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Effects of human marrow stromal cells on activation of microglial cells and production of inflammatory factors induced by lipopolysaccharide
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Effects of human marrow stromal cells on activation of microglial cells and production of inflammatory factors induced by lipopolysaccharide Chang Zhou a , Chen Zhang a,⁎, Song Chi a , Yiongfeng Xu b , Jijun Teng a , Haiping Wang a , Yuqiang Song a , Renliang Zhao a a Department of Neurology, The Affiliated Hospital of Medical College Qingdao University, 16 Jiangsu Road, Qingdao, Shandong, 266003, PR China b Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
There has been an increasing appreciation of the role that microglial cells play in neural
Accepted 22 February 2009
damage. Marrow stromal cells (MSCs) can dramatically lessen neural damage in animal
Available online 6 March 2009
models, but the mechanisms involved have not been defined. This study aimed to investigate the effects of human MSCs (hMSCs) on the activation of primary microglia and
Keywords:
the attendant production of pro-inflammatory factors stimulated by bacterial endotoxin
Microglial cell
lipopolysaccharide (LPS). Our study showed that hMSCs in co-cultures and in transwell
Human marrow stromal cell
cultures inhibited the activation of microglial cells, reduced the production of tumor
Lipopolysaccharide
necrosis factor-α (TNF-α) and nitric oxide (NO), downregulated the expression of inducible
Neurotrophic factor
nitric oxide synthase (iNOS) and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK), whereas hMSCs conditioned medium did not have any effect on microglial inflammation. To further investigate the mechanisms by which hMSCs exert antiinflammatory effects, we examined the production of neurotrophic factors by hMSCs with enzyme linked immunosorbent assay (ELISA). Our results showed that the production of insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and hepatocyte growth factor (HGF) was significantly increased by hMSCs when cultured in the conditioned medium from activated microglia. We conclude that hMSCs can inhibit microglial activation and the production of attendant inflammatory factors. In addition, hMSCs can interact with microglial cells through diffusible soluble factors, whereas cell contact is not a prerequisite for anti-inflammatory effects. Finally, hMSCs within inflammatory environment can significantly increase the production of neurotrophic factors, which may involve with the anti-inflammatory mechanisms. © 2009 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: +86 0532 8291 1999. E-mail address: [email protected] (C. Zhang). Abbreviations: BBB, blood-brain barrier; BDNF, brain-derived neurotrophic factor; CM, conditioned medium; CNS, central nervous system; ELISA, enzyme linked immunosorbent assay; HGF, hepatocyte growth factor; hMSCs, human MSCs; IGF-1, insulin-like growth factor-1; IFNγ, interferon γ; iNOS, inducible nitric oxide synthase; IL-1, interleukin-1β; LPS, lipopolysaccharide; MNCs, mononuclear cells; MSCs, marrow stromal cells; MTT, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; NO, nitric oxide; p38 MAPK, p38 mitogen-activated protein kinase; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.049
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1.
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Introduction
Microglial cells are the resident immune cells of the central nervous system (CNS). As the source of inflammation, activated microglia produce a variety of inflammatory factors, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and reactive oxygen species. Inflammation in the CNS has been closely associated with the
pathogenesis of neural damage resulting from cerebral ischemia and neurodegenerative diseases (Skaper, 2007; Wang, Tang and Yenari, 2007). Therefore, the effective control of microglial activation in these neurological diseases is regarded as an important therapeutic target. Stem cell transplantation is a promising therapeutic strategy for neural damage. Human marrow stromal cells (hMSCs) are isolated from adult bone marrow, and easy access suggests the feasibility in clinical therapies. These
Fig. 1 – Effects of hMSCs on LPS-induced activation of primary microglia. (A–E) Microglial cells were treated with vehicle (B), hMSCs co-cultures (C), hMSCs transwell cultures (D), and conditioned medium (CM) from hMSCs (E). Cells were stimulated with 1 μg/ml LPS for 6 h. Unstimulated microglia were as normal controls (A). Microglial cells were immunostained with an antibody against the F4/80 antigen, and hMSCs were stained with anti-HuNu antibody. (F) Quantitative cell counts of activated microglia per field (*p < 0.01 versus LPS alone).
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cells have the potential to differentiate to lineages of mesenchymal tissues — bone, cartilage, fat, and muscle. Autologous transplantation of hMSCs would circumvent potential ethical and immunological concerns. These advantages suggest that hMSCs may be the suitable candidates for human therapies. A number of studies in animal models suggest that infusion of marrow stromal cells (MSCs) can dramatically lessen neural damage (Chen et al., 2001; Honma et al., 2006; Park et al., 2008; Wu et al., 2007). Even with the encouraging results of cell transplantation, the mechanisms involved have not been defined. The functional replacement of lost motoneurons is proved very difficultly, and more likely cells exert their neuroprotective effects (Phinney and Prockop, 2007). Recently, the anti-inflammatory and immunomodulatory effects of MSCs have generated a great deal of interest (Iyer and Rojas, 2008; Uccelli et al., 2008). Thus, it would not be unexpected that the neuroprotective effects may involve with the anti-inflammatory activity of MSCs. Here, we investigated if hMSCs attenuate microglial activation and attendant inflammatory factor secretion stimulated by bacterial endotoxin lipopolysaccharide (LPS). In addition, we also focused on the mode of influence on microglial inflammation by hMSCs.
2.
Results
2.1. hMSCs inhibited the activation of microglial cells induced by LPS After stimulation with LPS for 6 h, activated microglia showed an enlarged shape and an intense immunoreactivity for F4/80 antigen, marker for murine microglia, whereas unstimulated microglia showed a small and rounded shape (Figs. 1A–E). The counts of enlarged microglia cultured in hMSCs conditioned medium were similar to that in vehicle-treated cultures (36.5 ± 1.3 versus 35.5 ± 2.1 per field; p > 0.05). Compared with vehicle-treated cultures, hMSCs significantly reduced the number of enlarged microglia in co-cultures (15.5 ± 5.3 per field; p < 0.01) and in transwell cultures (10.5 ± 3.2 per field; p < 0.01) after LPS stimulation for 6 h (Fig. 1F).
2.2. hMSCs reduced LPS-induced production of NO and TNF-α by microglial cells First, we investigated the effects of hMSCs on LPS-mediated NO production by microglial cells. Normal microglia produced very little amount of nitrite at 24 h (1.6 ± 0.6 μM). In contrast, LPS stimulation caused a moderate increase of nitrite accumulation at 12 h (3.6 ± 1.8 μM), and a dramatic increase of nitrite accumulation at 24 h (19.6 ± 1.2 μM). After stimulation of LPS for 24 h, NO production was significantly reduced by hMSCs in co-cultures (14.8 ± 2.3 μM; p < 0.01) and in transwell cultures (11.5 ± 1.2 μM; p < 0.01), whereas hMSCs conditioned medium was ineffective for NO production (18.6 ± 1.4 μM; p > 0.05; Fig. 2A). Additionally, nitrite accumulation in the medium of pure hMSCs cultures was insensitive to LPS stimulation. It is well known that LPS can induce inducible nitric oxide synthase (iNOS) and, in turn, the production of NO in microglial cells. To determine whether
Fig. 2 – Effects of hMSCs on LPS-induced nitrite production and iNOS expression by microglial cells. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglia were as normal controls. (A) Media were collected after stimulation for 24 h, and accumulated nitrite was detected with Greiss assay (*p < 0.01 versus LPS alone). (B) Protein was extracted after stimulation for 6 h. Protein expression of iNOS was analyzed through Western blot. (C) Quantitative evaluation through optical densitometry of iNOS blot and relative to β actin blot (*p < 0.01 versus LPS alone).
hMSCs could also inhibit LPS-mediated iNOS expression, Western blotting was carried out. The expression of iNOS was induced in microglial cells after 6 h exposure to LPS (Fig. 2B). A significant reduction in LPS-induced iNOS expression was observed in cultures treated with either hMSCs cocultures or hMSCs transwell cultures (p < 0.05; Fig. 2C). The reduction in LPS-induced iNOS expression was in line with the reduction of NO production in microglial cells by hMSCs. Next, we investigated the influence of hMSCs on the secretion of TNF-α by activated microglia. The secretion of TNF-α by microglial cells reached maximal level at 12 h (9.4 ± 0.8 ng/ml) and dropped at 24 h (4.2 ± 0.5 ng/ml) after
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Fig. 3 – Effects of hMSCs on LPS-induced TNF-α secretion by microglial cells. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglial cells were as normal controls. Media were collected after stimulation for 12 h, and TNF-α production was evaluated with ELISA (*p < 0.01 versus LPS alone).
Fig. 5 – Secretion of neurotrophic factors by hMSCs was evaluated with ELISA. Compared with cells cultured in normal condition, the productions of IGF-1, VEGF, BDNF, and HGF by hMSCs cultured in conditioned medium from activated microglia were significantly increased (*p < 0.01).
the level of phosphorylated p38 MAPK in microglial cells (p < 0.01; Fig. 4B). stimulation with LPS. Therefore the amount of TNF-α was determined at the time point of 12 h after stimulation with LPS to assess the effects of hMSCs. Compared to vehicle-treated microglia, hMSCs significantly reduced TNF-α secretion by 24% in co-cultures (p < 0.01) and by 35% in transwell cultures (p < 0.01), while hMSCs conditioned medium did not inhibit TNF-α secretion (p > 0.05; Fig. 3).
2.3. hMSCs downregulated the level of phosphorylated p38 MAPK We investigated whether the reduced secretion of NO and TNF-α by hMSCs involves with the downregulation of p38 mitogen-activated protein kinase (p38 MAPK) activation using Western blotting. Total p38 MAPK level was not significantly different between the groups (p > 0.05; Fig. 4A). Level of phosphorylated p38 MAPK in microglia was significantly increased after LPS stimulation for 6 h (p < 0.01). hMSCs in cocultures and in transwell cultures significantly downregulated
2.4. hMSCs produced more neurotrophic factors in inflammatory environment To understand the mechanisms by which hMSCs inhibited microglial activation, we examined the production of neurotrophic factors, including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) by hMSCs. Our results showed that the production of IGF-1, VEGF, BDNF and HGF by hMSCs cultured in conditioned medium from activated microglia was significantly increased compared to normal condition (Fig. 5; p < 0.01).
2.5.
hMSCs did not cause reduction of microglial viability
To verify that the decreased production of nitrite and TNF-α by microglial cells in co-cultures and in transwell cultures with hMSCs was not due to the reduction of microglial viability, we
Fig. 4 – Effects of hMSCs on the expression of phosphorylated p38 MAPK. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglia were as normal controls. Protein was extracted after stimulation for 6 h. (A) Protein expression of phosphorylated p38MAPK and total p38MAPK were analyzed through Western blot. (B) Quantitative evaluation through optical densitometry of phosphorylated p38MAPK blot and relative to total p38MAPK blot (*p < 0.01 versus LPS alone).
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Fig. 6 – MTT assay was performed to assess cell viability. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglia were as normal controls. Cell viability was examined after stimulation for 12 h (A) and 24 h (B). The basal level of absorbance value from untreated microglia was regarded as 100% (p < 0.05 versus LPS alone).
examined cell viability stimulated by 1 μg/ml LPS for 12 h and 24 h as measured by an MTT cell viability assay. The basal level of absorbance value from unstimulated microglia was regarded as 100%. LPS did not cause a reduction of microglial viability in pure cultures (p > 0.05). There was also no significant decrease in cell viability when microglial cells were treated with hMSCs in co-cultures or in transwell cultures (Fig. 6; p > 0.05).
3.
Discussions
In the present study we reported that hMSCs inhibited LPSstimulated microglial activation and the production of inflammatory factors. Furthermore, our results showed that hMSCs interacted with microglial cells through diffusible molecules. Finally, hMSCs within inflammatory environment could significantly increase the production of neurotrophic factors.
3.1. hMSCs inhibited LPS-stimulated microglial activation and the production of inflammatory factors The anti-inflammatory and immunomodulatory effects of MSCs have only been described recently in vivo and in vitro.
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In the mouse model of multiple sclerosis, MSCs delivered intravenously decrease the infiltration of the CNS by T cell, and inhibit the production of pathogenic proteolipid protein (PLP)-specific antibodies (Kassis et al., 2008). In the animal models of lung injury and myocardial infarction, MSCs have also been shown to decrease the inflammatory reaction (Guo et al., 2007; Iyer and Rojas, 2008). More recently, some studies showed that MSCs inhibit microglial activation and inflammatory response in the animal models of neural damages (Kim et al., 2009; Ohtaki et al., 2008; Vercelli et al., 2008). Moreover, MSCs can interact with cells of both the innate and adaptive immune system in vitro. MSCs can decrease the pro-inflammatory potential of dendritic cells and natural killer cells by inhibiting the production of inflammatory factors such as TNF-α and interferon γ (IFNγ) (Aggarwal and Pittenger, 2005; Spaggiari et al., 2006). MSCs inhibit T-cell proliferation, decrease IFNγ production, and increase IL-4 production, which indicate a shift in T cells from a pro-inflammatory (IFNγ-producing) state to an antiinflammatory (IL-4-producing) state (Aggarwal and Pittenger, 2005). Nevertheless the potential immunomodulatory effects of MSCs on primary microglia have to date not been fully evaluated. Microglial cells play a critical role as resident immunocompetent cells within the CNS. Upon activation, they produce a variety of pro-inflammatory and potentially cytotoxic factors, including NO, TNF-α, IL-1, and reactive oxygen species. It is now generally accepted that excessive production and/or accumulation of these factors are responsible for the neuronal death (Monk and Shaw, 2006; Sargsyan et al., 2005). Anti-inflammatory compounds such as minocycline and celecoxib have been shown to be neuroprotective in the animal models of neural damage (Drachman et al., 2002; Zhu et al., 2002). Here, we showed that hMSCs inhibited microglial activation and reduced the production of NO and TNF-α by activated microglia in vitro. Thus, it would not be unexpected that the anti-inflammatory effects of hMSCs may be a critical mechanism by which hMSCs administration can protect neurons. Additionally, the p38 MAPK signaling pathway plays an important role in inflammation. Inflammatory stimuli can activate p38 MAPK via phosphorylation in glial cells, and then activated p38 MAPK promotes inflammatory gene expression (Saklatvala, 2004). Selective p38 MAPK inhibitors can inhibit the production of inflammatory cytokine by LPS-stimulated cells (Kumar et al., 2008). Our study showed that hMSCs downregulated p38 MAPK activation in microglial cells. The downregulation of p38 MAPK activation may involve with the inhibitory mechanism of LPS-mediated inflammatory response by hMSCs. For the inhibitory effects of hMSCs on the production of inflammatory factors by LPS-stimulated microglia, we have considered a possibility that whether there was a reduction of microglial viability. However there was no significant difference of cell viability between hMSCs-treated cultures and vehicle-treated cultures. Additionally, we also observed that the presence of hMSCs markedly decreased the morphological change of microglial cells. These findings indicate that hMSCs definitely can inhibit inflammatory response, which is not due to the reduction of microglial viability.
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3.2. Mode of influence on microglial inflammation by hMSCs Although a large number of studies have documented the immunosuppressive activities of MSCs, the underlying mechanisms are only partially known (Uccelli et al., 2008). To investigate the mode of anti-inflammatory effects by hMSCs, microglial cells were treated with either hMSCs cocultures that two cells interacted through contact, hMSCs transwell cultures that two cells interacted through diffusible molecules only, or hMSCs conditioned media that two cells did not interacted. Our study showed that hMSCs could reduce microglial activation through either co-cultures or transwell cultures. Our results suggest that hMSCs can reduce microglial inflammation through diffusible soluble factors, whereas cell contact is not a prerequisite for antiinflammatory effects of hMSCs. Additionally, conditioned medium from hMSCs has no effect on microglial inflammation, which suggests that hMSCs exert anti-inflammatory effects through a positive process. Recent study showed that TNF-α could strongly stimulate the production of growth factors by hMSCs (Wang et al., 2006). Therefore, hMSCs may response to inflammatory cues and then secrete some diffusible soluble factors adjusted to the cues to exert antiinflammatory effects.
4.
Experimental procedures
4.1.
Cell cultures
4.1.1.
Preparation of hMSCs
After obtained informed consent, 2 ml bone marrow was aspirated from the iliac crest of healthy normal volunteers. The protocol was approved by the Institutional Review Board of Qingdao University. Mononuclear cells (MNCs) were isolated by Ficoll density gradient centrifugation (1.077 g/ ml, Sigma, Germany) at 400 ×g for 35 min. MNCs were resuspended in the culture medium composed of Dulbecco's modified Eagle's medium (DMEM) with low glucose and 10% fetal bovine serum (FBS). MNCs were plated at 1 × 106 cells/ 25 cm2 in culture flasks and the cultures were incubated at 37 °C in 5% CO2 in air and 95% humidity. The medium was exchanged after 48 h and every 3–4 days thereafter. When the cultures reached approximately 90% of confluence, cells were passaged with 0.25% trypsin and replated into passage culture at a density of 5000–10,000 cells per cm2. Cells at passage 3–4 were used for experiments. The identity of hMSCs was confirmed with flow cytometry by immunophenotypic criteria (CD34−/CD45−; CD29+/CD44+).
4.1.2. 3.3. Production of neurotrophic factors increased in inflammatory environment by hMSCs hMSCs can produce a variety of neurotrophic factors (Chen et al., 2002; Wang et al., 2006). Resent studies have shown that neurotrophic factors can inhibit inflammation (Flügel et al., 2001; Gong, 2008), (Makar et al., 2008; Linker et al., 2002), so we investigated the production of neurotrophic factors by hMSCs cultured in conditioned medium from activated microglia. Our results showed that the production of neurotrophic factors, including IGF-1, VEGF, BDNF and HGF, was significantly increased by hMSCs within inflammatory environment. Therefore, the anti-inflammatory effects of hMSCs may involve with the secretion of neurotrophic factors. The production of several trophic factors is all increased in our study, so we speculate that none of these molecules has an exclusive role, and the anti-inflammatory effects of hMSCs are mediated by several factors. Moreover, a cocktail of trophic factors are positively released by hMSCs adjusted to inflammatory cues, so MSCs may provide a superior strategy than administration of exogenous growth factors. Further study aimed at neutralizing trophic factors is needed to verify the hypotheses, with the hope of achieving some useful therapeutic intervention for neural injury by MSCs. In conclusion, hMSCs can inhibit microglial activation and the production of attendant inflammatory factors in cocultures and in transwell cultures. In addition, hMSCs can interact with microglial cells through diffusible soluble factors, whereas cell contact is not a prerequisite for antiinflammatory effects. Finally, hMSCs within inflammatory environment can significantly increase the production of neurotrophic factors, which may involve with the antiinflammatory mechanisms.
Microglial cultures
Primary microglial cells were cultured from newborn C57/BL6 mice by a method described previously with a mild modification (Liu et al., 2000). In brief, the meninges were removed from the forebrains, and tissues collected from forebrains were triturated into single cells using fire-polished long Pasteur pipettes in serum-free media. Cells were plated onto 75 cm2 flask in DMEM with high glucose and 10% FBS and incubated at 37 °C in 5% CO2 in air and 95% humidity. The culture medium was replenished after 24 h and incubated for 10 days. Microglial cells were then purified from the initial mixed culture by sequential shaking at 180 rpm for 30 min at room temperature. The resultant supernatant was collected and centrifuged at 150 ×g for 5 min. The pellet was suspended in complete medium and then plated in 24-well culture plates. After 1 h, microglial cells were further purified by washing twice with serum-free media and grown in new complete media. To determine the purity of the microglial cells, immunocytochemical analysis was carried out using rat anti-mouse F4/80 antibody. These cultures were >95% F4/80 positive as determined by immunocytochemistry indicating that they were composed of microglial cells.
4.2.
Treatment
2 × 105 hMSCs were plated in 25 cm2 flask in 5 ml of DMEM with high glucose and 10% FBS. The culture medium was replenished after 24 h and incubated for 24 h. The resultant supernatant was collected and centrifuged at 150 ×g for 5 min to remove cellular debris as hMSCs conditioned medium. Microglial cells were randomly divided into: 1) coculture group: 1 × 105 microglial cells and 5 × 104 hMSCs were plated in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS; 2) transwell culture group: 1 × 105 microglial cells were plated
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in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS, and 5 × 104 hMSCs were plated in cell culture inserts (1 μm pore size) in 300 μl of DMEM with high glucose and 10% FBS; 3) conditioned medium group: 1 × 105 microglial cells were cultured in 24 well plates in 1 ml of hMSCs conditioned medium; 4) vehicle group: 1 × 105 microglial cells were plated in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS. Microglial cells were stimulated with 1 μg/ml LPS based on the literature. Unstimulated microglia were as normal controls. The experiments were carried out in triplicate and each experiment was repeated three times.
4.3.
Immunocytochemistry
Microglial cells were stained with an antibody against F4/80. Human-specific antibody against HuNu was used as a marker for hMSCs. In brief, the cultures were washed with phosphatebuffered saline (PBS), fixed in 4% paraformaldehyde in PBS (PH 7.4) for 20 min, blocked with normal serum, and then incubated with primary antibody at 4 °C overnight. Primary antibodies included: rat anti-mouse F4/80 (1:50; Serotec), mouse anti-human HuNu (1:50; Chemicon). Cultures were washed and incubated at RT for 1 h with goat anti-rat FITCconjugated IgG (1:50; Serotec) or goat anti-mouse CY3conjugated IgG (1:200; Chemicon). These cells were counterstained with DAPI. All staining involved the use of a negative control (secondary antibody alone) in order to evaluate the specificity of the antibodies. The number of enlarged microglia in a defined field was manually counted under a microscope at 400× magnification. Five representative fields were counted for each well of the 24well plates. The results are the mean ± S.M.E. of three separate experiments performed in triplicate.
4.4.
Measurement of TNF-α
The culture medium was collected after stimulation for 12 h with LPS. TNF-α concentration in the culture medium was determined by mice specific enzyme linked immunosorbent assay (ELISA) kit (BioSource International) according to the manufacturer's instructions, and samples were diluted 50-fold to fit the standard curve.
4.5.
Nitrite assay
The accumulation of nitrite in the culture supernatant, a stable end product of NO, was determined by using a colorimetric reaction with the Griess reagent. Briefly, culture supernatant was mixed with an equal volume of the Griess reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4). The mixture was incubated for 10 min at room temperature, and the absorbance at 540 nm was determined with a microplate reader. The concentration of nitrite was determined from a sodium nitrite standard curve.
Signaling Technology) containing protease inhibitor. Cells were scraped, sonicated briefly, and centrifuged at 14,000 g for 30 min at 4 °C. Protein extracts (20 μg) of cells were boiled in loading buffer for 3 min and subsequently were loaded into wells of 12% SDS-polyacrylamide gels at 80 V for 30 min and 150 V for 90 min. Proteins were then transferred onto PVDF membranes (Minipore). The blots were blocked with 5%(w/v) defatted milk in TBS-Tween20 (PH8.0) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies: polyclonal rabbit anti-mouse iNOS (1:1000; Santa Cruz Biotechnology), polyclonal rabbit antimouse phosphospecific p38 MAPK (1:1000; Chemicon), polyclonal rabbit anti-mouse total p38 MAPK (1:1000; cell signaling Technology), and mouse anti-actin (1:1000; Santa Cruz Biotechnology). The blots were washed three times in TBST for 10 min each, incubated with a HRP-conjugated goat anti-rabbit IgG (1:5000; Chemicon) or a HRP-conjugated goat anti-mouse IgG (1:2000; Chemicon) for 1 h at room temperature, washed four times in TBST for 10 min each. Protein bands were visualized after incubation with SuperSignal west Pico chemiluminescent Substrate (Pierce Biotechnology) and exposure of high performance chemiluminescence film to the membrane surface in the dark. Quantitative analysis of the intensity of the bands was performed with Band Scan 5.0 software.
4.7.
Measurement of trophic factors
hMSCs were randomly divided into: 1) conditioned medium group: 2 × 106 microglial cells were plated in 25 cm2 flask in 5 ml of DMEM with high glucose and 10% FBS. The culture medium was replenished after 12 h, and then 1 μg/ml LPS was added to cultures. After incubation for 24 h, the resultant supernatant was collected and centrifuged at 150 ×g for 5 min to remove cellular debris as conditioned medium of activated microglia. 5 × 104 hMSCs were cultured in 24 well plates in 1 ml of conditioned medium; 2) control group: 5 × 104 hMSCs were plated in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS. The culture medium was collected after incubation for 48 h. Production of VEGF, BDNF, IGF-I, and HGF in the culture medium was determined with ELISA using a commercially available kit (R & D Systems) according to the manufacturer's instructions.
4.8.
MTT assay
Cell viability was measured using an MTT cell viability assay. Medium was removed, and 100 μl fresh medium was added to each well of 96-well plates. MTT (Sigma) was added to the cultures at a final concentration of 1 mg/ml, and the cells were incubated for 4 h. Cells were lysed in DMSO. Colorimetric determination of MTT reduction was measured on the plate reader at 490 nm.
4.9. 4.6.
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Statistical analysis
Western blotting
Cells in 24 well plates were washed with PBS, and then incubated for 5 min on ice in 40 μl of lysis buffer (Cell
Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. The results are presented as mean ± SD.
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Acknowledgment The work was supported by a grant from Natural Science Fund of Shandong Province (32693). REFERENCES
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neurotrophic cytokine as modulator in neuroinflammation. Nat. Med. 8, 620–624. Makar, T.K., Trisler, D., Sura, K.T., 2008. Brain derived neurotrophic factor treatment reduces inflammation and apoptosis in experimental allergic encephalomyelitis. J. Neurol. Sci. 270, 70–76. Monk, P.N., Shaw, P.J., 2006. ALS: life and death in a bad neighborhood. Nat. Med. 12, 885–887. Ohtaki, H., Ylostalo, J.H., Foraker, J.E., 2008. Stem/progenitor cells from bone marrow decrease neuronal death in global ischemia by modulation of inflammatory/immune responses. Proc. Natl. Acad. Sci. U. S. A. 105, 14638–14643. Park, H.J., Lee, P.H., Bang, O.Y., 2008. Mesenchymal stem cells therapy exerts neuroprotection in a progressive animal model of Parkinson's disease. J. Neurochem. 107, 141–151. Phinney, D.G., Prockop, D.J., 2007. Concise review: mesenchymal stem/multipotent stromal cells: the state of transdifferentiation and modes of tissue repair—current views. Stem Cells 25, 2896–2902. Saklatvala, J., 2004. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr. Opin. Pharmacol. 4, 372–377. Sargsyan, S.A., Monk, P.N., Shaw, P.J., 2005. Microglial cells as potential contributors to motor neuron injury in amyotrophic lateral sclerosis. Glia 51, 241–253. Skaper, S.D., 2007. The brain as a target for inflammatory processes and neuroprotective strategies. Ann. N. Y. Acad. Sci. 1122, 23–34. Spaggiari, G.M., Capobianco, A., Becchetti, S., 2006. Mesenchymal stem cell–natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 107, 1484–1490. Uccelli, A., Moretta, L., Pistoia, V., 2008. Mesenchymal stem cells in health and disease. Nat. Rev. Immunol. 8, 726–736. Vercelli, A., Mereuta, O.M., Garbossa, D., 2008. Human mesenchymal stem cell transplantation extends survival, improves motor performance and decreases neuroinflammation in mouse model of amyotrophic lateral sclerosis. Neurobiol. Dis. 31, 395–405. Wang, M., Crisostomo, P.R., Herring, C., 2006. Human progenitor cells from bone marrow or adipose tissue produce VEGF, HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R880–R884. Wang, Q., Tang, X.N., Yenari, M.A., 2007. The inflammatory response in stroke. J. Neuroimmunol. 184, 53–68. Wu, Q.Y., Li, J., Feng, Z.T., 2007. Bone marrow stromal cells of transgenic mice can improve the cognitive ability of an Alzheimer's disease rat model. Neurosci. Lett. 417, 281–285. Zhu, S., Stavrovskaya, I.G., Drozda, M., 2002. Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature 417, 74–78.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Effects of human marrow stromal cells on activation of microglial cells and production of inflammatory factors induced by lipopolysaccharide Chang Zhou a , Chen Zhang a,⁎, Song Chi a , Yiongfeng Xu b , Jijun Teng a , Haiping Wang a , Yuqiang Song a , Renliang Zhao a a Department of Neurology, The Affiliated Hospital of Medical College Qingdao University, 16 Jiangsu Road, Qingdao, Shandong, 266003, PR China b Department of Neurology, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
There has been an increasing appreciation of the role that microglial cells play in neural
Accepted 22 February 2009
damage. Marrow stromal cells (MSCs) can dramatically lessen neural damage in animal
Available online 6 March 2009
models, but the mechanisms involved have not been defined. This study aimed to investigate the effects of human MSCs (hMSCs) on the activation of primary microglia and
Keywords:
the attendant production of pro-inflammatory factors stimulated by bacterial endotoxin
Microglial cell
lipopolysaccharide (LPS). Our study showed that hMSCs in co-cultures and in transwell
Human marrow stromal cell
cultures inhibited the activation of microglial cells, reduced the production of tumor
Lipopolysaccharide
necrosis factor-α (TNF-α) and nitric oxide (NO), downregulated the expression of inducible
Neurotrophic factor
nitric oxide synthase (iNOS) and phosphorylated p38 mitogen-activated protein kinase (p38 MAPK), whereas hMSCs conditioned medium did not have any effect on microglial inflammation. To further investigate the mechanisms by which hMSCs exert antiinflammatory effects, we examined the production of neurotrophic factors by hMSCs with enzyme linked immunosorbent assay (ELISA). Our results showed that the production of insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), and hepatocyte growth factor (HGF) was significantly increased by hMSCs when cultured in the conditioned medium from activated microglia. We conclude that hMSCs can inhibit microglial activation and the production of attendant inflammatory factors. In addition, hMSCs can interact with microglial cells through diffusible soluble factors, whereas cell contact is not a prerequisite for anti-inflammatory effects. Finally, hMSCs within inflammatory environment can significantly increase the production of neurotrophic factors, which may involve with the anti-inflammatory mechanisms. © 2009 Elsevier B.V. All rights reserved.
⁎ Corresponding author. Fax: +86 0532 8291 1999. E-mail address: [email protected] (C. Zhang). Abbreviations: BBB, blood-brain barrier; BDNF, brain-derived neurotrophic factor; CM, conditioned medium; CNS, central nervous system; ELISA, enzyme linked immunosorbent assay; HGF, hepatocyte growth factor; hMSCs, human MSCs; IGF-1, insulin-like growth factor-1; IFNγ, interferon γ; iNOS, inducible nitric oxide synthase; IL-1, interleukin-1β; LPS, lipopolysaccharide; MNCs, mononuclear cells; MSCs, marrow stromal cells; MTT, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide; NO, nitric oxide; p38 MAPK, p38 mitogen-activated protein kinase; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.02.049
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1.
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Introduction
Microglial cells are the resident immune cells of the central nervous system (CNS). As the source of inflammation, activated microglia produce a variety of inflammatory factors, including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and reactive oxygen species. Inflammation in the CNS has been closely associated with the
pathogenesis of neural damage resulting from cerebral ischemia and neurodegenerative diseases (Skaper, 2007; Wang, Tang and Yenari, 2007). Therefore, the effective control of microglial activation in these neurological diseases is regarded as an important therapeutic target. Stem cell transplantation is a promising therapeutic strategy for neural damage. Human marrow stromal cells (hMSCs) are isolated from adult bone marrow, and easy access suggests the feasibility in clinical therapies. These
Fig. 1 – Effects of hMSCs on LPS-induced activation of primary microglia. (A–E) Microglial cells were treated with vehicle (B), hMSCs co-cultures (C), hMSCs transwell cultures (D), and conditioned medium (CM) from hMSCs (E). Cells were stimulated with 1 μg/ml LPS for 6 h. Unstimulated microglia were as normal controls (A). Microglial cells were immunostained with an antibody against the F4/80 antigen, and hMSCs were stained with anti-HuNu antibody. (F) Quantitative cell counts of activated microglia per field (*p < 0.01 versus LPS alone).
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cells have the potential to differentiate to lineages of mesenchymal tissues — bone, cartilage, fat, and muscle. Autologous transplantation of hMSCs would circumvent potential ethical and immunological concerns. These advantages suggest that hMSCs may be the suitable candidates for human therapies. A number of studies in animal models suggest that infusion of marrow stromal cells (MSCs) can dramatically lessen neural damage (Chen et al., 2001; Honma et al., 2006; Park et al., 2008; Wu et al., 2007). Even with the encouraging results of cell transplantation, the mechanisms involved have not been defined. The functional replacement of lost motoneurons is proved very difficultly, and more likely cells exert their neuroprotective effects (Phinney and Prockop, 2007). Recently, the anti-inflammatory and immunomodulatory effects of MSCs have generated a great deal of interest (Iyer and Rojas, 2008; Uccelli et al., 2008). Thus, it would not be unexpected that the neuroprotective effects may involve with the anti-inflammatory activity of MSCs. Here, we investigated if hMSCs attenuate microglial activation and attendant inflammatory factor secretion stimulated by bacterial endotoxin lipopolysaccharide (LPS). In addition, we also focused on the mode of influence on microglial inflammation by hMSCs.
2.
Results
2.1. hMSCs inhibited the activation of microglial cells induced by LPS After stimulation with LPS for 6 h, activated microglia showed an enlarged shape and an intense immunoreactivity for F4/80 antigen, marker for murine microglia, whereas unstimulated microglia showed a small and rounded shape (Figs. 1A–E). The counts of enlarged microglia cultured in hMSCs conditioned medium were similar to that in vehicle-treated cultures (36.5 ± 1.3 versus 35.5 ± 2.1 per field; p > 0.05). Compared with vehicle-treated cultures, hMSCs significantly reduced the number of enlarged microglia in co-cultures (15.5 ± 5.3 per field; p < 0.01) and in transwell cultures (10.5 ± 3.2 per field; p < 0.01) after LPS stimulation for 6 h (Fig. 1F).
2.2. hMSCs reduced LPS-induced production of NO and TNF-α by microglial cells First, we investigated the effects of hMSCs on LPS-mediated NO production by microglial cells. Normal microglia produced very little amount of nitrite at 24 h (1.6 ± 0.6 μM). In contrast, LPS stimulation caused a moderate increase of nitrite accumulation at 12 h (3.6 ± 1.8 μM), and a dramatic increase of nitrite accumulation at 24 h (19.6 ± 1.2 μM). After stimulation of LPS for 24 h, NO production was significantly reduced by hMSCs in co-cultures (14.8 ± 2.3 μM; p < 0.01) and in transwell cultures (11.5 ± 1.2 μM; p < 0.01), whereas hMSCs conditioned medium was ineffective for NO production (18.6 ± 1.4 μM; p > 0.05; Fig. 2A). Additionally, nitrite accumulation in the medium of pure hMSCs cultures was insensitive to LPS stimulation. It is well known that LPS can induce inducible nitric oxide synthase (iNOS) and, in turn, the production of NO in microglial cells. To determine whether
Fig. 2 – Effects of hMSCs on LPS-induced nitrite production and iNOS expression by microglial cells. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglia were as normal controls. (A) Media were collected after stimulation for 24 h, and accumulated nitrite was detected with Greiss assay (*p < 0.01 versus LPS alone). (B) Protein was extracted after stimulation for 6 h. Protein expression of iNOS was analyzed through Western blot. (C) Quantitative evaluation through optical densitometry of iNOS blot and relative to β actin blot (*p < 0.01 versus LPS alone).
hMSCs could also inhibit LPS-mediated iNOS expression, Western blotting was carried out. The expression of iNOS was induced in microglial cells after 6 h exposure to LPS (Fig. 2B). A significant reduction in LPS-induced iNOS expression was observed in cultures treated with either hMSCs cocultures or hMSCs transwell cultures (p < 0.05; Fig. 2C). The reduction in LPS-induced iNOS expression was in line with the reduction of NO production in microglial cells by hMSCs. Next, we investigated the influence of hMSCs on the secretion of TNF-α by activated microglia. The secretion of TNF-α by microglial cells reached maximal level at 12 h (9.4 ± 0.8 ng/ml) and dropped at 24 h (4.2 ± 0.5 ng/ml) after
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Fig. 3 – Effects of hMSCs on LPS-induced TNF-α secretion by microglial cells. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglial cells were as normal controls. Media were collected after stimulation for 12 h, and TNF-α production was evaluated with ELISA (*p < 0.01 versus LPS alone).
Fig. 5 – Secretion of neurotrophic factors by hMSCs was evaluated with ELISA. Compared with cells cultured in normal condition, the productions of IGF-1, VEGF, BDNF, and HGF by hMSCs cultured in conditioned medium from activated microglia were significantly increased (*p < 0.01).
the level of phosphorylated p38 MAPK in microglial cells (p < 0.01; Fig. 4B). stimulation with LPS. Therefore the amount of TNF-α was determined at the time point of 12 h after stimulation with LPS to assess the effects of hMSCs. Compared to vehicle-treated microglia, hMSCs significantly reduced TNF-α secretion by 24% in co-cultures (p < 0.01) and by 35% in transwell cultures (p < 0.01), while hMSCs conditioned medium did not inhibit TNF-α secretion (p > 0.05; Fig. 3).
2.3. hMSCs downregulated the level of phosphorylated p38 MAPK We investigated whether the reduced secretion of NO and TNF-α by hMSCs involves with the downregulation of p38 mitogen-activated protein kinase (p38 MAPK) activation using Western blotting. Total p38 MAPK level was not significantly different between the groups (p > 0.05; Fig. 4A). Level of phosphorylated p38 MAPK in microglia was significantly increased after LPS stimulation for 6 h (p < 0.01). hMSCs in cocultures and in transwell cultures significantly downregulated
2.4. hMSCs produced more neurotrophic factors in inflammatory environment To understand the mechanisms by which hMSCs inhibited microglial activation, we examined the production of neurotrophic factors, including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-1) and brain-derived neurotrophic factor (BDNF) by hMSCs. Our results showed that the production of IGF-1, VEGF, BDNF and HGF by hMSCs cultured in conditioned medium from activated microglia was significantly increased compared to normal condition (Fig. 5; p < 0.01).
2.5.
hMSCs did not cause reduction of microglial viability
To verify that the decreased production of nitrite and TNF-α by microglial cells in co-cultures and in transwell cultures with hMSCs was not due to the reduction of microglial viability, we
Fig. 4 – Effects of hMSCs on the expression of phosphorylated p38 MAPK. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglia were as normal controls. Protein was extracted after stimulation for 6 h. (A) Protein expression of phosphorylated p38MAPK and total p38MAPK were analyzed through Western blot. (B) Quantitative evaluation through optical densitometry of phosphorylated p38MAPK blot and relative to total p38MAPK blot (*p < 0.01 versus LPS alone).
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Fig. 6 – MTT assay was performed to assess cell viability. Microglial cells were treated with vehicle, hMSCs co-cultures, hMSCs transwell cultures, and CM from hMSCs. Cells were stimulated with 1 μg/ml LPS. Unstimulated microglia were as normal controls. Cell viability was examined after stimulation for 12 h (A) and 24 h (B). The basal level of absorbance value from untreated microglia was regarded as 100% (p < 0.05 versus LPS alone).
examined cell viability stimulated by 1 μg/ml LPS for 12 h and 24 h as measured by an MTT cell viability assay. The basal level of absorbance value from unstimulated microglia was regarded as 100%. LPS did not cause a reduction of microglial viability in pure cultures (p > 0.05). There was also no significant decrease in cell viability when microglial cells were treated with hMSCs in co-cultures or in transwell cultures (Fig. 6; p > 0.05).
3.
Discussions
In the present study we reported that hMSCs inhibited LPSstimulated microglial activation and the production of inflammatory factors. Furthermore, our results showed that hMSCs interacted with microglial cells through diffusible molecules. Finally, hMSCs within inflammatory environment could significantly increase the production of neurotrophic factors.
3.1. hMSCs inhibited LPS-stimulated microglial activation and the production of inflammatory factors The anti-inflammatory and immunomodulatory effects of MSCs have only been described recently in vivo and in vitro.
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In the mouse model of multiple sclerosis, MSCs delivered intravenously decrease the infiltration of the CNS by T cell, and inhibit the production of pathogenic proteolipid protein (PLP)-specific antibodies (Kassis et al., 2008). In the animal models of lung injury and myocardial infarction, MSCs have also been shown to decrease the inflammatory reaction (Guo et al., 2007; Iyer and Rojas, 2008). More recently, some studies showed that MSCs inhibit microglial activation and inflammatory response in the animal models of neural damages (Kim et al., 2009; Ohtaki et al., 2008; Vercelli et al., 2008). Moreover, MSCs can interact with cells of both the innate and adaptive immune system in vitro. MSCs can decrease the pro-inflammatory potential of dendritic cells and natural killer cells by inhibiting the production of inflammatory factors such as TNF-α and interferon γ (IFNγ) (Aggarwal and Pittenger, 2005; Spaggiari et al., 2006). MSCs inhibit T-cell proliferation, decrease IFNγ production, and increase IL-4 production, which indicate a shift in T cells from a pro-inflammatory (IFNγ-producing) state to an antiinflammatory (IL-4-producing) state (Aggarwal and Pittenger, 2005). Nevertheless the potential immunomodulatory effects of MSCs on primary microglia have to date not been fully evaluated. Microglial cells play a critical role as resident immunocompetent cells within the CNS. Upon activation, they produce a variety of pro-inflammatory and potentially cytotoxic factors, including NO, TNF-α, IL-1, and reactive oxygen species. It is now generally accepted that excessive production and/or accumulation of these factors are responsible for the neuronal death (Monk and Shaw, 2006; Sargsyan et al., 2005). Anti-inflammatory compounds such as minocycline and celecoxib have been shown to be neuroprotective in the animal models of neural damage (Drachman et al., 2002; Zhu et al., 2002). Here, we showed that hMSCs inhibited microglial activation and reduced the production of NO and TNF-α by activated microglia in vitro. Thus, it would not be unexpected that the anti-inflammatory effects of hMSCs may be a critical mechanism by which hMSCs administration can protect neurons. Additionally, the p38 MAPK signaling pathway plays an important role in inflammation. Inflammatory stimuli can activate p38 MAPK via phosphorylation in glial cells, and then activated p38 MAPK promotes inflammatory gene expression (Saklatvala, 2004). Selective p38 MAPK inhibitors can inhibit the production of inflammatory cytokine by LPS-stimulated cells (Kumar et al., 2008). Our study showed that hMSCs downregulated p38 MAPK activation in microglial cells. The downregulation of p38 MAPK activation may involve with the inhibitory mechanism of LPS-mediated inflammatory response by hMSCs. For the inhibitory effects of hMSCs on the production of inflammatory factors by LPS-stimulated microglia, we have considered a possibility that whether there was a reduction of microglial viability. However there was no significant difference of cell viability between hMSCs-treated cultures and vehicle-treated cultures. Additionally, we also observed that the presence of hMSCs markedly decreased the morphological change of microglial cells. These findings indicate that hMSCs definitely can inhibit inflammatory response, which is not due to the reduction of microglial viability.
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3.2. Mode of influence on microglial inflammation by hMSCs Although a large number of studies have documented the immunosuppressive activities of MSCs, the underlying mechanisms are only partially known (Uccelli et al., 2008). To investigate the mode of anti-inflammatory effects by hMSCs, microglial cells were treated with either hMSCs cocultures that two cells interacted through contact, hMSCs transwell cultures that two cells interacted through diffusible molecules only, or hMSCs conditioned media that two cells did not interacted. Our study showed that hMSCs could reduce microglial activation through either co-cultures or transwell cultures. Our results suggest that hMSCs can reduce microglial inflammation through diffusible soluble factors, whereas cell contact is not a prerequisite for antiinflammatory effects of hMSCs. Additionally, conditioned medium from hMSCs has no effect on microglial inflammation, which suggests that hMSCs exert anti-inflammatory effects through a positive process. Recent study showed that TNF-α could strongly stimulate the production of growth factors by hMSCs (Wang et al., 2006). Therefore, hMSCs may response to inflammatory cues and then secrete some diffusible soluble factors adjusted to the cues to exert antiinflammatory effects.
4.
Experimental procedures
4.1.
Cell cultures
4.1.1.
Preparation of hMSCs
After obtained informed consent, 2 ml bone marrow was aspirated from the iliac crest of healthy normal volunteers. The protocol was approved by the Institutional Review Board of Qingdao University. Mononuclear cells (MNCs) were isolated by Ficoll density gradient centrifugation (1.077 g/ ml, Sigma, Germany) at 400 ×g for 35 min. MNCs were resuspended in the culture medium composed of Dulbecco's modified Eagle's medium (DMEM) with low glucose and 10% fetal bovine serum (FBS). MNCs were plated at 1 × 106 cells/ 25 cm2 in culture flasks and the cultures were incubated at 37 °C in 5% CO2 in air and 95% humidity. The medium was exchanged after 48 h and every 3–4 days thereafter. When the cultures reached approximately 90% of confluence, cells were passaged with 0.25% trypsin and replated into passage culture at a density of 5000–10,000 cells per cm2. Cells at passage 3–4 were used for experiments. The identity of hMSCs was confirmed with flow cytometry by immunophenotypic criteria (CD34−/CD45−; CD29+/CD44+).
4.1.2. 3.3. Production of neurotrophic factors increased in inflammatory environment by hMSCs hMSCs can produce a variety of neurotrophic factors (Chen et al., 2002; Wang et al., 2006). Resent studies have shown that neurotrophic factors can inhibit inflammation (Flügel et al., 2001; Gong, 2008), (Makar et al., 2008; Linker et al., 2002), so we investigated the production of neurotrophic factors by hMSCs cultured in conditioned medium from activated microglia. Our results showed that the production of neurotrophic factors, including IGF-1, VEGF, BDNF and HGF, was significantly increased by hMSCs within inflammatory environment. Therefore, the anti-inflammatory effects of hMSCs may involve with the secretion of neurotrophic factors. The production of several trophic factors is all increased in our study, so we speculate that none of these molecules has an exclusive role, and the anti-inflammatory effects of hMSCs are mediated by several factors. Moreover, a cocktail of trophic factors are positively released by hMSCs adjusted to inflammatory cues, so MSCs may provide a superior strategy than administration of exogenous growth factors. Further study aimed at neutralizing trophic factors is needed to verify the hypotheses, with the hope of achieving some useful therapeutic intervention for neural injury by MSCs. In conclusion, hMSCs can inhibit microglial activation and the production of attendant inflammatory factors in cocultures and in transwell cultures. In addition, hMSCs can interact with microglial cells through diffusible soluble factors, whereas cell contact is not a prerequisite for antiinflammatory effects. Finally, hMSCs within inflammatory environment can significantly increase the production of neurotrophic factors, which may involve with the antiinflammatory mechanisms.
Microglial cultures
Primary microglial cells were cultured from newborn C57/BL6 mice by a method described previously with a mild modification (Liu et al., 2000). In brief, the meninges were removed from the forebrains, and tissues collected from forebrains were triturated into single cells using fire-polished long Pasteur pipettes in serum-free media. Cells were plated onto 75 cm2 flask in DMEM with high glucose and 10% FBS and incubated at 37 °C in 5% CO2 in air and 95% humidity. The culture medium was replenished after 24 h and incubated for 10 days. Microglial cells were then purified from the initial mixed culture by sequential shaking at 180 rpm for 30 min at room temperature. The resultant supernatant was collected and centrifuged at 150 ×g for 5 min. The pellet was suspended in complete medium and then plated in 24-well culture plates. After 1 h, microglial cells were further purified by washing twice with serum-free media and grown in new complete media. To determine the purity of the microglial cells, immunocytochemical analysis was carried out using rat anti-mouse F4/80 antibody. These cultures were >95% F4/80 positive as determined by immunocytochemistry indicating that they were composed of microglial cells.
4.2.
Treatment
2 × 105 hMSCs were plated in 25 cm2 flask in 5 ml of DMEM with high glucose and 10% FBS. The culture medium was replenished after 24 h and incubated for 24 h. The resultant supernatant was collected and centrifuged at 150 ×g for 5 min to remove cellular debris as hMSCs conditioned medium. Microglial cells were randomly divided into: 1) coculture group: 1 × 105 microglial cells and 5 × 104 hMSCs were plated in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS; 2) transwell culture group: 1 × 105 microglial cells were plated
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in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS, and 5 × 104 hMSCs were plated in cell culture inserts (1 μm pore size) in 300 μl of DMEM with high glucose and 10% FBS; 3) conditioned medium group: 1 × 105 microglial cells were cultured in 24 well plates in 1 ml of hMSCs conditioned medium; 4) vehicle group: 1 × 105 microglial cells were plated in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS. Microglial cells were stimulated with 1 μg/ml LPS based on the literature. Unstimulated microglia were as normal controls. The experiments were carried out in triplicate and each experiment was repeated three times.
4.3.
Immunocytochemistry
Microglial cells were stained with an antibody against F4/80. Human-specific antibody against HuNu was used as a marker for hMSCs. In brief, the cultures were washed with phosphatebuffered saline (PBS), fixed in 4% paraformaldehyde in PBS (PH 7.4) for 20 min, blocked with normal serum, and then incubated with primary antibody at 4 °C overnight. Primary antibodies included: rat anti-mouse F4/80 (1:50; Serotec), mouse anti-human HuNu (1:50; Chemicon). Cultures were washed and incubated at RT for 1 h with goat anti-rat FITCconjugated IgG (1:50; Serotec) or goat anti-mouse CY3conjugated IgG (1:200; Chemicon). These cells were counterstained with DAPI. All staining involved the use of a negative control (secondary antibody alone) in order to evaluate the specificity of the antibodies. The number of enlarged microglia in a defined field was manually counted under a microscope at 400× magnification. Five representative fields were counted for each well of the 24well plates. The results are the mean ± S.M.E. of three separate experiments performed in triplicate.
4.4.
Measurement of TNF-α
The culture medium was collected after stimulation for 12 h with LPS. TNF-α concentration in the culture medium was determined by mice specific enzyme linked immunosorbent assay (ELISA) kit (BioSource International) according to the manufacturer's instructions, and samples were diluted 50-fold to fit the standard curve.
4.5.
Nitrite assay
The accumulation of nitrite in the culture supernatant, a stable end product of NO, was determined by using a colorimetric reaction with the Griess reagent. Briefly, culture supernatant was mixed with an equal volume of the Griess reagent (0.1% N-(1-naphthyl) ethylenediamine dihydrochloride, dihydrochloride, 1% sulfanilamide, and 2.5% H3PO4). The mixture was incubated for 10 min at room temperature, and the absorbance at 540 nm was determined with a microplate reader. The concentration of nitrite was determined from a sodium nitrite standard curve.
Signaling Technology) containing protease inhibitor. Cells were scraped, sonicated briefly, and centrifuged at 14,000 g for 30 min at 4 °C. Protein extracts (20 μg) of cells were boiled in loading buffer for 3 min and subsequently were loaded into wells of 12% SDS-polyacrylamide gels at 80 V for 30 min and 150 V for 90 min. Proteins were then transferred onto PVDF membranes (Minipore). The blots were blocked with 5%(w/v) defatted milk in TBS-Tween20 (PH8.0) for 1 h at room temperature and then incubated overnight at 4 °C with primary antibodies: polyclonal rabbit anti-mouse iNOS (1:1000; Santa Cruz Biotechnology), polyclonal rabbit antimouse phosphospecific p38 MAPK (1:1000; Chemicon), polyclonal rabbit anti-mouse total p38 MAPK (1:1000; cell signaling Technology), and mouse anti-actin (1:1000; Santa Cruz Biotechnology). The blots were washed three times in TBST for 10 min each, incubated with a HRP-conjugated goat anti-rabbit IgG (1:5000; Chemicon) or a HRP-conjugated goat anti-mouse IgG (1:2000; Chemicon) for 1 h at room temperature, washed four times in TBST for 10 min each. Protein bands were visualized after incubation with SuperSignal west Pico chemiluminescent Substrate (Pierce Biotechnology) and exposure of high performance chemiluminescence film to the membrane surface in the dark. Quantitative analysis of the intensity of the bands was performed with Band Scan 5.0 software.
4.7.
Measurement of trophic factors
hMSCs were randomly divided into: 1) conditioned medium group: 2 × 106 microglial cells were plated in 25 cm2 flask in 5 ml of DMEM with high glucose and 10% FBS. The culture medium was replenished after 12 h, and then 1 μg/ml LPS was added to cultures. After incubation for 24 h, the resultant supernatant was collected and centrifuged at 150 ×g for 5 min to remove cellular debris as conditioned medium of activated microglia. 5 × 104 hMSCs were cultured in 24 well plates in 1 ml of conditioned medium; 2) control group: 5 × 104 hMSCs were plated in 24 well plates in 1 ml of DMEM with high glucose and 10% FBS. The culture medium was collected after incubation for 48 h. Production of VEGF, BDNF, IGF-I, and HGF in the culture medium was determined with ELISA using a commercially available kit (R & D Systems) according to the manufacturer's instructions.
4.8.
MTT assay
Cell viability was measured using an MTT cell viability assay. Medium was removed, and 100 μl fresh medium was added to each well of 96-well plates. MTT (Sigma) was added to the cultures at a final concentration of 1 mg/ml, and the cells were incubated for 4 h. Cells were lysed in DMSO. Colorimetric determination of MTT reduction was measured on the plate reader at 490 nm.
4.9. 4.6.
29
Statistical analysis
Western blotting
Cells in 24 well plates were washed with PBS, and then incubated for 5 min on ice in 40 μl of lysis buffer (Cell
Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. The results are presented as mean ± SD.
30
BR A IN RE S EA RCH 1 2 69 ( 20 0 9 ) 2 3 –30
Acknowledgment The work was supported by a grant from Natural Science Fund of Shandong Province (32693). REFERENCES
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