Human mesenchymal stem cells protect neutrophils from serum-deprived cell death

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Cell Biol. Int. (2011) 35, 1247–1251 (Printed in Great Britain)

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Human mesenchymal stem cells protect neutrophils from serum-deprived cell death Maryam Maqbool*, Sharmili Vidyadaran*, Elizabeth George{ and Rajesh Ramasamy1* * Immunology Laboratory, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, {

Malaysia Haematology Unit, Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Abstract We have previously shown that human MSC (mesenchymal stem cells) inhibit the proliferation of most of the immune cells. However, there are innate immune cells such as neutrophils and other PMN (polymorphonuclear) cells that do not require an extensive proliferation prior to their effector function. In this study, the effect of MSC on neutrophils in the presence of complete and serum-deprived culture media was investigated. In the presence of MSC, the viability of neutrophils increase as measured in 24 h of incubation at various supplementation of serum concentration. We have utilized Annexin V and PI (propidium iodide) staining to confirm whether the enhancement of neutrophil’s viability is due to a reduction in PCD (programmed cell death). MSC significantly rescue neutrophils from apoptosis at 1, 5 and 10% of FBS (fetal bovine serum) supplementation. The fractions of viable and dead cells were increased and decreased respectively in the presence of MSC. Our results indicate MSC rescue neutrophils from nutrient- or serum-deprived cell death. However, whether this effect is exerted through a specific signalling pathway or confining neutrophils in resting state by MSC requires further investigation. Keywords: apoptosis; mesenchymal stem cells; neutrophils; serum deprivation and cell death

1. Introduction MSC (mesenchymal stem cells) are one of the main adult stem cells that are found in bone marrow beside HSC (haematopoietic stem cells). The presence of MSC in bone marrow niche is to provide the necessary scaffold and growth factors to the expanding and differentiating HSC through terminally differentiated stromal cells, osteoblasts and cytokines (Blair and Thomas, 1997; Devine and Hoffman, 2000; Angelopoulou et al., 2003; Dazzi et al., 2005). MSC also showed a potent immunomodulatory effect on highly proliferating mature immune cells due to their close interaction at bone marrow niche. Our earlier studies have shown that MSC suppress proliferation of T-, B- and NK (natural killer) cells via a cell-cycle arrest by preventing the target cells from entering into S phase of cells cycle (Ramasamy et al., 2007a, 2008). The generalised anti-proliferative activity of MSC is confined mostly to all cell types that were actively in cell cycle such as normal and malignant immune cells (Ramasamy et al., 2007b). However, the effect of MSC on naturally nonproliferating cells at periphery such as neutrophils is not well defined although MSC is known to halt differentiation of dendritic cells that are non-proliferating cells (Ramasamy et al., 2007a). Neutrophils, also known as PMN (polymorphonuclear) leucocytes are the major cell type that constitutes innate immunity. They comprise approx. 50–70% of leucocytes and predominate in eliminating pathogens that induce acute inflammation (Greenberg and Grinstein, 2002). Neutrophils contain or produce an array of cytotoxic molecules, including numerous proteases and ROS (reactive oxygen species), which have potential to cause damage

to host tissues during inflammation processes. Therefore it is critical that neutrophil homoeostasis and turnover are highly regulated. The neutrophils turnover is approx. 1011 per day in the average human adult and this dramatic PMN turnover is controlled by apoptosis (Kobayashi et al., 2005). It has been proposed that MSC-mediated immunomodulation represents an important defence mechanism against harmful immune activation at the interface between blood and mesenchymal compartment in vivo (Rasmusson, 2006). MSC interact with the cells of the immune system, inducing energy in vivo and modulating their functional activities in vitro (Uccelli et al., 2006). However, most studies on MSC-mediated immunosuppression were conducted in adaptive immune cells with T- and B-cells models. Recently many studies have looked on the effects of MSC on innate immune cells. Studies on macrophages and dendritic cells showed active cell proliferation and antigen presentation were inhibited by MSC. To the best of our knowledge, in this study the effect of MSC on neutrophil survival with or without serum at varying concentration has been evaluated for the first time.

2. Materials and methods 2.1. Isolation of neutrophils Peripheral blood was collected from healthy volunteers after obtaining written informed consent from volunteers and ethical clearance of Faculty of Medicine and Health Sciences, Putra University, Malaysia. Then 20 ml of venous blood was collected


To whom correspondence should be addressed (email [email protected]). Abbreviations: FBS, fetal bovine serum; HBSS, Hanks balanced salt solution; HSC, haematopoietic stem cells; IL, interleukin; LPS, lipopolysaccharide; MSC, mesenchymal stem cells; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PI, propidium iodide; PMN, polymorphonuclear; PS, phosphatidylserine; RBC, red blood cell.

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and diluted in 16HBSS (Hanks balanced salt solution; Gibco) at 1:1 ratio and layered over 5 ml Ficoll-Paque plus (GE Healthcare); centrifuged at 1800 rev./min for 20 min at room temperature. Plasma and the mononuclear cell layers were discarded. The red cell pellet that contains the PMN and RBC (red blood cell) were suspended in 5 ml HBSS and layered on 3% dextran (Fisher Scientific) for 40 min at room temperature for further sedimentation of RBC. Supernatant from dextran sedimentation was collected and centrifuged at 1800 rev./min for 10 min at room temperature. The remnant RBC was lysed using hypotonic lysing procedure to obtain the pure PMN population (Ramasamy et al., 2010). The morphological examination and cell count were performed to determine the cell count and purity of the PMN.

2.2. Generation of human umbilical cord derived MSC One inch of human umbilical cord samples were obtained from full-term pregnancy after the written informed consent from volunteers following ethical clearance from Faculty of Medicine and Health Sciences, University Putra Malaysia. Umbilical cord samples were disassociated into single-cell suspension using a combination of enzymatic digestion and mechanical dissociation method (Tong et al., 2010). Cells were cultured and characterized as previously described (Ramasamy et al, 2007b; Tong et al., 2010).

Figure 1 Determination of neutrophil viability through MTS Neutrophils were cultured for 24 (A) and 48 h (B) with or without MSC in various concentrations of serum at 1:10 ratio. MSC significantly enhanced neutrophil viability. The results are expressed as means¡S.D. from three independent experiments (*P,0.05).

2.3. Cell culture Neutrophils were cultured in RPMI 1640+Glutamax (GIBCO/ Invitrogen) supplemented with 1% penicillin/streptomycin and varying levels of FBS (foetal bovine serum; Gibco) markedly 0, 1, 5 and 10% (v/v) with or without MSC. PMA (Sigma) at 50 nM was used to stimulate neutrophils.

2.4. Determination of neutrophil viability The viability of neutrophils was determined by the MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium] assay [CellTiter 96j AQueous Non-Radioactive cell viability kit (Promega)]. Briefly 56104 neutrophils were incubated in the presence or absence of MSC in 96-well plate at ratio 10:1. At scheduled times, 20 ml of MTS reagent was added to each well and further incubated at 37uC in a humidified, 5% CO2 for 2 h. The conversion of MTS into aqueous, soluble formazan is accomplished by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan formed is directly proportional to the number of viable cells in the culture and was determined by a microplate reader (Dynex MRX II) at 450 nm.

2.5. Apoptosis assay Neutrophils were cultured at different time points (4, 8 and 12 h) and at varying serum concentrations in the presence or absence of MSC. At end of culture, neutrophils were washed twice using PBS and re-suspended in 500 ml of isotonic-binding buffer. After

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15 min of incubation with Annexin V and PI (propidium iodide) (Annexin V-FITC Apoptosis Detection Kit; BD Biosciences), the cells were analysed by flow cytometer (LSR Fortessa, BD Biosciences); 104 events were acquired and analysed using the FACS Diva software.

2.6. Statistical analysis Results were expressed as means¡S.D. Differences were considered significant at P,0.05. Statistical analyses were conducted using student’s t test using Microsoft Office 2007 (Excel).

3. Results 3.1. MSC increase the viability of resting and activated neutrophils in various culture serum concentrations Circulating neutrophils have a short life span of 6–10 h after which the cell undergoes apoptosis (Coxon et al., 1999). The effects of MSC on neutrophil survival at varying serum concentrations, over a period of time were assessed. MSC significantly enhanced the survival of both resting and PMA-activated neutrophil at 24 h at all FBS concentrations including in no FBS (0%) (Figure 1). The overall results indicate that MSC are effective in rescuing the resting and

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Figure 3 Figure 2

Flow cytometry evaluation of viability of resting and activated neutrophils cultured with or without MSC in various concentrations of serum (A)–(C) Neutrophils were cultured for 4, 8 and 24 h respectively at 1:10 ratio. MSC posed no effect on neutrophil viability at early time points (4 and 8 h; A, B) but significantly enhanced the neutrophil viability at 24 h (C). The results are expressed as means¡S.D. from three independent experiments (*P,0.05).

stimulated neutrophils in serum-deprived or -reduced culture compared with neutrophil culture alone at respective serum levels.

3.2. MSC inhibit apoptosis of both resting and stimulated neutrophils To confirm whether the increased viability of neutrophils in the presence of MSC is due to an anti-apoptotic activity, flowcytometry analysis using Annexin V and PI was utilized. Annexin V is calcium-dependant anti-coagulant protein that binds to PS (phosphatidylserine) on cells undergoing apoptotic process. The early apoptosis could be determined by labelling cells with Annexin V and PI dye, whereby the necrotic cells that express PS can easily distinguish with positive staining of PI (Vermes et al., 1995). Apoptosis assay was conducted at 4, 8 and 24 h time points and apoptotic cells were measured as Annexin V+/PI2 population. MSC neither increase nor decrease apoptosis in resting and PMAstimulated neutrophils at 4 and 8 h time points in any serum concentrations. However, a significant reduction in apoptosis is observed in 1, 5 and 10% serum concentrations at 24 h (Figure 2C).

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Flow cytometry evaluation of early apoptosis in resting or activated neutrophils cultured with or without human MSC in various concentrations of serum Neutrophils were cultured for 4 (A), 8 (B) and 24 h (C) at 1:10 ratio. MSC posed no effect on neutrophil apoptosis at early hour (4 and 8 h) but significantly inhibited the neutrophil apoptosis at 24 h. The results are expressed as means¡S.D. from three independent experiments (*P,0.05).

3.3. Effect of MSC on viable and dead cells fraction at varying serum concentration Using Annexin V and PI staining, we have assessed the effect of MSC on viable and dead cell population of neutrophils at different time points and serum concentration. Viable and dead cells were defined as Annexin V2/PI2 and Annexin V+/PI+ respectively. Similar to early apoptosis, the fraction of viable and dead cells populations was unaffected at 4 and 8 h of time points in all serum concentration. However, MSC at 24 h incubation significantly increased the cell viability (Figure 3C) along with decreased number of cell death (Figure 4C) in all serum concentration.

4. Discussion We have previously shown that MSC protect and enhance the survival of T-cells and haematopoietic tumour cells by reducing the rate of apoptosis and preserve the target cells in transient growth arrest condition via cell cycle block (Ramasamy et al., 2007b, 2008). This effect may arise from the nature of MSC in bone

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Figure 4

Flow cytometry evaluation of dead cells in resting or activated neutrophils cultured with or without human MSC in various concentrations of serum Neutrophils were cultured for 4 (A), 8 (B) and 24 h (C) at 1:10 ratio. MSC posed no effect on neutrophil death at early hour (4 and 8 h) but significantly inhibited the neutrophil death at 24 h. The results are expressed as means¡S.D. from three independent experiments (*P,0.05).

marrow niche whereby the stromal cell compartment that is formed by MSC nurture preserves the HSC from exhaustion of stem cell pool and regulate haematopoiesis. Here, we postulate that MSC also exert a similar effect on mature neutrophils by protecting them from cell death due to external stimulation or insult. Neutrophils have a limited lifespan at in vitro where approx. 50% of cell death occurred if cultured for more than 48 h (data not shown). Here, we evaluated the effect of MSC on resting and PMA-stimulated neutrophils at 4, 8 and 24 h time points in various serum concentrations. Serum or serum-fractions have universal applications in cell culture as an indispensable supplement for classical cell-culture media. In adherent cell culture, serum facilitates the attachment and spreading of cells on plastic surface thus increasing the viability of cells (Horiuti et al., 1982). Our work was conducted at 0, 1, 5 and 10% of serum concentration where the 10% serum level is a standard concentration used in classical cell-culture system.

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In order to explore the effect of MSC on neutrophils viability, tissue culture insults such as reduction in serum supplementation was imposed to neutrophil culture to induce apoptosis. Our result showed the viability of resting and stimulated neutrophils were increased in the presence of MSC even at 0% of serum concentration (Figure 1). Brandau et al. (2010) also have showed that neutrophils recruited by parotid gland derived MSC in the presence of LPS (lipopolysaccharide) exhibited a prolonged lifespan. The prolongation of neutrophil survival was further studied by apoptosis assays. Apoptosis is PCD (programmed cell death) invoked by cells due to internal and external insults. The concentration of serum has a potent impact in inducing apoptosis in neutrophils due to lack of essential components or sustainable survival signals. Furthermore, withdrawal of serum has been documented to induce intrinsic pathway-mediated apoptosis in corpus luteum cells via the caspase cascade (Goyeneche et al., 2006). Although the inhibition was not statistically significant at 0% of serum, however, a noticeable reduction of apoptosis was observed. The common pattern of apoptosis reduction in all serum concentrations reflects the capability of MSC to inhibit neutrophils’ apoptosis (Figure 2). This is the first report that shows, MSC could protect neutrophils from apoptosis-mediated cell death due to declining serum supplementation. However, this effect is not unique for neutrophils, as we have previously demonstrated that bone marrow derived MSC reduced the apoptosis of BV173 tumour cell line at 1 and 5% of serum concentration (Ramasamy et al. 2007b). MSC enhanced the lifespan of resting and PMA-activated neutrophils through their cellular and/or humoral communication. In resting neutrophils, MSC were capable to restrain neutrophils from serum-deprived cell death. This anti-apoptotic effect is profoundly significant at incubation period of 24 h. The required minimal time for the anti-apoptotic effect indirectly shows that the communication among neutrophils and MSC occurs with particular signalling pathways and secretion of soluble factors. In line with this, Brandau et al. (2010) showed that incubation of MSC supernatant at 24 h reduced the rate of apoptosis in LPS-stimulated neutrophils by secretion of IL-8 (interleukin 8)/MIF (macrophage inhibitory factor). The viability of the neutrophils not only increased in the presence of MSC, but also their effector function such as expression of inflammatory cytokines and enhanced chemotaxis was ameliorated. It is reported that, bone marrow derived MSC inhibits apoptosis of resting and IL-8 activated neutrophils in normal serum supplemented culture. They also demonstrated that MSC via IL-6 signalling decreased the mitochondrial pro-apoptotic protein, Bax and increased mitochondrial anti-apoptotic protein MCL-1 (Raffaghello et al., 2008). Although a direct interface between MSC and mature neutrophils could not be elucidated in the bone marrow compartment, recent research has suggested that tissue resident MSC localized in perivascular and periendothelial areas provide a venue for neutrophils and MSC interaction (Crisan et al., 2008). The close interaction among tissue resident MSC and neutrophils at the peripheral vicinity improve the neutrophils survival thus enhance the immune function as neutrophils are one of the first defence cells recruited at tissue microenvironment due to infection and inflammation.

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5. Conclusion Although our work did not elucidate the molecular mechanisms that govern protection of neutrophils due to serum-induced apoptosis, it verified a fundamental role of MSC in supporting neutrophils function at the periphery.

Author Contribution Maryam Maqbool performed collection and assembly of data, data analysis and interpretation and wrote the manuscript. Sharmili Vidyadaran performed data analysis and wrote the manuscript. Elizabeth George performed administrative support and wrote the manuscript. Rajesh Ramasamy performed the conception and design, financial support, data analysis and interpretation, manuscript writing and final approval of the manuscript.

Funding This work was supported by Research University Grant Scheme, Universiti Putra Malaysia [grant number 04-01-09-0781RU] and Science Fund, Ministry of Science, Technology and Innovation (MOSTI), Malaysia [grant number 02-01-04-SF1022].

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Received 3 February 2011/ 29 April 2011; accepted 8 June 2011 Published as Immediate Publication 8 June 2011, doi 10.1042/CBI20110070

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