Chemokines Promote Quiescence and Survival of Human Neural Progenitor Cells
http://www.100md.com
《干细胞学杂志》
Department of Medicine, Center for Immunology and Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA
Key Words. Neural stem cell ? Proliferation ? Chemokines ? Cycle regulation
Mitchell D. Krathwohl, M.D., University of Minnesota, MMC 250, 420 Delaware St. SE., Minneapolis, Minnesota 55455, USA. Telephone: 612-625-2618; Fax: 612-625-4410; e-mail: krath001@umn.edu
ABSTRACT
Neural progenitor cells (NPCs) are capable of forming both neurons and glia upon differentiation, and are present both during development of the brain and in adulthood in a broad range of mammals including humans . The most primitive neural stem cells (NSCs) appear to undergo asymmetric cell division, forming a daughter stem cell (SC) and a more restricted progenitor cell . These progenitor cells are then capable of forming neurons and astrocytes . However, not all progenitor cells in the adult brain enter the cell cycle. Some progenitor cells exist in a quiescent state that is regulated by the local environment . Some molecules implicated in the regulation of NPC proliferation include cortisol , retinoic acid , opioids , and glutamate . The extent to which these molecules play a role in vivo is not well described. Because of the need to tightly control cell division in the adult, other molecules likely play a role in the regulation of cell division under homeostatic conditions.
One group of molecules that has been shown to regulate cell division in other systems is the chemokine group of cytokines. These are small peptides secreted by a variety of cell types, originally described because of their ability to induce chemotaxis in inflammatory cells. They have been shown to regulate diverse processes such as hematopoiesis and angiogenesis . Interestingly, several cell types in the mammalian brain have been shown to possess chemokine receptors . This includes CCR3, CXCR4, CXCR2, and CX3CR1 expression on neurons, CXCR4 expression on astrocytes, and CCR3 and CCR5 expression on microglia. The actual function of these receptors in the developed nervous system is not known. However, several parallels between hematopoiesis and the development of NPCs have been noted, including the involvement of many of the same cytokines in both systems . Because quiescence is forced upon proliferating hematopoietic stem cells (HSCs) by the chemokine family of molecules , one possible role of chemokine on NPC development might be the control of cellular proliferation. However, no similar studies with chemokines have been undertaken with NPCs. Additionally, some chemokines support the survival of (HSCs) but have unknown effects on NSC survival. Because the chemokines stromal derived factor-1 (SDF-1) and fractalkine are produced constitutively in the brain, and several others are produced during inflammation , we hypothesized that chemokines would affect neural SC survival and proliferation. To test this hypothesis, we studied the effects of chemokines on isolated colonies of human NPCs and slice cultures of human hippocampus. We found that indeed specific chemokines promote both survival and quiescence of NPCs.
MATERIALS AND METHODS
Expression of Chemokine Receptors on NPC
We first sought to establish which chemokine receptors are present on NPCs. Both hippocampal neurons and HSCs have been shown to express CCR5, CXCR4, and CCR3 . We therefore analyzed isolated human NPCs by flow cytometry to determine whether they would similarly express CCR3, CCR5, or CXCR4. In multiple experiments, surface expression of CCR3 was seen on 55%–69% of cells, while CXCR4 was seen on 65%–85% of cells (Fig. 1). In contrast, CCR5 expression was detected on less than 5% of cells. These results show that like HSCs, NPCs express CCR3 and CXCR4 but unlike HSCs, NPCs do not express appreciable levels of CCR5.
Figure 1. Human NPCs express CCR3 and CXCR4. Chemokine receptor expression was detected by flow cytometry.
Chemokines Promote Quiescence of NPC
To determine the role of these chemokine receptors on NPCs, we tested the ability of chemokines to affect the proliferation of NPCs. Isolated NPCs were cultured by limiting dilution with and without chemokines that bind to CCR3, CCR5 or CXCR4; namely, eotaxin, microphage inhibitory protein 1 alpha (MIP-1), or SDF-1. We found that eotaxin and SDF-1 prevented cell division by NPCs (Fig. 2A) while MIP-1 allowed cells to proliferate and form colonies of tightly packed cells similar to controls. In order to determine whether cells treated with SDF-1 and eotaxin were quiescent, cells were washed and placed in fresh media without chemokines. After 5 days, individual cells showed evidence of proliferation (Fig. 2A). To confirm that cells ceased proliferation in response to chemokines, NPCs were cultured with or without chemokines and BrdU was added on day 2 or day 7 of culture. The incorporation of BrdU was analyzed 24 hours after addition using flow cytometric detection of BrdU. We found that on day 2, chemokines that can activate CCR3 or CXCR4 inhibited BrdU incorporation, while chemokines that activate CCR5 did not (Fig. 2B). By day 7, this inhibitory effect was no longer seen, and chemokine-treated cultures showed no difference in BrdU uptake compared to proliferating controls, indicating that this inhibition is transient (not shown). Thus chemokines that bind to the CCR3 or CXCR4 receptors present on NPCs inhibit cellular proliferation and induce cellular quiescence, but allow cells to maintain their proliferative potential.
Figure 2. Chemokines inhibit NPC proliferation and promote cellular quiescence. A) Proliferation of NPCs in limiting dilution cultures. Original magnification in all photos is 400x. B) Removal of chemokines allows isolated NPCs to resume proliferation. Individual cells treated with either eotaxin or SDF-1 were washed and placed in fresh media, then followed for 5 days. Removal of chemokines resulted in cell division. C) BrdU uptake in chemokine-treated NPC cultures, plotted as n of cells versus BrdU fluorescence. Shaded area = control cultures, solid line = chemokine-treated cells. D) Chemokine-treated NPCs maintain multipotential ability. Chemokine-treated cells were washed and plated onto laminin-coated plates. Expression of NeuN after 7 days was detected in differentiated cells in all chemokine-treatment groups by a monoclonal antibody. E) Chemokine-treated NPCs maintain their multipotential ability. The ability to form GFAP+ astrocytes was assessed by staining plated cells with GFAP followed by a Texas Red labeled secondary antibody.
To further confirm that chemokines were inducing NPCs to become quiescent, we tested whether NPCs exposed to chemokines maintained their multipotential ability. NPCs that had been treated with chemokines were washed and plated onto laminin-coated plates to induce differentiation. We found that once chemokines were removed, even SDF-1- or eotaxin-treated NPCs could still form neurons as determined by NeuN staining (Fig. 2C) and GFAP staining (Fig. 2E).
NPCs can also be grown as clusters of cells termed neurospheres. These spherules of cells have been used as models of NPC biology in vitro and in vivo . Growing cells as spherules maintains cell-to-cell contact, tight junctions, and prevents the trauma of passaging monolayers of cells. Thus these neurospheres may be more representative of cells in vivo. We tested whether chemokines would also affect NPCs grown as spherules, reasoning that if chemokines induced quiescence in these cells, the spherules would fail to attach and begin a program of differentiation into neurons and astrocytes. Counting the number of attached and differentiated colonies then allows a measure of the ability of each chemokine to induce cellular quiescence. We again tested chemokines that bind to CCR3 and CXCR4 receptors as well as several chemokines that do not. We found that incubation of NPCs with the chemokines MCP-4, eotaxin, and RANTES, all of which can bind to CCR3 , decreased the number of colonies formed by 77%, 68% and 79%, respectively (Fig. 3). The CXCR4-binding chemokine SDF-1 decreased the number of colonies formed by 61%. In contrast, the chemokines MIP-1 and MCP-1, which can bind to CCR1, CCR2, or CCR5, had no effect on colony formation. The ability of the CCR3 receptor to decrease colony formation was confirmed by using a monoclonal antibody to CCR3 in the NPC culture, which also decreased colony formation by 50%. To rule out the possibility that chemokines were cytotoxic, we removed unattached spherules from SDF-1 and eotaxin-treated cultures after plating. These spherules were washed and replated on coated plates in fresh media. We found that these cells were able to attach and differentiate (not shown), thus confirming that the inhibition by chemokines is transient. These studies show that chemokines that activate either the CCR3 or CXCR4 receptor can inhibit the growth and differentiation of NPCs in neurospheres.
Figure 3. Chemokines promote cellular quiescence of cells within cultured neurospheres. The number of attached neurospheres that form colonies of differentiated cells on coated plates after treatment with various chemokines is shown. OA = okadaic acid. Bars represent standard error of the mean. Each chemokine was tested in triplicate. p < 0.001 for RANTES, SDF-1, eotaxin and anti-CCR3 versus control.
Effects of Chemokines on Reelin Expression
Because Reelin has been shown to prevent attachment of NPCs to fibronectin , we reasoned that chemokines might be inducing Reelin expression in NPCs that would then prevent the attachment of neurospheres to coated surfaces. We tested chemokine-treated NPCs for Reelin expression and found that SDF-1 increased Reelin expression compared to controls, but MIP-1 did not (Fig. 4). This result provides further mechanistic evidence for how chemokines induce quiescence in NPCs.
Figure 4. Chemokines affect Reelin expression on NPCs. Reelin expression (green) on chemokine-treated NPCs. Nuclei are counterstained with propidium iodide (red).
Chemokines Reduce Phosphorylation of ERK
We next asked what signaling pathway might be activated by chemokines to result in reduced proliferation. Because the ERK inhibitor PD98059 has been shown to block the proliferation of NPCs , we hypothesized that the MAPK pathway is affected by chemokines. Therefore, NPCs were incubated with chemokines for 10 minutes then assayed for intracellular expression of the phosphorylated MAPK family member ERK. Consistent with ongoing proliferation in these cells, we found a high basal level of p-ERK (Fig. 5A, 5B) and p38 (not shown). We found that eotaxin reduced p-ERK by 42%. SDF-1 reduced p-ERK by 37%. In contrast, MIP-1 showed a non-significant reduction of 20%. Because a reduction of phosphorylation might imply an increase in phosphatase activity, the PP2A phosphatase inhibitor OA was added to the cultures prior to the addition of chemokines. The phosphatase inhibitor blocked the reduction in p-ERK induced by eotaxin and SDF-1 (Fig. 5B), suggesting that phosphatases are activated by these chemokines. Neither chemokine, however, had any effect on the level of p38 phosphorylation (not shown). We also tested the ability of OA to block the inhibitory effects of eotaxin in cultured NPCs. We found that OA completely reversed the inhibitory effects of eotaxin on cultured NPCs (Fig. 3). These results demonstrate that specific chemokines can decrease the p-ERK by activating cellular phosphatases.
Figure 5. Chemokines promote ERK dephosphorylation. A) Detection of p-ERK in chemokine-treated NPCs, plotted as n of cells versus p-ERK fluorescence. Shaded area = control cells, solid line = chemokine-treated cells. B) Percentage of cells expressing p-ERK in chemokine-treated cultures with and without OA. Bars represent standard error of the mean. p < 0.01 for SDF-1 and eotaxin.
Fractalkine Promotes Survival of NPC
Chemokines have been shown to act as survival factors for other types of SCs . In agreement with this role, we did not detect an increase in apoptosis in chemokine-treated, isolated NPCs (95% versus 96% viability by trypan blue exclusion), or in neurospheres. To determine whether chemokines would promote survival of NPCs during growth factor withdrawal, we tested SDF-1 and fractalkine, both of which are constitutively expressed in the brain, as well as the chemokines that failed to inhibit proliferation. We grew NPCs in media without FGF-B, EGF, and N2 to induce apoptosis, adding chemokines to some cultures to determine whether they would promote survival. As measured by trypan blue analysis, fractalkine increased viability of NPCs under these conditions from 50% in control cultures to 75% in fractalkine-treated cultures. The chemokines MCP-1, SDF-1, and MIP-1 showed 48%, 53%, and 44% viability in cultures, respectively, that were not statistically different than control cultures. We confirmed this activity of fractalkine by measuring Annexin V staining in chemokine-treated NPC cultures by flow cytometry. Fractalkine-treated cultures showed only 50% of cells expressing Annexin V, compared to 69.1% of cells in the control (Fig. 6). This demonstrates that chemokines also act as survival agents for NPCs, similar to the survival role ascribed to fractalkine on mature neurons.
Figure 6. Chemokines control survival of human NPC. Expression of annexin V on NPC grown without neural growth factors for 5 days as determined by flow cytometry is shown.
Apolipoproteins and Heparan Sulfate Modulate the Effects of Chemokines on NPC
If chemokines promote quiescence of NPCs, then a mechanism must exist to counteract this inhibition under conditions requiring new neuron formation. Conditions such as development, injury or inflammation, where new neurons are needed, have been associated with alterations in HS expression and apolipoprotein expression . We hypothesized that HS might bind directly to chemokines, while the apolipoproteins might compete indirectly with chemokines for HS on the cell surface. Thus alterations in either HS or apolipoproteins could abrogate the inhibitory effect of chemokines. We tested the ability of exogenous HS to prevent the inhibitory effects of chemokines on NPCs (Fig. 7). We found that HS prevented SDF-1 (17% of control with SDF-1 alone versus 86% of control with HS + SDF-1, p < 0.001) and eotaxin (29% of control with eotaxin alone versus 71% of control with HS + eotaxin, p < 0.001)-induced inhibition of NPC proliferation in isolated colonies as well as in hippocampal slice cultures with eotaxin (21% of control with eotaxin alone versus 207% of control with HS + eotaxin, p < 0.01). A closely related glycosaminoglycan, heparin, could not block the effect of eotaxin in isolated colonies (29% of control versus 23% of control with heparin + eotaxin, p = not significant ) or in slice cultures (21% of control versus 29% of control with heparin + eotaxin, p = n.s.). We also tested recombinant apolipoproteins E3 and E4 for their ability to block the effects of chemokines on NPCs (Fig. 7). We found that while apolipoprotein E3 prevented chemokine inhibition of NPC proliferation by SDF-1 in isolated colonies (17% of control with SDF-1 alone versus 148% of control with SDF-1 + Apo E3, p < 0.001), apolipoprotein E4 did not (17% of control alone versus 42% of control with SDF-1 + Apo E4, p = n.s.). We conclude that HS and apolipoproteins can modulate the effects of chemokines on NPCs, although not all apolipoproteins share this ability.
Figure 7. HS and apolipoproteins modulate effects of chemokines on NPCs. Proliferation of NPCs in cultures treated with apolipoproteins or HS and the inhibitory chemokine SDF-1. Only cultures treated with HS or apolipoprotein E3 show clumps of proliferating cells despite SDF-1 treatment.
Chemokines Promote Quiescence of NPC in Human Hippocampal Slices
The local environment has been shown to be an important factor in the behavior of SCs in vivo . We therefore sought to determine whether chemokines affect NPCs in the complex environment of the whole brain. Human hippocampal tissue was obtained from patients aged 21–50 years and grown as thin slices in tissue culture. Cultures were treated with a variety of chemokines, and pulsed with BrdU as a marker for proliferating NPCs. Cultures were harvested and IHC for BrdU-positive cells was performed. We found that the number of BrdU positive cells was decreased in cultures treated with eotaxin and SDF-1 by 79% and 71%, respectively, but not with MIP-1 or fractalkine (Fig. 8). To determine whether chemokines act only to induce quiescence of NPCs or are actively inducing apoptosis in slice cultures, a TUNEL assay was performed on the chemokine-treated hippocampal slices. No difference in apoptosis was seen in treated versus untreated cultures (MIP-1, eotaxin, and SDF-1 showed 20.5, 29.5 and 29 apoptotic cells per high power field, respectively, versus 27.5 for control). To confirm that BrdU-stained cells were in fact NPCs, we tested cells for expression of the NPC-associated marker Msi-1. We stained slices with fluorescent monoclonal antibodies to BrdU and either Msi-1, GFAP, or isolectin B4. When slices were examined, the vast majority of BrdU-labeled cells were also Msi-1+ (Fig. 9) but were GFAP and isolectin B4 negative (not shown), thus confirming their status as NPCs and not astrocytes or microglia. Thus, chemokines act specifically on NPCs in whole brain cultures to decrease proliferation.
Figure 8. Chemokines inhibit proliferation of NPCs in hippocampal slice cultures. Number of BrdU+ cells in five consecutive 5 μm sections in five high-power fields in chemokine-treated hippocampal slices is shown, with each chemokine tested in triplicate. Bars represent standard error of the mean. p < 0.05 for SDF-1 and eotaxin.
Figure 9. Proliferation of NPCs in hippocampal slice cultures. Shown are hippocampal slices with BrdU (green) and Musashi (red) merged together. Yellow cells represent doubly labeled BrdU+Mushashi+ cells.
DISCUSSION
We gratefully acknowledge the technical help of Diane Trussoni in preparing the tissue sections, and the surgical expertise of Dr. Robert Maxwell in obtaining the hippocampal specimens. This work was supported in part by NIH grant K08 01544 and the Great Lakes Regional Center for AIDS Research Developmental Award.
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Key Words. Neural stem cell ? Proliferation ? Chemokines ? Cycle regulation
Mitchell D. Krathwohl, M.D., University of Minnesota, MMC 250, 420 Delaware St. SE., Minneapolis, Minnesota 55455, USA. Telephone: 612-625-2618; Fax: 612-625-4410; e-mail: krath001@umn.edu
ABSTRACT
Neural progenitor cells (NPCs) are capable of forming both neurons and glia upon differentiation, and are present both during development of the brain and in adulthood in a broad range of mammals including humans . The most primitive neural stem cells (NSCs) appear to undergo asymmetric cell division, forming a daughter stem cell (SC) and a more restricted progenitor cell . These progenitor cells are then capable of forming neurons and astrocytes . However, not all progenitor cells in the adult brain enter the cell cycle. Some progenitor cells exist in a quiescent state that is regulated by the local environment . Some molecules implicated in the regulation of NPC proliferation include cortisol , retinoic acid , opioids , and glutamate . The extent to which these molecules play a role in vivo is not well described. Because of the need to tightly control cell division in the adult, other molecules likely play a role in the regulation of cell division under homeostatic conditions.
One group of molecules that has been shown to regulate cell division in other systems is the chemokine group of cytokines. These are small peptides secreted by a variety of cell types, originally described because of their ability to induce chemotaxis in inflammatory cells. They have been shown to regulate diverse processes such as hematopoiesis and angiogenesis . Interestingly, several cell types in the mammalian brain have been shown to possess chemokine receptors . This includes CCR3, CXCR4, CXCR2, and CX3CR1 expression on neurons, CXCR4 expression on astrocytes, and CCR3 and CCR5 expression on microglia. The actual function of these receptors in the developed nervous system is not known. However, several parallels between hematopoiesis and the development of NPCs have been noted, including the involvement of many of the same cytokines in both systems . Because quiescence is forced upon proliferating hematopoietic stem cells (HSCs) by the chemokine family of molecules , one possible role of chemokine on NPC development might be the control of cellular proliferation. However, no similar studies with chemokines have been undertaken with NPCs. Additionally, some chemokines support the survival of (HSCs) but have unknown effects on NSC survival. Because the chemokines stromal derived factor-1 (SDF-1) and fractalkine are produced constitutively in the brain, and several others are produced during inflammation , we hypothesized that chemokines would affect neural SC survival and proliferation. To test this hypothesis, we studied the effects of chemokines on isolated colonies of human NPCs and slice cultures of human hippocampus. We found that indeed specific chemokines promote both survival and quiescence of NPCs.
MATERIALS AND METHODS
Expression of Chemokine Receptors on NPC
We first sought to establish which chemokine receptors are present on NPCs. Both hippocampal neurons and HSCs have been shown to express CCR5, CXCR4, and CCR3 . We therefore analyzed isolated human NPCs by flow cytometry to determine whether they would similarly express CCR3, CCR5, or CXCR4. In multiple experiments, surface expression of CCR3 was seen on 55%–69% of cells, while CXCR4 was seen on 65%–85% of cells (Fig. 1). In contrast, CCR5 expression was detected on less than 5% of cells. These results show that like HSCs, NPCs express CCR3 and CXCR4 but unlike HSCs, NPCs do not express appreciable levels of CCR5.
Figure 1. Human NPCs express CCR3 and CXCR4. Chemokine receptor expression was detected by flow cytometry.
Chemokines Promote Quiescence of NPC
To determine the role of these chemokine receptors on NPCs, we tested the ability of chemokines to affect the proliferation of NPCs. Isolated NPCs were cultured by limiting dilution with and without chemokines that bind to CCR3, CCR5 or CXCR4; namely, eotaxin, microphage inhibitory protein 1 alpha (MIP-1), or SDF-1. We found that eotaxin and SDF-1 prevented cell division by NPCs (Fig. 2A) while MIP-1 allowed cells to proliferate and form colonies of tightly packed cells similar to controls. In order to determine whether cells treated with SDF-1 and eotaxin were quiescent, cells were washed and placed in fresh media without chemokines. After 5 days, individual cells showed evidence of proliferation (Fig. 2A). To confirm that cells ceased proliferation in response to chemokines, NPCs were cultured with or without chemokines and BrdU was added on day 2 or day 7 of culture. The incorporation of BrdU was analyzed 24 hours after addition using flow cytometric detection of BrdU. We found that on day 2, chemokines that can activate CCR3 or CXCR4 inhibited BrdU incorporation, while chemokines that activate CCR5 did not (Fig. 2B). By day 7, this inhibitory effect was no longer seen, and chemokine-treated cultures showed no difference in BrdU uptake compared to proliferating controls, indicating that this inhibition is transient (not shown). Thus chemokines that bind to the CCR3 or CXCR4 receptors present on NPCs inhibit cellular proliferation and induce cellular quiescence, but allow cells to maintain their proliferative potential.
Figure 2. Chemokines inhibit NPC proliferation and promote cellular quiescence. A) Proliferation of NPCs in limiting dilution cultures. Original magnification in all photos is 400x. B) Removal of chemokines allows isolated NPCs to resume proliferation. Individual cells treated with either eotaxin or SDF-1 were washed and placed in fresh media, then followed for 5 days. Removal of chemokines resulted in cell division. C) BrdU uptake in chemokine-treated NPC cultures, plotted as n of cells versus BrdU fluorescence. Shaded area = control cultures, solid line = chemokine-treated cells. D) Chemokine-treated NPCs maintain multipotential ability. Chemokine-treated cells were washed and plated onto laminin-coated plates. Expression of NeuN after 7 days was detected in differentiated cells in all chemokine-treatment groups by a monoclonal antibody. E) Chemokine-treated NPCs maintain their multipotential ability. The ability to form GFAP+ astrocytes was assessed by staining plated cells with GFAP followed by a Texas Red labeled secondary antibody.
To further confirm that chemokines were inducing NPCs to become quiescent, we tested whether NPCs exposed to chemokines maintained their multipotential ability. NPCs that had been treated with chemokines were washed and plated onto laminin-coated plates to induce differentiation. We found that once chemokines were removed, even SDF-1- or eotaxin-treated NPCs could still form neurons as determined by NeuN staining (Fig. 2C) and GFAP staining (Fig. 2E).
NPCs can also be grown as clusters of cells termed neurospheres. These spherules of cells have been used as models of NPC biology in vitro and in vivo . Growing cells as spherules maintains cell-to-cell contact, tight junctions, and prevents the trauma of passaging monolayers of cells. Thus these neurospheres may be more representative of cells in vivo. We tested whether chemokines would also affect NPCs grown as spherules, reasoning that if chemokines induced quiescence in these cells, the spherules would fail to attach and begin a program of differentiation into neurons and astrocytes. Counting the number of attached and differentiated colonies then allows a measure of the ability of each chemokine to induce cellular quiescence. We again tested chemokines that bind to CCR3 and CXCR4 receptors as well as several chemokines that do not. We found that incubation of NPCs with the chemokines MCP-4, eotaxin, and RANTES, all of which can bind to CCR3 , decreased the number of colonies formed by 77%, 68% and 79%, respectively (Fig. 3). The CXCR4-binding chemokine SDF-1 decreased the number of colonies formed by 61%. In contrast, the chemokines MIP-1 and MCP-1, which can bind to CCR1, CCR2, or CCR5, had no effect on colony formation. The ability of the CCR3 receptor to decrease colony formation was confirmed by using a monoclonal antibody to CCR3 in the NPC culture, which also decreased colony formation by 50%. To rule out the possibility that chemokines were cytotoxic, we removed unattached spherules from SDF-1 and eotaxin-treated cultures after plating. These spherules were washed and replated on coated plates in fresh media. We found that these cells were able to attach and differentiate (not shown), thus confirming that the inhibition by chemokines is transient. These studies show that chemokines that activate either the CCR3 or CXCR4 receptor can inhibit the growth and differentiation of NPCs in neurospheres.
Figure 3. Chemokines promote cellular quiescence of cells within cultured neurospheres. The number of attached neurospheres that form colonies of differentiated cells on coated plates after treatment with various chemokines is shown. OA = okadaic acid. Bars represent standard error of the mean. Each chemokine was tested in triplicate. p < 0.001 for RANTES, SDF-1, eotaxin and anti-CCR3 versus control.
Effects of Chemokines on Reelin Expression
Because Reelin has been shown to prevent attachment of NPCs to fibronectin , we reasoned that chemokines might be inducing Reelin expression in NPCs that would then prevent the attachment of neurospheres to coated surfaces. We tested chemokine-treated NPCs for Reelin expression and found that SDF-1 increased Reelin expression compared to controls, but MIP-1 did not (Fig. 4). This result provides further mechanistic evidence for how chemokines induce quiescence in NPCs.
Figure 4. Chemokines affect Reelin expression on NPCs. Reelin expression (green) on chemokine-treated NPCs. Nuclei are counterstained with propidium iodide (red).
Chemokines Reduce Phosphorylation of ERK
We next asked what signaling pathway might be activated by chemokines to result in reduced proliferation. Because the ERK inhibitor PD98059 has been shown to block the proliferation of NPCs , we hypothesized that the MAPK pathway is affected by chemokines. Therefore, NPCs were incubated with chemokines for 10 minutes then assayed for intracellular expression of the phosphorylated MAPK family member ERK. Consistent with ongoing proliferation in these cells, we found a high basal level of p-ERK (Fig. 5A, 5B) and p38 (not shown). We found that eotaxin reduced p-ERK by 42%. SDF-1 reduced p-ERK by 37%. In contrast, MIP-1 showed a non-significant reduction of 20%. Because a reduction of phosphorylation might imply an increase in phosphatase activity, the PP2A phosphatase inhibitor OA was added to the cultures prior to the addition of chemokines. The phosphatase inhibitor blocked the reduction in p-ERK induced by eotaxin and SDF-1 (Fig. 5B), suggesting that phosphatases are activated by these chemokines. Neither chemokine, however, had any effect on the level of p38 phosphorylation (not shown). We also tested the ability of OA to block the inhibitory effects of eotaxin in cultured NPCs. We found that OA completely reversed the inhibitory effects of eotaxin on cultured NPCs (Fig. 3). These results demonstrate that specific chemokines can decrease the p-ERK by activating cellular phosphatases.
Figure 5. Chemokines promote ERK dephosphorylation. A) Detection of p-ERK in chemokine-treated NPCs, plotted as n of cells versus p-ERK fluorescence. Shaded area = control cells, solid line = chemokine-treated cells. B) Percentage of cells expressing p-ERK in chemokine-treated cultures with and without OA. Bars represent standard error of the mean. p < 0.01 for SDF-1 and eotaxin.
Fractalkine Promotes Survival of NPC
Chemokines have been shown to act as survival factors for other types of SCs . In agreement with this role, we did not detect an increase in apoptosis in chemokine-treated, isolated NPCs (95% versus 96% viability by trypan blue exclusion), or in neurospheres. To determine whether chemokines would promote survival of NPCs during growth factor withdrawal, we tested SDF-1 and fractalkine, both of which are constitutively expressed in the brain, as well as the chemokines that failed to inhibit proliferation. We grew NPCs in media without FGF-B, EGF, and N2 to induce apoptosis, adding chemokines to some cultures to determine whether they would promote survival. As measured by trypan blue analysis, fractalkine increased viability of NPCs under these conditions from 50% in control cultures to 75% in fractalkine-treated cultures. The chemokines MCP-1, SDF-1, and MIP-1 showed 48%, 53%, and 44% viability in cultures, respectively, that were not statistically different than control cultures. We confirmed this activity of fractalkine by measuring Annexin V staining in chemokine-treated NPC cultures by flow cytometry. Fractalkine-treated cultures showed only 50% of cells expressing Annexin V, compared to 69.1% of cells in the control (Fig. 6). This demonstrates that chemokines also act as survival agents for NPCs, similar to the survival role ascribed to fractalkine on mature neurons.
Figure 6. Chemokines control survival of human NPC. Expression of annexin V on NPC grown without neural growth factors for 5 days as determined by flow cytometry is shown.
Apolipoproteins and Heparan Sulfate Modulate the Effects of Chemokines on NPC
If chemokines promote quiescence of NPCs, then a mechanism must exist to counteract this inhibition under conditions requiring new neuron formation. Conditions such as development, injury or inflammation, where new neurons are needed, have been associated with alterations in HS expression and apolipoprotein expression . We hypothesized that HS might bind directly to chemokines, while the apolipoproteins might compete indirectly with chemokines for HS on the cell surface. Thus alterations in either HS or apolipoproteins could abrogate the inhibitory effect of chemokines. We tested the ability of exogenous HS to prevent the inhibitory effects of chemokines on NPCs (Fig. 7). We found that HS prevented SDF-1 (17% of control with SDF-1 alone versus 86% of control with HS + SDF-1, p < 0.001) and eotaxin (29% of control with eotaxin alone versus 71% of control with HS + eotaxin, p < 0.001)-induced inhibition of NPC proliferation in isolated colonies as well as in hippocampal slice cultures with eotaxin (21% of control with eotaxin alone versus 207% of control with HS + eotaxin, p < 0.01). A closely related glycosaminoglycan, heparin, could not block the effect of eotaxin in isolated colonies (29% of control versus 23% of control with heparin + eotaxin, p = not significant ) or in slice cultures (21% of control versus 29% of control with heparin + eotaxin, p = n.s.). We also tested recombinant apolipoproteins E3 and E4 for their ability to block the effects of chemokines on NPCs (Fig. 7). We found that while apolipoprotein E3 prevented chemokine inhibition of NPC proliferation by SDF-1 in isolated colonies (17% of control with SDF-1 alone versus 148% of control with SDF-1 + Apo E3, p < 0.001), apolipoprotein E4 did not (17% of control alone versus 42% of control with SDF-1 + Apo E4, p = n.s.). We conclude that HS and apolipoproteins can modulate the effects of chemokines on NPCs, although not all apolipoproteins share this ability.
Figure 7. HS and apolipoproteins modulate effects of chemokines on NPCs. Proliferation of NPCs in cultures treated with apolipoproteins or HS and the inhibitory chemokine SDF-1. Only cultures treated with HS or apolipoprotein E3 show clumps of proliferating cells despite SDF-1 treatment.
Chemokines Promote Quiescence of NPC in Human Hippocampal Slices
The local environment has been shown to be an important factor in the behavior of SCs in vivo . We therefore sought to determine whether chemokines affect NPCs in the complex environment of the whole brain. Human hippocampal tissue was obtained from patients aged 21–50 years and grown as thin slices in tissue culture. Cultures were treated with a variety of chemokines, and pulsed with BrdU as a marker for proliferating NPCs. Cultures were harvested and IHC for BrdU-positive cells was performed. We found that the number of BrdU positive cells was decreased in cultures treated with eotaxin and SDF-1 by 79% and 71%, respectively, but not with MIP-1 or fractalkine (Fig. 8). To determine whether chemokines act only to induce quiescence of NPCs or are actively inducing apoptosis in slice cultures, a TUNEL assay was performed on the chemokine-treated hippocampal slices. No difference in apoptosis was seen in treated versus untreated cultures (MIP-1, eotaxin, and SDF-1 showed 20.5, 29.5 and 29 apoptotic cells per high power field, respectively, versus 27.5 for control). To confirm that BrdU-stained cells were in fact NPCs, we tested cells for expression of the NPC-associated marker Msi-1. We stained slices with fluorescent monoclonal antibodies to BrdU and either Msi-1, GFAP, or isolectin B4. When slices were examined, the vast majority of BrdU-labeled cells were also Msi-1+ (Fig. 9) but were GFAP and isolectin B4 negative (not shown), thus confirming their status as NPCs and not astrocytes or microglia. Thus, chemokines act specifically on NPCs in whole brain cultures to decrease proliferation.
Figure 8. Chemokines inhibit proliferation of NPCs in hippocampal slice cultures. Number of BrdU+ cells in five consecutive 5 μm sections in five high-power fields in chemokine-treated hippocampal slices is shown, with each chemokine tested in triplicate. Bars represent standard error of the mean. p < 0.05 for SDF-1 and eotaxin.
Figure 9. Proliferation of NPCs in hippocampal slice cultures. Shown are hippocampal slices with BrdU (green) and Musashi (red) merged together. Yellow cells represent doubly labeled BrdU+Mushashi+ cells.
DISCUSSION
We gratefully acknowledge the technical help of Diane Trussoni in preparing the tissue sections, and the surgical expertise of Dr. Robert Maxwell in obtaining the hippocampal specimens. This work was supported in part by NIH grant K08 01544 and the Great Lakes Regional Center for AIDS Research Developmental Award.
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