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-induced chemokine production and apoptosis in human neural precursor cells
,1
* Neuroimmunology Laboratory, Minneapolis Medical Research Foundation, Minnesota;
Department of Medicine, University of Minnesota Medical School, Minneapolis; and
Stem Cell Group, R&D Systems, Inc., Minneapolis, Minnesota
1 Correspondence: Department of Medicine, University of Minnesota Medical School, 420 Delaware Street, S.E., Minneapolis, MN 55455. E-mail: peter137{at}umn.edu
ABSTRACT
Recent studies have shown that proinflammatory cytokines damage rodent neural precursor cells (NPCs), a source of self-renewing, multipotent cells that play an important role in the developing as well as adult brain. In this study, the effects of tumor necrosis factor 
(TNF-) on cytokine and chemokine production by human NPCs (>98% nestin- and >90% A2B5-positive), obtained from 6- to 8-week-old fetal brain specimens, were evaluated. NPCs stimulated with this proinflammatory cytokine were found to produce abundant amounts of the chemokines monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL2) and interferon-inducible protein 10 (IP-10)/CXC chemokine ligand 10 (CXCL10) in a time- and concentration-dependent manner. TNF- treatment also induced NPC apoptosis. Receptors for TNF [TNFRI (p55) and TNFRII (p75)] mRNA were constitutively expressed on NPCs. However, only TNFRI was involved in TNF--induced chemokine production and apoptosis by NPCs, as anti-TNFRI but not anti-TNFRII antibodies blocked the stimulatory effect. TNF- treatment induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in NPCs, and SB202190, an inhibitor of p38 MAPK, blocked TNF--induced chemokine production. Thus, this study demonstrated that NPCs constitutively express receptors for TNF-, which when activated, trigger via a p38 MAPK signaling pathway production of two chemokines, MCP-1/CCL2 and IP-10/CXCL10, which are involved in infectious and inflammatory diseases of the brain.
Key Words: cytokines • MCP-1 • IP-10 • CCL2 • CXCL10
INTRODUCTION
Neural precursor cells (NPCs), like neural stem or progenitor cells, are a self-renewing, multipotent population of cells, which are capable of differentiating into neurons, astrocytes, and oligodendrocytes [1 2 3 4 ]. Localized within specific areas of the developing as well as the adult brain, these cells contribute importantly to brain patterning, memory formation, and brain repair [5 6 7 8 9 ]. It has been reported that activation of NPCs can regenerate hippocampal pyramidal neurons after ischemic brain injury [10 ], and transplanted NPCs are able to migrate toward inflammatory sites within the central nervous system (CNS) [11 ]. Transplanted NPCs also have been tracked by luciferase imaging to study their in vivo migration toward ischemic infarcts in a murine model [12 ]. The migratory process was found to be mediated by cytokines and chemokines released in the area of inflammation. In vitro studies have revealed that NPCs are able to migrate toward the chemokine stromal cell-derived factor-1 (SDF-1)/CXC chemokine ligand 12 (CXCL12), and the cells were shown to express high levels of CXC chemokine receptor 4 (CXCR4) [3 , 13 ], the cognate receptor for this chemokine.
Proinflammatory cytokines are known to have detrimental and beneficial effects in the CNS under different circumstances. When they are activated, microglia (the resident macrophages of the brain) and astrocytes are the main sources of proinflammatory cytokines such as tumor necrosis factor (TNF-) [14
15
16
17
] within the brain parenchyma. Implicated in the pathogenesis of many CNS disorders, TNF- can elicit autocrine and paracrine effects on surrounding cells [18
], which may lead to further cytokine and chemokine production. Besides inflammation, TNF- treatment can also trigger apoptosis through its receptor and intracellular signaling [19
20
21
22
]. Recent studies have demonstrated that intracortical [23
] or intraperitoneal [24
] challenge of rats with the gram-negative bacterial cell wall component lipopolysaccharide (LPS) resulted in marked impairment of neurogenesis through a mechanism involving activation of microglial cells. TNF-, interleukin (IL)-1ß, and IL-6, released from LPS-activated microglia, were implicated in the damage done to NPCs [24
]. Under physiological conditions, conversely, hippocampal astrocytes promote neurogenesis [25
], and microglia direct migration and influence differentiation of NPCs through release of soluble factors [26
], further demonstrating the importance of interactions between glial cells and NPCs.
Work in our laboratory and that of other investigators has shown that TNF- is released from human microglial cells stimulated by LPS [14
, 27
] as well as in response to viral pathogens, such as human immunodeficiency virus type 1 (HIV-1) [28
], cytomegalovirus [29
], and herpes simplex virus type 1 [15
]. As activated astrocytes are also known to generate proinflammatory cytokines [27
, 30
], we were interested in the present study to test the hypothesis that TNF- would induce cytokine production by human NPCs, which could thereby theoretically exacerbate the damage done to NPCs by activated microglia. We found instead that NPCs release substantial amounts of the proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 (CCL2) and interferon (IFN)-inducible protein 10 (IP-10)/CXCL10, which have been implicated in a number of neurodegenerative diseases, such as HIV-associated dementia [31
, 32
], multiple sclerosis [33
34
35
], Alzheimer’s disease [36
, 37
], and Parkinson’s disease [38
]. We also found that TNF- induced apoptosis of NPCs, further supporting the neuropathogenic role of the interaction of this proinflammatory cytokine with these neural progenitor cells.
MATERIALS AND METHODS
Reagents
The following reagents were purchased from the indicated sources: recombinant human TNF-, IL-1ß, IFN-
, and SDF-1/CXCL12; antibodies to human TNF-, IL-1ß, IL-6, IL-10, MCP-1/CCL2, regulated on activation of normal T cell expressed and secreted (RANTES)/CCL5, IP-10/CXCL10, TNF receptors I and II [TNFRI (p55), TNFRII (p75)], nestin, A2B5, and CD133; N2 plus supplement, human fibroblast growth factor-basic (hFGFb), human epidermal growth factor (hEGF), platelet-derived growth factor (PDGF), and brain-derived neurotrophic factor (BDNF; R&D Systems, Minneapolis, MN); anti-p38 mitogen-activated protein kinase (MAPK) antibody (Cell Signaling, Beverly, MA); Texas red-conjugated avidin D, rhodamine red-conjugated donkey anti-mouse immunoglobulin G (IgG), biotin-conjugated donkey anti-goat IgG, and biotin-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA); fluorescein isothiocyanate (FITC)-streptavidin (Vector Laboratories, Burlingame, CA); 4'-6-diamidino-2-phenylindole (DAPI; Intergen, Purchase, NY); SB202190 (an inhibitor of p38 MAPK) and SB202474 (an inactive inhibitor of p38 MAPK, EMD Biosciences, La Jolla, CA); cell death detection enzyme-linked immunosorbent assay (ELISA; Roche Applied Science, Indianapolis, IN); Dulbecco’s modified Eagle’s medium (DMEM)/F12 and gentamicin (Invitrogen, Carlsbad, CA); penicillin, streptomycin, poly-D-lysine, trypsin, polyoxyethylenesorbitan monolaurate (Tween 20), phosphate-buffered saline (PBS), Tris, and LPS (Sigma Chemical Co., St. Louis, MO); acrylamide/bis solution (Bio-Rad, Hercules, CA) and heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT).
NPC cultures
NPCs were prepared from 6- to 8-week-old aborted human fetal brain tissues obtained under a protocol approved by the Human Subjects Research Committee at Hennepin County Medical Center (Minneapolis, MN). Human fetal brain tissues were mechanically dissociated and resuspended in DMEM/F12 media [containing 8 mM glucose and glutamine, N2 plus supplement, penicillin (100 U/ml), and streptomycin (100 µg/ml), gentamicin (50 µg/ml), and hFGFb and hEGF (20 ng/ml each)] and plated onto poly-D-lysine (50 µg/ml)-coated, 10 cm tissue-culture dishes. This stage is considered as passage 0. When cell cultures reached 60% confluence, they were subcultured by treatment with trypsin (0.0125%) and deposited onto poly-D-lysine-coated plates (considered as passage 1). Culture media were replaced every other day. NPCs at passages 1–3 were used in the studies. Cells were plated onto coated 24-, 12-, or 6-well plates or 100 mm petri dishes at a density of 1 x 104, 2 x 104, 3 x 104, or 5 x 104 cells per well, respectively, and were ready for experiments 4–5 days after plating. These NPCs express the neural stem cell markers nestin (>90% positive) and CD133 (>80% positive) and are glial fibrillary acidic protein (GFAP; a marker for astrocytes)- and microtubule-associated protein 2 (a neuronal cell marker)-negative [3
]. When subjected under differentiation culture conditions (i.e., withdrawal of hEGF and hFGFb growth factors and addition of serum or PDGF plus BDNF), the NPCs are capable of differentiating into neurons, astrocytes, and oligodendrocytes [3
].
Flow cytometry [fluorescein-activated cell sorter (FACS)]
To measure cytokine receptor expression on NPCs, staining with mouse anti-human TNFRI- and TNFRII-phycoerythrin antibodies (R&D Systems) was performed with FACS analysis according to the manufacturer’s suggested procedures.
Immunocytochemical staining
To verify the expression of nestin and A2B5 in NPCs, cells plated onto four-well chamber slides (2x102 cells/well) were fixed with 4% paraformaldehyde followed by washing with PBS. After blocking with 10% normal goat serum in PBS, cells were incubated with mouse anti-human nestin (10 µg/ml) or anti-human A2B5 (10 µg/ml) antibodies. After washing with PBS, cells were incubated with secondary biotin-conjugated goat anti-mouse IgG (5 µg/ml) for 60 min at room temperature followed by addition of FITC-streptavidin or Texas red-conjugated avidin D and counter-stained with DAPI (1 µg/ml) for nuclei. Cells were examined under fluorescent microscope for nestin (green) or A2B5 (red) expression.
To confirm the expression of nestin and CXCL10 in NPCs, cells were stimulated 4–5 days after plating with TNF- (20 ng/ml) for 48 h and fixed, followed by washing with PBS as described above. After blocking with 10% normal donkey serum in PBS, cells were incubated with mouse anti-human nestin (10 µg/ml) and goat anti-human CXCL10 (1 µg/ml) antibodies. After washing with PBS, cells were incubated with secondary biotin-conjugated donkey anti-goat IgG (5 µg/ml) for 60 min at room temperature followed by addition of rhodamine red-conjugated donkey anti-mouse IgG and FITC-streptavidin. Cells were examined under a fluorescent microscope for nestin (red) and CXCL10 (green) expression.
RNase protection assay (RPA)
To assess cytokine, chemokine, and cytokine receptor mRNA expression, total RNA isolated from NPC cultures was used in the multiprobe RPA according to the manufacturer’s protocol (BD Biosciences PharMingen, San Diego, CA).
ELISA
To measure IL-6, IL-10, TNF-, IL-1ß, MCP-1/CCL2, RANTES/CCL5, and IP-10/CXCL10 in NPC culture supernatants, ELISA plates (96-well) were coated with corresponding mouse anti-human antibodies (1–2 µg/ml) overnight at 4°C. The plates were blocked with 1% bovine serum albumin in PBS for 1 h at 37°C. After washing with PBS with Tween 20, culture supernatants and a series of dilutions of IL-6, IL-10, TNF-, IL-1ß, MCP-1/CCL2, RANTES/CCL5, or IP-10/CXCL10 (as standards) were added to wells for 2 h at 37°C. Following washing, detection antibody (goat anti-human IL-6, IL-10, TNF-, IL-1ß, MCP-1/CCL2, RANTES/CCL5, or IP-10/CXCL10 antibodies, 1–2 µg/ml) was added for 90 min at 37°C followed by donkey anti-goat IgG horseradish-peroxidase conjugate (1:10,000) for 45 min. A chromogen substrate K-blue was then added at room temperature for color development, which was stopped with 1 M H2SO4. The plate was read at 450 nm to generate standard concentration curves for IL-6, IL-10, TNF-, IL-1ß, MCP-1/CCL2, RANTES/CCL5, or IP-10/CXCL10 concentration extrapolation.
Cell death detection ELISA
To measure apoptosis, cell lysates from culture medium- or TNF--treated NPC cultures in 24-well plates were collected for histone-associated DNA fragmentation measurement according to the manufacturer’s protocol. Briefly, cell lysates were added to the streptavidin-coated 96-well ELISA plates together with anti-histone biotinylated and anti-DNA peroxidase antibodies. After incubation and washing, DNA fragments were captured and detected by a chromogenic enzyme-substrate reaction.
Western blot analysis
To assess whether TNF- stimulates p38 MAPK phosphorylation and whether TNFRI and TNFRII are involved in TNF--induced signaling, cell lysates from untreated and treated NPCs were electrophoresed on 12% bis/acrylamide gel followed by transblotting to nitrocellulose membrane. After incubation in blocking buffer [5% milk in Tris-buffered saline with Tween 20 (TTBS)], the membrane was probed with rabbit anti-phospho p38 MAPK polyclonal antibody (in 1% blocking buffer) overnight. After washing with TTBS, the membrane was incubated with secondary antibody (goat anti-rabbit), conjugated with alkaline phosphatase, followed by chemiluminescent detection (CDP-Star substrates, Applied Biosystems, Bedford, MA) under an image station (Kodak, New Haven, CT). Membrane blot was stripped and reprobed with rabbit anti-p38 MAPK antibody.
Statistical analysis
Data are expressed as mean ± SEM. For comparison of means of multiple groups, ANOVA was used, followed by Scheffe’s test.
RESULTS
NPC cultures
After plating dispersed brain cells onto poly-D-lysine-coated petri dishes, proliferated clones of cells were often observed by Day 2. The NPCs cultured have a proliferation rate of 5–7 days to reach 50–60% confluency of culture plates or petri dishes, and we elected to use NPC cultures from passages 1 to 3 in this study. By immunocytochemical staining, these NPCs were stained positive for nestin (>98%) and A2B5 (>90%; Fig. 1
). By FACS analysis, these NPCs were nestin (>90%)-, CD133 (>80%)- [3
], and A2B5 (>94%)- [39
] positive for these stem cell markers. Thus, the NPCs we have isolated and subjected to study are relatively homogenous. When subjected to differentiation medum (i.e., withdrawing the growth factors hEGF and hFGFb and adding PDGF/BDNF or 1% FBS) for 3 weeks, NPCs were able to differentiate mostly into neurons and a lesser extent into astrocytes. However, if 10% FBS instead of 1% FBS were added, NPCs differentiated mostly into astrocytes (>80% GFAP-positive) and fewer numbers of neurons (<20%) and oligodendrocytes (<1%) [3
, 39
].
|
(20 ng/ml), IL-1ß (10 ng/ml), IFN- (200 U/ml), and SDF-1/CXCL12 (100 ng/ml). The concentrations of LPS and cytokines used were based on our previous studies of glial cells [14
, 40
41
42
] and that of SDF-1/CXCL12, on our studies of NPCs [3
]. Levels of the cytokines IL-6, IL-10, TNF-, and IL-1ß and of the chemokines MCP-1/CCL2, RANTES/CCL5, and IP-10/CXCL10 released by stimulated NPCs for 24 h are shown in Table 1
. Although LPS is able to induce robust cytokine and chemokine production by human microglia [14
, 27
, 43
], it had no effect on NPCs in this regard. Similarly, IFN- and SDF-1/CXCL12 elicited no cytokine or chemokine production by NPCs, although these cells express high levels of CXCR4 [3
], the receptor for SDF-1/CXCL12. However, treatment with TNF- or IL-1ß induced production of the chemokines MCP-1/CCL2 and IP-10/CXCL10.
|
induced MCP-1/CCL2 and IP-10/CXCL10 mRNA expression (Fig. 2
) and protein production (Figs. 3
and 4
). Production of CCL2 and CXCL10 in response to TNF- was time (Fig. 3
)- and concentration-dependent (Fig. 4
), and peak production levels were at 72 h, with TNF- concentrations between 20 ng/ml and 100 ng/ml. To demonstrate that chemokine was produced in undifferentiated NPCs, immunocytochemical staining of NPCs stimulated with TNF- for 48 h showed the expression of the NPC marker nestin (red) and intracellular IP-10/CXCL10 (green; Fig. 5
). No significant changes of nestin expression in NPCs treated with TNF- for 72 h were found by flow cytometry analysis (data not shown).
|
|
|
|
, we next investigated whether these cells express receptors for TNF-. NPCs constitutively expressed TNFRI and TNFRII mRNA (Fig. 6
). Flow cytometry analysis of NPCs also revealed the expression of TNFRI to be 43% (Fig. 7A
), and only minimal expression of TNFRII was detectable (Fig. 7B)
.
|
|
-induced chemokine production, anti-TNFRI and -TNFRII antibodies were used to block these receptors. The production of MCP-1/CCL2 and IP-10/CXCL10 by NPCs stimulated with TNF- for 72 h was blocked by pretreatment with anti-TNFRI but not by antibody to TNFRII (Fig. 8
) or by goat IgG isotype antibody (as a negative control; data not shown).
|
elicits chemokine production by NPCs, we examined the involvement of p38 MAPK, as this MAPK is commonly activated by cytokines. We found that treatment of NPCs with TNF- induced p38 MAPK phosphorylation (Fig. 9A
). No effect on total p38 MAPK protein by TNF- treatment was found (Fig. 9A)
. Pretreatment of NPCs with anti-TNFRI but not anti-TNFRII or mouse IgG isotype antibodies suppressed TNF--induced p38 MAPK phosphorylation (Fig. 9B)
, suggesting that TNFRI and not TNFRII is involved in the signaling of TNF- in these cells.
|
-induced MCP-1/CCL2 and IP-10/CXCL10 production in a concentration-dependent manner (Fig. 10
), and pretreatment with the inactive compound SB202474 (as a negative control) had no effect (data not shown).
|
induction of chemokines in NPCs, we observed morphological differences in cultures between untreated (control) and TNF--treated NPCs over 72 h. Thus, we sought to investigate if TNF- induced apoptosis of NPCs. We found that apoptosis was triggered by TNF- treatment for 72 h, and the apoptotic effect was blocked by pretreatment of NPCs with anti-TNFRI but not anti-TNFRII antibodies (Fig. 11
), suggesting that TNF--induced apoptosis of NPCs was mediated through TNFRI.
|
We have demonstrated for the first time in this study that TNF- treatment induced production of two biologically important chemokines, MCP-1/CCL2 and IP-10/CXCL10, by human NPCs. Expression of TNFRI and TNFRII mRNA was also shown in NPCs. Using antibodies against TNFR, we found that anti-TNFRI but not anti-TNFRII blocked TNF--induced chemokine production and apoptosis, suggesting that MCP-1/CCL2 and IP-10/CXCL10 production and apoptosis were mediated through TNFRI. Evidence of phospho p38 MAPK induction in NPCs by TNF- treatment was also presented, suggesting that the p38 MAPK intracellular signaling pathway is involved in the action of this cytokine. The finding that treatment of NPCs with the p38 MAPK inhibitor SB202190 abrogated chemokine production in response to TNF- provided support for the involvement of p38 MAPK in the induction of MCP-1/CCL2 and IP-10/CXCL10. Expression of the NPC marker, nestin, was not affected significantly by TNF-, suggesting that these cells maintained their progenitor status during the 72-h treatment period.
Although the neuropathological or neurophysiological consequences of chemokine generation by TNF--stimulated NPCs are unknown, MCP-1/CCL2 and IP-10/CXCL10 have been shown to be involved in many CNS diseases [44
], such as AIDS dementia [32
, 45
], multiple sclerosis [33
], and Alzheimer’s disease [36
, 37
]. MCP-1/CCL2 or IP-10/CXCL10 also plays a role in many inflammatory disorders outside the CNS, including lung disease [46
], myasthenia gravis [47
], type 1 diabetes [48
], rheumatoid arthritis [49
], periapical granulomas [50
], proliferative glomerulonephritis [51
], and delayed-type hypersensitivity [52
], involving their ability to recruit leukocytes to sites of inflammation. MCP-1/CCL2 has also been shown to regulate T cell differentiation through CC chemokine receptor 2 expressed on T cells [53
]. Thus, an overzealous proinflammatory chemokine reaction could trigger a pathogenic outcome. However, beneficial roles of these chemokines are also found in angiogenesis and hematopoiesis [54
].
Although our initial hypothesis that TNF- would induce production of proinflammatory cytokines by NPCs turned out not to be supported by our results, nonetheless, release of MCP-1/CCL2 and IP-10/CXCL10 from cytokine-activated NPCs could serve as signals for recruitment of microglia, peripheral blood monocytes, and T lymphocytes for the purposes of defense or brain repair. However, an escalated cytokine response from migratory microglia and cells from the peripheral blood could also inflict further damage to NPCs. Such a deleterious effect is supported by reports of inflammation-induced microglial activation, resulting in impaired neurogenesis [23
, 24
].
Human astrocytes respond to TNF-, IL-1ß, or IFN- treatment by producing cytokines and chemokines [29
, 55
56
57
58
], but NPCs only responded to TNF- and IL-1ß and not IFN- treatment. Although the NPCs used in this study are capable of differentiating into astrocytes under differentiation conditions [3
], a large majority of these cells bears the neural stem cell markers nestin and CD133, and they are uniformly negative for the astrocyte marker GFAP. Also, we have found that in contrast to astrocytes [29
, 35
], NPCs do not produce nitric oxide in response to IL-1ß treatment (unpublished observation). Thus, the functional capacity of NPCs to produce chemokines appears to precede their commitment to an astrocyte lineage.
It is interesting that two potent stimuli of microglial cell cytokine and chemokine production, i.e., LPS and IFN- [27
, 43
, 56
], had no effect on NPCs, suggesting that human NPCs either do not express receptors for LPS and IFN- or that these stimuli do not trigger intracellular signaling mechanisms, which lead to cytokine or chemokine generation. Support for that latter notion is provided by rodent systems, demonstrating that neurosphere generation by murine stem cells is inhibited by IFN- treatment [59
], although IFN- had no effect on neurogenesis in rat brain [24
]. However, rat NPCs have been demonstrated to express receptors for IFN- but not for IL-1, and IFN- inhibited proliferation and induced migration of rat NPC neurospheres [60
].
Taken together, our results demonstrate that human NPCs are capable of producing the chemokines MCP-1/CCL2 and IP-10/CXCL10 in response to a cytokine signal, which is known to be released from activated microglia and astrocytes. The produced proinflammatory chemokines MCP-1/CCL2 and IP-10/CXCL10 could play pathogenic or beneficial roles for the host under different circumstances. It is interesting that NPCs have been shown to express abundant amounts of CXCR4, yet the natural ligand for this receptor (CXCL12/SDF-1), which directs a migratory response by NPCs [3 , 13 ], did not elicit chemokine expression. Further studies of NPC cytokine/chemokine networks and of the intracellular signaling pathways triggered by inflammatory mediators should help delineate the dynamic interaction between glial cells and NPCs and may foster development of neural stem cell transplantation for the treatment of neuroinflammatory diseases of the brain.
ACKNOWLEDGEMENTS
This study was supported in part by United States Public Health Service Grant DA 09924. We are grateful to Drs. R. McKay and E. Major for their valuable advice and assistance.
Received April 24, 2005; revised August 25, 2005; accepted September 8, 2005.
REFERENCES
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in brain cultures by human immunodeficiency virus type 1 J. Virol. 66,2217-2225-inducible protein 10 (IP-10, CXCL10) in HIV-1 infection J. Neuroimmunol. Epub ahead of print.
upregulates human microglial cell production of interleukin-10 in vitro Clin. Diagn. Lab. Immunol. 2,604-608[Abstract]
inducible protein-10 expression in rheumatoid arthritis Arthritis Res. Ther. 5,R74-R81[CrossRef][Medline]
but not TNF promotes neuronal differentiation and neurite outgrowth of murine adult neural stem cells Exp. Neurol. 187,171-177[CrossRef][Medline]
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