation and that chemokines are a major regulator of this process. Migration of NPs was observed after injecting an inflammatory stimulus into the area of the fimbria and transplanting enhanced green fluorescent protein (EGFP)-labeled NPs into the dentate gyrus of cultured hippocampal slices. Three to 7 d after transplantation, EGFP-NPs in control slices showed little tendency to migrate and had differentiated into neurons and glia. In contrast, in slices injected with inflammatory stimuli, EGFP-NPs migrated toward the site of the injection. NPs in these slices also survived less well. The inflammatory stimuli used were a combination of the cytokines tumor necrosis factor-alpha and interferon-gamma, the bacterial toxin lipopolysaccharide, the human immunodeficiency virus-1 coat protein glycoprotein 120, or a beta-amyloid-expressing adenovirus. We showed that these inflammatory stimuli increased the synthesis of numerous chemokines and cytokines by hippocampal slices. When EGFP-NPs from CC chemokine receptor CCR2 knock-out mice were transplanted into slices, they exhibited little migration toward sites of inflammation. Similarly, wild-type EGFP-NPs exhibited little migration toward inflammatory sites when transplanted into slices prepared from monocyte chemoattractant protein-1 (MCP-1) knock-out mice. These data indicate that factors secreted by sites of neuroinflammation are attractive to neural progenitors and suggest that chemokines such as MCP-1 play an important role in this process.

Figure 1.

Developmental potential of NPs expressing EGFP (green cells). A, Confocal image of EGFP–NPs transplanted into the area of the dentate gyrus (dg) of a cultured mouse hippocampal slice after 18 h in culture, illustrating the spherical morphology of cells, similar to that of dissociated neurospheres (as illustrated in C). Scale bar, 500 μm. A is magnified in B. Scale bar, 100 μm. C, Confocal image showing grafted EGFP–NPs have differentiated into cells with neuronal and glial morphology after 3–5 d in cultures.

Figure 2.

Undifferentiated phenotype typical of EGFP–NPs 48 h after transplantation into the dentate gyrus of 7-d-old hippocampal slice cultures. A, B, Double-fluorescent confocal images of neural progenitor cells: EGFP (A, green) and anti-nestin (B, red). C, DNA was visualized by staining with DAPI. D, Three-dimensional analysis of a double-fluorescent confocal image of an NP: EGFP (green) and anti-nestin (red). The x–z and y–z images are shown. n = 6 and 9 slices per experiment were used. Scale bar, 50 μm.

Figure 3.

Confocal images showing neuronal differentiation of grafted NPs after transplantation of EGFP–NPs into the dentate gyrus of 7-d-old hippocampal slice cultures. After 3–7 d, some grafted EGFP–NPs (green, arrowhead in A) stained for NeuN (red, arrowhead in B and in merged image, yellow, in C). C, Three-dimensional analysis of double-fluorescent confocal image of NPs: EGFP (green) and anti-NeuN (red). In control slices, 9 of 230 EGFP-labeled cells were NeuN positive and 3 of 98 EGFP cells were NeuN positive in TNF-α–IFN-γ-injected slices. n = 3, 6 slices per group per experiment. Scale bars, 20 μm.

Figure 4.

Confocal images showing astrocyte differentiation of grafted NPs after transplantation of EGFP–NPs into the dentate gyrus of 7-d-old hippocampal slice cultures. After 3–7 d, some grafted EGFP–NPs (green, arrowhead in A) stained for GFAP (red, arrowhead in B and in merged image, yellow, in C). C, Three-dimensional analysis of a double-fluorescent confocal image of NPs: EGFP (green) and anti-GFAP (red). The x–z and y–z images are shown. One hundred fifty-five of 253 EGFP cells were GFAP positive (61 ± 5%) in control slices, and 106 of 138 EGFP cells were GFAP positive (77 ± 7%) in TNF-α–IFN-γ-injected slices. n = 3, 6 slices/ per group per experiment. Scale bar, 50 μm.

Figure 5.

Patterns of migration of EGFP–NPs 3–5 d after injection of an inflammatory stimulus into the area of the fimbria (red dot in C), followed by transplantation of EGFP–NPs (green dot in B and C) into the dentate gyrus of 7-d-old hippocampal slice cultures. Confocal images were further processed for assessment of cell migration using MetaMorph software, and the extent of cell migration was determined as the average distance between the site of injection and 25–50 green cells in each slice as shown in B and C. The accuracy of the injection is indicated by the green spot in A, which illustrates EGFP expression after the injection of an EGFP-adenovirus into the slice. Scale bar, 500 μm.

Figure 6.

Survival of grafted migrating neural progenitors in response to TNF-α–IFN-γ injection into hippocampal slice cultures. TUNEL assays for grafted migrating NPs (red in B, C and green in B′, C′) were used to assess cell survival. Arrowhead in A indicates an EGFP–NP cell with reduced fluorescence that is TUNEL negative (arrowhead in B and C). Arrows in A indicate EGFP–NPs that are TUNEL positive (arrows in B, red and in merged image, yellow in C). In cytokine-injected slices, 70 of 167 cells (42 ± 10%) were TUNEL positives versus 26 of 194 cells (13 ± 2.5%) in control slices (p < 0.01; n = 2 and 10–11 slices per group). Scale bar, 50 μm. Confocal images in A′, B′, and C′ show migrating PKH26–NPs (red cells) 3 d after injection of TNF-α–IFN-γ into the area of the fimbria, after transplantation of PKH26–NPs into the dentate gyrus of cultured slices. Images in A′ and B′ were merged to show dead and dying cells (arrowheads in C′). Scale bars, 20 μm. Arrowheads in A′, B′, and C′ show PKH26-labeled NPs (A′, C′) and TUNEL staining (B′, C′). In cytokine-injected slices, 46 of 114 cells were TUNEL positive compare with 23 of 175 cells in control slices (p < 0.0001; n = 9 slices per group). Quantification of the average fluorescence of cells (D) (*p < 0.0001; n = 3 and 17 slices per group) was generated from images such as those shown in A using MetaMorph software. When PKH26-labeled cells were used, the average fluorescence in both groups was in the range 339 ± 15 (arbitrary fluorescence units).

Figure 7.

Migration of NPs in response to different inflammatory stimuli. EGFP-labeled NPs from wild-type CD1 mice were grafted into the area of the DG of 7-d-old hippocampal slice in control cultures (A, A′, A″). After 3 d, cells migrated toward the inflammatory site (red spot) of slices treated with TNFα–IFN-γ (B), gp120–CD4 (B′), or LPS (B″). After 72 h of transplantation, the extent of cell “migration” (ECM) was found in the range of 584 ± 18 μm in control slices. It increased by 54% in LPS-injected slices (p < 0.001; n = 3 and 9 slices per group), 43% in TNF-α–IFN-γ-injected slices (p < 0.01; n = 5 and 9 slices per group), and 39% in HIV-1–gp120-injected slices (p < 0.001; n = 3 and 9 slices per group). Confocal images in A‴, B‴, and C‴ show PKH26-labeled NPs (red) grafted into the area of the DG of 7-d-old slice cultures injected with an EGFP– β-amyloid-expressing adenovirus [fimbria (fi), green cells in A‴]. After 3 d, cells migrated toward the inflammatory site (red spot in B‴, C‴). Scale bar, 500 μm.

Figure 8.

Activation of macrophages/microglia and migration of transplanted cells in response to inflammatory stimuli in 7-d-old hippocampal slice cultures. In A and B, hippocampal slices were generated from CX3CR₁–EGFP transgenic mice, in which EGFP–CX3CR₁ is mostly expressed in microglia. When injected with the inflammatory stimulus (TNF-α–IFN-γ) into the dentate gyrus (dg), slice shows activation of EGFP–CX3CR₁-expressing microglia at the site of the inflammatory stimulus (B) and in control slice after saline injection (A). In A′, B′, and C′, slices were injected with EGFP–β-amyloid-expressing adenovirus as an inflammatory stimulus. A′ is magnified in B′ (scale bar, 200 μm) and C′ (scale bar, 100 μm), showing CD11b- and F4/80-stained microglia/macrophages at the injection site of the inflammatory stimulus (red in A′, B′, C′). In A″ and B″, grafted GFP–NPs in slices injected with TNF-α–IFN-γ were immunostained for macrophage/microglia markers using a combined cyanine 3-conjugated monoclonal to CD11b⁺ and F4/80⁺. Box in A″ is magnified in B″ (scale bar, 200 μm). Box in B″ is magnified in C″, D″, and E″ (scale bar, 20 μm), which shows CD11b/F4/80-stained macrophage/microglia (red in D″, E″). Images in C″ and D″ were merged to show that there is no colocalization between migrating EGFP–NPs and activated macrophage/microglia. n = 1, 9 slices were used.

Figure 9.

Migration of EGFP–NPs in response to inflammation depends on MCP-1/CCR₂ signaling. In A, B, and C, EGFP–CCR₂ KO labeled NPs (B6 × 129 strain) were transplanted into the area of the DG of 7-d-old hippocampal slice cultures from wild-type mice (B6 × 129 strain). In A′, B′, and C′, wild-type EGFP-labeled NPs were transplanted into the area of the DG of 7-d-old slice cultures from MCP-1 KO mice. After 3 d, cells failed to show oriented migration toward the inflammatory site (red spot) in TNF-α–IFN-γ-treated slices (B, B′). The extent of cell “migration” (ECM) in cytokine-treated group was found in the same range as in control group. In A and B, the ECM was 612 ± 40 versus 562 ± 31 μm in control group. In A′ and B′, the ECM was 875 ± 45 versus 824 ± 87 μm in control group. However, when wild-type EGFP-labeled NPs were transplanted into the area of the DG of wild-type slices (all from B6 × 129 strain), the ECM increased by 34% over control values (p < 0.01; n = 2 and 9 slices per group). Scale bar, 500 μm.

Figure 10.

Role of MCP-1/CCR₂ signaling on cell survival of migrating EGFP–NPs. EGFP–CCR₂ KO-labeled NPs were transplanted into the area of the DG of 7-d-old hippocampal slice cultures from wild-type mice: A, control slices; B, TNF-α–IFN-γ-injected slices. In A and B, double-fluorescent confocal images of neural progenitor cells: EGFP (green) and TUNEL (red). After 3 d, cells failed to show an oriented migration toward the inflammatory site in TNF-α–IFN-γ-treated slices. In TNF-α–IFN-γ-injected slices, the number of TUNEL-positive cells was in the range of 3.6 ± 0.3% of the cells versus 3.3 ± 0.4% in control cultures (n = 2, 9–12 slices per group). Scale bar, 100 μm.