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First published online October 11, 2007
Stem Cells Vol. 26 No. 1 January 2008, pp. 151 -162
doi:10.1634/stemcells.2007-0416; www.StemCells.com
© 2008 AlphaMed Press

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THE STEM CELL NICHE

Human Mesenchymal Stem Cells Inhibit Neutrophil Apoptosis: A Model for Neutrophil Preservation in the Bone Marrow Niche

Lizzia Raffaghelloa, Giordano Bianchib, Maria Bertolottob, Fabrizio Montecuccob, Alessandro Buscac, Franco Dallegrib, Luciano Ottonellob, Vito Pistoiaa

aLaboratory of Oncology, G. Gaslini Institute, Genoa, Italy;
bClinic of Internal Medicine I, Department of Internal Medicine, University of Genoa Medical School, Genoa, Italy;
cAz. Ospedaliera S. Giovanni Battista (Molinette), Turin, Italy

Key Words. Mesenchymal stem cells • Neutrophils • Interleukin-6R/Interleukin-6 fusion protein • Bone marrow

Correspondence: Correspondence: Lizzia Raffaghello, Ph.D., Laboratory of Oncology, G. Gaslini Institute, Genova 15145, Italy. Telephone: 0039-010-5636342; Fax: 0039-010-3779820; e-mail: lizziaraffaghello{at}ospedale-gaslini.ge.it

Received on May 29, 2007; accepted for publication on October 3, 2007.

First published online in STEM CELLS EXPRESS  October 11, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mesenchymal stem cells (MSC) establish close interactions with bone marrow sinusoids in a putative perivascular niche. These vessels contain a large storage pool of mature nonproliferating neutrophils. Here, we have investigated the effects of human bone marrow MSC on neutrophil survival and effector functions. MSC from healthy donors, at very low MSC:neutrophil ratios (up to 1:500), significantly inhibited apoptosis of resting and interleukin (IL)-8-activated neutrophils and dampened N-formyl-L-methionin-L-leucyl-L-phenylalanine (f-MLP)-induced respiratory burst. The antiapoptotic activity of MSC did not require cell-to-cell contact, as shown by transwell experiments. Antibody neutralization experiments demonstrated that the key MSC-derived soluble factor responsible for neutrophil protection from apoptosis was IL-6, which signaled by activating STAT-3 transcription factor. Furthermore, IL-6 expression was detected in MSC by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Finally, recombinant IL-6 was found to protect neutrophils from apoptosis in a dose-dependent manner. MSC had no effect on neutrophil phagocytosis, expression of adhesion molecules, and chemotaxis in response to IL-8, f-MLP, or C5a. These results support the following conclusions: (a) in the bone marrow niche, MSC likely protect neutrophils of the storage pool from apoptosis, preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism, and (b) a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and reactive oxygen species production without impairing phagocytosis and chemotaxis has been identified.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Different types of stromal cells, including endothelial cells, smooth muscle cells, reticular cells, and osteoblasts, are found within the bone marrow cavity, where they form a niche that plays an indispensable role in supporting survival, growth, and differentiation of hematopoietic progenitor cells [1–3]. A subset of mesodermal progenitor cells named multipotent mesenchymal stromal cells [4, 5] or, more commonly, mesenchymal stem cells (MSC) is found in the stromal component of the bone marrow and of many other tissues [6, 7]. MSC are difficult to isolate ex vivo but can be expanded in culture from the bone marrow of humans and animals.

MSC can differentiate along several lineages: osteoblasts, chondrocytes, adipocytes, smooth muscle cells, endothelial cells, and reticular cells [7–9]. MSC have successfully been used to treat patients with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, leading to osteopenia, multiple fractures, severe bony deformities, and shortened stature [10–12]. Other potential applications of MSC in tissue engineering are the matter of active investigation [13].

MSC have been shown to interact with the cells of the immune system, inducing anergy in vivo and modulating their functional activities in vitro [14]. A number of groups in the last few years have extensively reported that human MSC inhibit the effector functions of both T and B cells, the generation of dendritic cells, and the proliferation of natural killer (NK) cells in response to interleukin (IL)-2 [15–21].

It has been proposed that MSC-mediated immunomodulation represents an important defense mechanism against harmful immune activation at the interfaces between blood and mesenchymal compartments in vivo [22]. The immunosuppressive activities of MSC have been exploited for the treatment of human graft-versus-host disease following allogeneic hematopoietic stem cell transplantation. The first results have been encouraging [23], and clinical trials are ongoing.

The bone marrow is not only the organ where hematopoiesis occurs but also the site where a large amount of nonproliferating neutrophils are retained in the storage pool of bone marrow sinusoids [24, 25]. The bone marrow reserve, whose mass has been estimated to be 25–30 times larger than the circulating mass of granulocytes, represents in conditions of increased demand a readily available source of neutrophils that possess the same functional properties as their peripheral counterparts [24, 26, 27]. Interestingly, MSC, which exert their homeostatic functions through both paracrine mechanisms involving the release of soluble factors and contact-dependent mechanisms, would line the bone marrow extravascular space, forming a network that interpolates with the sinusoids, where neutrophils of the bone marrow reserve reside [28]. The postulated close proximity of the latter cells and MSC in the bone marrow niche led us to hypothesize that MSC could modulate survival and function of neutrophils belonging to the storage pool, in analogy to the trophic effects exerted by MSC on hematopoietic progenitors.

Here, we provide evidence that MSC, even at very low concentrations, inhibit in vitro apoptosis of resting and IL-8-activated neutrophils and reduce N-formyl-L-methionin-L-leucyl-L-phenylalanine (f-MLP)-induced respiratory burst while not affecting phagocytosis, expression of adhesion molecules, or the migration capability of neutrophils in response to classic stimuli. MSC rescued neutrophils from apoptosis by constitutive release of IL-6, which is signaled through a STAT-3-dependent mechanism.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mesenchymal Stem Cell Isolation and Culture
Human MSC were expanded in vitro from the bone marrow of healthy donors after informed consent was obtained. Mononuclear cells were isolated by gradient centrifugation at 2,500 rpm for 30 minutes on Ficoll-Hystopaque 1077 (1,077 g/ml density; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), washed twice with phosphate-buffered saline (PBS; Sigma-Aldrich), counted, and plated at a concentration of 20–30 x 106 cells per 75-cm2 flask in MesenCult basal medium supplemented with mesenchymal Stem Cell Stimulatory Supplement (StemCell Technologies, Vancouver, BC, Canada, http://www.stemcell.com). After 1 week of culture at 37°C in an atmosphere of 5% CO2, nonadherent cells were removed, and the medium was replaced every other day. MSC were trypsinized when the cultures reached 80%–100% confluence. The purity of MSC suspensions was assessed by flow cytometry using the following monoclonal antibodies: anti-CD34 fluorescein isothiocyanate (FITC) (BD Pharmingen, Franklin Lakes, NJ, http://www.bdbiosciences.com/index_us.shtml), CD45 FITC (Caltag Laboratories, Burlingame, CA, http://www.caltag.com), CD14 phycoerythrin (PE) (BD Pharmingen), CD73 PE (BD Pharmingen), CD105 FITC (Diaclone, Besançon, France, http://www.diaclone.com/anglais), CD44 FITC (BD Pharmingen), and CD29 PE (BD Pharmingen). As expected, the immunophenotype of MSC, after two to three passages in culture, was as follows: CD34– (0%), CD14– (1.5%), CD45– (0%), CD105+ (96%), CD73+ (99%), CD44+ (98%), CD29+ (73%) cells (Fig. 1). MSC used for all of the following experiments were cultured for no longer than four passages in vitro. In neutralization experiments, neutrophils were incubated with anti-human IL-6R (10 {gamma}/ml; Bender MedSystems Inc., Burlingame, CA, http://www.bendermedsystems.com) or isotype-matched monoclonal antibody (mAb) (SouthernBiotech, Birmingham, AL, http://www.southernbiotech.com) for 18 hours in the presence or absence of MSC-derived supernatants. In additional experiments, neutrophils were cultured with MSC-derived supernatants preincubated with anti-IL-6 neutralizing mAb (Peprotech, Rocky Hill, NJ, http://www.peprotech.com) or an isotype-matched (SouthernBiotech) mAb for 2 hours at room temperature (RT).


Figure 1
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  		Figure 1. Immunophenotype of human mesenchymal stem cells. MSC were stained with antibodies against surface specific markers (shaded profile) or an isotype-matched monoclonal antibody (open profile) and analyzed by flow cytometry. This experiment is representative of the four performed.

 
Neutrophil Isolation and Culture
Neutrophils were isolated from human heparinized (10 U/ml heparin; Vister; Pfizer Italia, Latina, Italy, http://www.pfizer.it) venous blood from healthy volunteers (ages 24–48 years old) after informed consent was obtained. Neutrophilic polymorphonuclear leukocytes (here referred to as neutrophils or polymorphonuclear neutrophils [PMN]) were prepared by Dextran 70,000 (Fresenius Kabi Italia, Verona, Italy, http://www.fresenius-kabi.com) sedimentation followed by centrifugation (400g, 30 minutes) on a Ficoll-Hypaque density gradient, as previously described [29]. Plasma and the mononuclear cell layer were discarded, and contaminant erythrocytes were removed by hypotonic lysis [29]. Neutrophils were then resuspended in RPMI 1640 (Sigma-Aldrich) supplemented with L-glutamine, penicillin/streptomycin, nonessential amino acids, and 10% fetal bovine serum (Sigma-Aldrich). Neutrophils were on average 97% pure, as determined by morphologic analysis of May-Grumwald-Giemsa-stained cytopreps (Merck, Darmstadt, Germany, http://www.merck.com).

Neutrophil suspensions were incubated with MSC at different ratios in 12-well tissue culture plates (Corning Costar, Cambridge, MA, http://www.corning.com/lifesciences) for the indicated times. In some experiments, neutrophils were incubated with MSC supernatants obtained from MSC grown at confluence. Moreover, in other experiments, neutrophils were cultured with MSC suspensions using 24-transwell plates with a 0.4-µm pore size polycarbonate membrane (Corning Costar). In some experiments, neutrophils were incubated with different concentrations of human recombinant (r)IL-6 (Sigma-Aldrich) for 18 hours, and apoptosis was evaluated by morphological analysis. Neutrophils were activated with IL-8 (BioSource, Camarillo, CA, http://www.invitrogen.com) (10–8 M) for 30 minutes.

Morphological Assessment of Neutrophil Apoptosis
Neutrophils were cultured for different times with or without MSC suspensions. Cytocentrifuged cell preparations were fixed and stained with May-Grunwald-Giemsa. Thereafter, cytopreps were read by oil immersion light microscopic examination of at least 500 cells per slide (magnification, x1,000) (Leitz Laborlux, Esselte Italia, Milan, Italy, http://www.esselte.com). Cells showing apoptotic morphology were identified according to cell shrinking, nuclear condensation and fragmentation, plasma membrane ruffling, and blebbing, as described previously [30].

Flow Cytometric Analysis of Neutrophil Apoptosis
Neutrophils, cultured at different times with or without MSC suspensions, were washed and resuspended in 500 µl of isotonic binding buffer. After 15 minutes of incubation with annexin V and propidium iodide (annexin V-FITC Apoptosis Detection Kit; MBL International Corporation, Woburn, MA, http://www.mblintl.com), the cells were run on a fluorescence-activated cell sorter (FACScan; Becton, Dickinson and Company, San Jose, CA, http://www.bd.com) [30], and 104 events were acquired and analyzed using the CellQuest software.

Immunocytochemistry
Bax and MCL-1 protein expression was investigated by immunocytochemistry as described by Dibbert et al. [31] with slight modification. Briefly, neutrophils, collected after incubation with or without MSC suspensions, were cytospun. After rehydration in PBS, spots were submerged in peroxidase-quenching solution for 10 minutes to neutralize endogenous peroxidase activity. Then, the slides were incubated with anti-human Bax polyclonal antibody (1 mg/ml; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) or with anti-human MCL-1 mAb (1 mg/ml; Santa Cruz Biotechnology) diluted 1:200 and 1:500 in PBS, respectively. The secondary biotinylated IgG antibody (Kit Histostain SP; Zymed Laboratories, San Francisco, http://www.invitrogen.com) was used. After washing and subsequent incubation with biotin-streptavidin-peroxidase (Kit Histostain SP), slides were incubated at RT for 5 minutes with the peroxidase-substrate solution (Histostain SP), rinsed with PBS, and counterstained with hematoxylin. Then, cytospins were mounted in Eukitt (Merck), examined by light microscopy (Leica, Cambridge, U.K., http://www.leica.com), and evaluated by image analysis (Leica). Isotype-matched mAbs (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) of irrelevant specificity were tested as a negative control.

Image Analysis
Image analysis was performed by the Leica Q500 MC image analysis system (Leica). For each sample, 100 cells were randomly analyzed, and the optical density of the signal was quantified by computer analysis. The video image was digitalized for image analysis at 256 gray levels. Imported data were analyzed quantitatively by Q500 MC Software-Qwin (Leica). The operator randomly selected single cells using the cursor. Then, the positive area was analyzed automatically. The same optical threshold and filter combination were used throughout all of these experiments.

Flow Cytometric Assessment of Neutrophil-Oxidative Metabolism
Flow cytometric analysis of neutrophil-oxidative metabolism was carried out according to Ottonello et al. [32]. Briefly, neutrophils were cocultured for 1 hour with or without MSC suspensions. Thereafter, neutrophils were treated with 2'-7'-dichlorofluorescein-diacetate (Sigma-Aldrich) (5 µM) and then stimulated with f-MLP (Sigma-Aldrich) (1 µM) for 30 minutes. The reaction was stopped by keeping the samples on ice before they were analyzed by flow cytometry. Controls were treated in the same way, without f-MLP.

Flow Cytometric Analysis of CD95 Neutrophil Expression
Neutrophils were incubated with or without MSC suspensions for 18 hours at 37°C and then stained with FITC-conjugated CD95 mAb (R&D Systems). An isotype-matched, FITC-conjugated mAb (R&D Systems) of irrelevant specificity was tested as negative control.

Flow Cytometric Analysis of CD11b and CD62L Neutrophil Expression
Neutrophils were incubated with or without MSC suspensions for 1 hour at 37°C. Neutrophils were then exposed to f-MLP (1 µM) or medium for 5 minutes and subsequently stained with FITC-conjugated anti-CD11b mAb (Caltag) or FITC-conjugated anti-CD62L mAb (R&D Systems). Isotype-matched, FITC- and PE-conjugated mAbs (R&D Systems) of irrelevant specificity were tested as negative control. The reaction was stopped by keeping the samples on ice before they were analyzed by flow cytometry.

Migration Assay
Neutrophils were incubated with or without MSC suspensions for 1 hour at 37°C, washed, and resuspended in medium containing one of the following chemotactic stimuli: C5a (Sigma-Aldrich) (1 nM), IL-8 (1 nM), or f-MLP (10 nM). Neutrophil migration was assessed in a 48-well microchemotaxis chamber (Neuro Probe, Gaithersburg, MD, http://www.neuroprobe.com) with a polycarbonate filter with a 5-µm pore size and 5-µm-thick polycarbonate polyvinylpyrrolidone-free filter. The lower wells of chemotaxis chamber were filled with chemoattractant solutions or control medium and the upper wells with 50 µl of cell suspension (2 x 106/ml). After incubation (60 minutes, 37°C), the filters were removed from the chambers, washed, and stained with Diff-Quick (Baxter, Rome, http://www.baxter.com). Each combination was tested in duplicate. Cells in five different oil-immersion fields were counted, and the chemotaxis index was obtained dividing the number of cells that migrated to the stimulus by the number of cells that migrated to the control medium.

Neutrophil Phagocytosis
Neutrophil phagocytosis was tested as the release of superoxide anion triggered by opsonized zymozan (OPZ). OPZ was obtained from whole human serum, pooled from 8 or 10 healthy donors.

Zymosan A from Saccharomyces cerevisiae (Sigma-Aldrich), diluted in distilled water, was boiled for 30 minutes, recovered by centrifugation (10 minutes at 1,500 rpm), and resuspended in PBS (10 mg/ml). One volume of pooled human serum and one volume of zymosan suspension were then incubated for 30 minutes at 37°C in a shaking water bath. The OPZ was recovered by centrifugation and resuspended in PBS (10 mg/ml) for use as a stimulus.

Briefly, neutrophils (5 x 105) were incubated with MSC for 1 hour at 37°C under the same conditions used for the apoptosis assay. Thereafter, the cells were incubated for 20 minutes at 37°C with 80 µM ferricytochrome c (Sigma-Aldrich) in the absence or presence of 300 U/ml superoxide dismutase (Sigma-Aldrich) and then stimulated for 20 minutes at 37°C with or without OPZ (1 mg/ml). The reactions were then stopped by adding 2 ml of ice-cold 1 mM N-ethylmaleimide (Sigma-Aldrich), and superoxide production was determined in neutrophil supernatants at a 550-nm wavelength using an extinction coefficient of 2.1 x 104 M/cm, as previously described [25]. The optical density changes were monitored with a PerkinElmer 576 ST (PerkinElmer Life and Analytical Sciences, Waltham, MA, http://www.perkinelmer.com) spectrophotometer.

Western Blot Analysis
Proteins extracts were prepared from cultured neutrophils as follows: 107 cells were pelleted, washed in PBS, and lysed in 70 µl of a buffer containing 20 mM Hepes, 0.15 M NaCl, 10% glycerol, 0.25% Nonidet P40, 1 mm EDTA, 2.5 mM dithiothreitol (DTT), 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na3VO4. All products were purchased from Sigma-Aldrich. The pellets were resuspended by vortex, kept on ice for 10 minutes, frozen and thawed twice, and centrifuged (12,000 rpm) at 4°C for 15 minutes. Protein concentration was determined in lysates by Coomassie (Bradford) Protein Assay Kit (Pierce, Rockford, IL, http://www.piercenet.com).

Twenty micrograms of protein per sample were diluted with reducing sample buffer and separated by electrophoresis. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and blotted onto Hybond C-nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) by electrotransfer. Membranes were blocked with 5% (vol/vol) nonfat powdered milk; stained with monoclonal phospho-Stat-3{alpha} antibody (Upstate, Charlottesville, VA, http://www.upstate.com; Millipore, Billerica, MA, http://www.millipore.com) overnight at 4°C with shaking; washed in PBS/Tween 20 0.05% (vol/vol), pH 7.4 (PBST); and, finally, incubated in secondary horseradish peroxidase-labeled goat anti-mouse antibody (Amersham Biosciences) for 1 hour at room temperature. After three additional washes with PBST, secondary antibody was detected using Enhanced Chemiluminescent Assay (Amersham Biosciences).

Quantitative Polymerase Chain Reaction Analysis of IL-6 mRNA Expression in Mesenchymal Stem Cells
RNA isolation, reverse transcription, and real-time polymerase chain reaction (PCR) were performed as described [33]. Mononuclear cells, obtained after informed consent was obtained from 10 healthy volunteers, were used as a negative control. Briefly, total RNA was isolated using RNeasy Micro Kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) according to the manufacturer's instructions. cDNA was synthesized by incubating 1 µg of total RNA for 45 minutes at 42°C in a thermal cycler (Genenco; MJ Research Inc., Waltham, MA, http://www.mjr.com) in the presence of 200 U of murine Moloney leukemia virus enzyme (Applera-Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com), 20 U of RNase inhibitor (Applera-Applied Biosystems), 1 mM dNTPs, 10 mM DTT solution, 500 pM random hexamers (Amersham Pharmacia Biotech), and 2.5 mM MgCl2 solution, in a final volume of 20 µl. Subsequently, 30 µl of diethyl pyrocarbonate-water was added. For real-time PCR, 5 µl of the resulting cDNA dilution was amplified using the TaqMan system (HT7900 Fast Real Time AB) for IL-6 and the ribosomal protein large P0 (RPLP0), tested as control gene. Primers and probes were from Applied Biosystems (cod HS00174131_ml and Hs99999902_m1). All assays were performed in duplicate. Serial dilutions (from 106 to 10) of control plasmids containing cloned Abelson sequences (Ipsogen, Marseille, France, http://www.ipsogen.com) were used to generate the standard curve. The relative levels of IL-6 transcript in MSC versus normal peripheral blood mononuclear cells were calculated using 2–{Delta}{Delta}Ct according to the following formula: {Delta}{Delta}Ct = (CtIL-6 – CtRPL0)MSC – (CtIL-6 – CtRPL0)mononuclear cells. A value of 2–{Delta}{Delta}Ct > 2 is indicative of IL-6 amplification.

The PCR products were analyzed by electrophoresis on a 2% agarose gel containing ethidium bromide (AMRESCO Inc., Solon, OH, http://www.amresco-inc.com). The ladder PhiXi 174 DNA/BsuRI (HaeIII) (Bio-Rad, Hercules, CA, http://www.bio-rad.com) was used.

Enzyme-Linked Immunosorbent Assay
Supernatants from MSC grown at confluence were tested for IL-6 by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D Systems).

Statistical Analysis
Data were expressed as mean ± SD. Differences were determined by one-way or repeated-measure analysis of variance with Bonferroni's post-test using GraphPad InStat version 3.05 for Windows 95 (GraphPad Software, Inc., San Diego, http://www.graphpad.com). Differences were accepted as significant when p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
MSC Inhibit Apoptosis of Resting and IL-8-Activated Neutrophils
We first investigated the effects of MSC on spontaneous neutrophil apoptosis. Circulating neutrophils from healthy donors were cultured in the absence or presence of MSC suspensions for different times and at different ratios. MSC exerted a significant antiapoptotic effect, as assessed by morphological evaluation. Inhibition of neutrophil apoptosis occurred at an MSC:neutrophil ratios ranging from 1:1 to 1:500 and disappeared at higher ratios (1:1,000 and 1:2,000) (Fig. 2A). Based upon these results, all of the subsequent experiments were performed using an MSC:neutrophil ratio of 1:50.


Figure 2
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  		Figure 2. Evaluation of apoptosis in resting or activated neutrophils cultured with or without human mesenchymal stem cells. (A): PMN were cultured either alone or with MSC for 18 h at different ratios. Apoptosis was evaluated morphologically on cytospins stained with May-Grumwald-Giemsa. The results are expressed as mean ± SD from four independent experiments (*, p < .05; **, p < .001). Insets show the morphological features of neutrophils incubated without (Aa) or with (Ab) MSC. Cells showing apoptotic features such as condensed nuclei and apoptotic bodies are indicated by arrows (original magnification, x1,000). (B): Neutrophils were cultured either alone or with MSC for 18 or 40 h at a 50:1 ratio, and apoptosis was determined by morphological analysis. The results are expressed as mean ± SD from seven independent experiments (p < .001 for both culture times). (C): Neutrophils were cultured without or with MSC at a 50:1 ratio for 18 h, double-stained with annexin V-FITC (FL1) to detect phosphatidylserine exposure and propidium iodide (FL2) to detect DNA, and analyzed by flow cytometry. The lower left quadrant shows viable cells; the lower right quadrant shows cells in early stages of apoptosis; the upper right quadrant shows cells in later stage of apoptosis; and the upper left quadrant shows necrotic cells. This experiment is representative of the 10 performed. (D): Neutrophils were activated with IL-8 and cultured either alone or with MSC for 18 h at a 50:1 ratio. Apoptosis was evaluated morphologically on cytospins stained with May-Grumwald-Giemsa. The results are expressed as mean ± SD from four independent experiments (p < .001). Abbreviations: FITC, fluorescein isothiocyanate; h, hours; IL, interleukin; PMN, polymorphonuclear neutrophils.

 
Figure 2A, inset, shows that the vast majority of neutrophils cultured with medium alone displayed the typical morphological features of programmed cell death, including condensed nuclei and apoptotic bodies. In contrast, most neutrophils incubated in the presence of MSC were viable.

Figure 2B shows that MSC significantly inhibited neutrophil apoptosis after both 18- and 40-hour coincubation. These findings were confirmed by flow cytometric analysis of neutrophils stained with propidium iodide (PI) and annexin V (p < .002 for 18- and 40-hour cocultures). As shown in the representative experiment of Figure 2C, neutrophils incubated with MSC contained a lower percentage of annexin+ PI– early apoptotic cells than neutrophils cultured with medium alone. Notably, the low proportion of PI+ cells present both in control PMN and in PMN incubated with MSC may be attributable to the timing of the assay (18 hours after isolation). Indeed, following 40-hour culture, PI+ PMN increased up to 30% (data not shown).

Activated neutrophils play a fundamental role in the clearance of bacterial infections but, in a variety of disease states, can contribute to chronic inflammation and tissue damage [34]. We therefore investigated whether MSC also exerted an antiapoptotic effect on neutrophils that had been preactivated with IL-8. Indeed, MSC were found to mediate a significant antiapoptotic effect on IL-8-activated neutrophils, as shown by morphological analysis (Fig. 2D) and confirmed by flow cytometry (data not shown).

Effects of MSC on Bax and MCL-1 Expression in Neutrophils
Bax, a member of the Bcl-2 family, is a proapoptotic mitochondrial protein modulated during spontaneous neutrophil apoptosis [35]. Here, we evaluated by immunocytochemistry and digital image analysis the expression of Bax in neutrophils cultured in the absence or presence of MSC. As shown in Figure 3A, Bax was expressed in neutrophils cultured for 18 hours. However, upon incubation with MSC for the same time interval, neutrophils displayed significantly decreased Bax expression. Figure 3A (insets) shows a representative microscopic field from a cytospin where downregulation of Bax in neutrophils cultured with MSC (Fig. 3Ab) versus neutrophils cultured without MSC (Fig. 3Aa) is apparent.


Figure 3
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  		Figure 3. Modulation of Bax and MCL-1 expression in neutrophils cultured with or without human mesenchymal stem cells. Neutrophils were incubated for 18 hours with or without MSC. Bax (A) and MCL-1 (B) expression was determined by immunocytochemistry and digital image analysis. The results are expressed as mean ± SD from four independent experiments (p < .05). Insets show representative stainings of neutrophils coincubated in the absence (Aa, Ba) or presence (Ab, Bb) of MSC for Bax (Aa, Ab) and MCL-1 (Ba, Bb), respectively. Abbreviation: PMN, polymorphonuclear neutrophils.

 
In contrast, as shown in Figure 3B, we found that MCL-1, a well-known mitochondrial antiapoptotic protein, was significantly upregulated in neutrophils after 18-hour culture with MSC compared with neutrophils cultured in medium alone for the same time interval. Representative microscopic fields are shown in Figure 3Bb and 3Ba, respectively.

FAS (CD95) is a cell surface protein belonging to the tumor necrosis factor (TNF) superfamily membrane receptors that induces ligand-dependent apoptosis through the extrinsic pathway in many cell types, including neutrophils [36]. Here, we evaluated the expression of CD95 in neutrophils cultured in the presence or absence of MSC for 18 hours by flow cytometry. Constitutive neutrophil CD95 expression was unaffected by MSC incubation (PMN alone, 30% CD95+ cells; PMN cultured with MSC, 34% CD95+ cells; mean values from three independent experiments).

Effects of MSC on Neutrophil Oxidative Metabolism
Neutrophils undergo a spontaneous oxidative response that can be increased by different stimuli, such as the bacterial-derived peptide f-MLP [37]. Several studies indicate a close relationship between neutrophil apoptosis and oxidant production [38–40].

To investigate the effects of MSC on neutrophil oxidative metabolism, we assessed the oxidative status of neutrophils incubated with or without MSC for 1 hour and then exposed to f-MLP or medium for 30 minutes. Intracellular hydrogen peroxide levels were detected by flow cytometry. As shown in Figure 4A, MSC significantly downregulated intracellular hydrogen peroxide production by both unstimulated and f-MLP-stimulated neutrophils.


Figure 4
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  		Figure 4. Modulation of neutrophil oxidative metabolism by human mesenchymal stem cells. In (A), neutrophils were incubated with or without MSC for 1 h and then exposed to FMLP or medium for 30 minutes. MFI of 2'-7'-dichlorofluorescein-diacetate was then evaluated by flow cytometry. The results are expressed as mean ± SD from four independent experiments (p < .05). In (B), neutrophils were coincubated with (white columns) or without (black columns) MSC for 1 h and then exposed for 30 minutes to nil, OPZ, or PMA, tested as positive control. Superoxide production was then determined spectrophotometrically as measure of phagocytic activity. A representative experiment of three performed is shown. Abbreviations: FMLP, N-formyl-L-methionin-L-leucyl-L-phenylalanine; h, hours; MFI, mean fluorescence intensity; nil, medium; OPZ, opsonized zymozan; PMA, phorbol myristate acetate; PMN, polymorphonuclear neutrophils.

 
We next investigated the effects of MSC on neutrophil phagocytosis by evaluating superoxide anion production in neutrophils incubated 30 minutes with medium, OPZ, or phorbol myristate acetate tested as positive control. As shown in Figure 4B, neutrophil phagocytosis was unaffected by incubation with MSC.

Effects of MSC on Neutrophil Adhesion and Migration
To investigate the effects of MSC on neutrophil adhesion, we first addressed the expression of the integrin CD11b and the selectin CD62L. Neutrophils were incubated with or without MSC for 1 hour, exposed to f-MLP or medium alone, stained, and analyzed by flow cytometry. As shown in Figure 5A, MSC did not modulate neutrophil expression of CD11b or CD62L, irrespective of whether or not the latter cells had been treated with f-MLP.


Figure 5
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  		Figure 5. Expression of adhesion molecules and chemotaxis of neutrophils cultured with or without mesenchymal stem cells. In (A) and (B), neutrophils were incubated with or without MSC for 1 h and then exposed or not to FMLP for 30 minutes. CD62L (A) and CD11b (B) expression was evaluated by flow cytometry. The results are expressed as mean ± SD from four independent experiments (p > .05). In (C), neutrophils were incubated with or without MSC suspensions for 1 h, washed, and resuspended in medium containing C5a, IL-8, or FMLP. Neutrophil migration was assessed in 48-well microchemotaxis chambers and expressed as chemotaxis index, obtained by dividing the number of cells that migrated to the stimulus by the number of cells that migrated to the ctr medium. The results are expressed as mean ± SD from four independent experiments (p > .05). Abbreviations: ctr, control; FMLP, N-formyl-L-methionin-L-leucyl-L-phenylalanine; h, hours; IL, interleukin; MFI, mean fluorescence intensity; PMN, polymorphonuclear neutrophils.

 
We next investigated the effects of MSC on neutrophil chemotaxis. Neutrophils were cultured with or without MSC and subsequently incubated in a transwell system with C5a, IL-8, or f-MLP. Figure 5B shows that MSC did not exert any significant effect on neutrophil chemotaxis in response to the above stimuli.

Mechanisms of MSC-Mediated Protection of Neutrophils from Apoptosis
To identify the mechanisms underlying the MSC-mediated protective effect on neutrophil apoptosis, we performed transwell experiments in which resting neutrophils, seeded in the lower chamber, were physically separated from MSC plated in the upper chamber. Figure 6A shows that MSC exerted a significant antiapoptotic effect on resting neutrophils, irrespective of the presence of the filter separating MSC from neutrophils. Paired cultures in which neutrophils were seeded in the upper chamber and MSC in the lower one were performed with superimposable results.


Figure 6
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  		Figure 6. Human mesenchymal stem cells inhibit neutrophil apoptosis through the release of soluble factors. (A): Percentage of apoptotic cells evaluated by morphological analysis in a transwell system. MSC and neutrophils at a 1:50 ratio were seeded in six-well plates on the opposite sides (medium/neutrophil: medium in the upper chamber/neutrophils plated in the lower chamber; MSC/neutrophil: MSC in the upper chamber/neutrophils plated in the lower chamber) of 0.5-mm pore size polycarbonate membrane (transwell) for 18 h and analyzed for apoptosis. The results are expressed as mean ± SD from four independent experiments (p < .005). (B): Percentage of apoptotic cells evaluated by morphological analysis of neutrophils incubated for 18 h in the absence or presence of different dilutions of MSC supernatant. The results are expressed as mean ± SD from five independent experiments (*, p < .05; **, p < .001). (C): Percentage of apoptotic cells evaluated by morphological analysis of neutrophils incubated in the absence or presence of MSC supernatant (diluted 1:1) for 18 or 40 h. The results are expressed as mean ± SD from five independent experiments (p < .001). Abbreviations: h, hours; PMN, polymorphonuclear neutrophils.

 
Next, we investigated whether soluble factors produced constitutively by MSC were responsible for protection of neutrophils from apoptosis. Supernatants from confluent MSC diluted in the range between 1:1 and 1:100 were tested for inhibition of neutrophil apoptosis. As shown in Figure 6B, supernatant dilutions up to 1:5 significantly inhibited resting neutrophil apoptosis in a dose-dependent manner after 18-hour culture, as assessed by morphological analysis. Inhibition of neutrophil apoptosis by MSC supernatant was detected after both 18- and 40-hour culture (Fig. 6C). The above findings were confirmed by flow cytometric analysis of neutrophils stained with propidium iodide and annexin V (data not shown). Finally, MSC supernatants significantly inhibited neutrophil oxidative metabolism but did not affect phagocytosis, expression of adhesion molecules, or chemotaxis (data not shown).

MSC-Derived IL-6 Inhibits Neutrophil Apoptosis Through a STAT-3-Dependent Mechanism
To characterize the soluble factor(s) responsible for MSC-mediated inhibition of neutrophil apoptosis, we investigated the role of IL-6, which is constitutively produced by MSC [41, 42] and inhibits neutrophil programmed cell death [35, 43, 44]. First, we investigated IL-6 gene expression in MSC versus normal peripheral blood mononuclear cells by real-time PCR. The change of amplification of IL-6 was normalized to the RPL0 gene. We obtained a –{Delta}{Delta}Ct value of –8.2, corresponding to a relative fold increase in IL-6 gene expression of 29,407. After real-time PCR, the expected IL-6 band represented by a 100-bp fragment was run on a gel. A representative experiment is shown in Figure 7A.


Figure 7
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  		Figure 7. Mesenchymal stem cell-derived IL-6 rescues neutrophils from apoptosis. (A): IL-6 gene expression in MSC and normal mononuclear cells as assessed by reverse transcription-polymerase chain reaction. Lanes 1 and 2, amplification products of RPL0 and IL-6 genes, respectively, from MSC cells; lanes 4 and 5, amplification products of the RPL0 and IL-6 genes, respectively, from mononuclear cells; lanes 3 and 6, water in the place of cDNA. Arrow indicates a 100-bp fragment corresponding to the amplification product of both IL-6 and RPL0 genes. (B): Quantification of IL-6 in MSC sup as determined by enzyme-linked immunosorbent assay. Sample 1 and sample 2 represent the sup harvested from MSC isolated from two donors. Experiments were performed in quadruplicate, and results are shown as mean ± SD. (C): Percentage of apoptotic cells evaluated by morphological analysis of neutrophils incubated in the absence or presence of MSC sup, alone or in combination with an anti-human-IL-6 receptor or an isotype-matched monoclonal Ab (mAb), for 18 hours (**, p < .001). (D): Percentage of apoptotic neutrophils evaluated by morphological analysis following 18-hour incubation with or without human rIL-6 at different concentrations (**, p < .001). (E): Phosphorylation of STAT3, as assessed by Western blot analysis, in neutrophils incubated for 10 minutes in the absence or presence of MSC sups with an anti-human-IL-6 receptor mAb or an isotype-matched ctr. Lane 1, neutrophil control; lane 2, neutrophils + MSC sup with isotype-matched mAb; lane 3, neutrophils + MSC sup with anti-human-IL-6 receptor mAb. The specific 92-kDa band is shown. This experiment is representative of the three performed with similar results. Abbreviations: Ab, antibody; bp, base pair; ctr, control; IL, interleukin; M, marker; PMN, polymorphonuclear neutrophils; rIL, recombinant interleukin; sup, supernatant.

 
Next, IL-6 was quantified by ELISA in two supernatants (sample 1 and sample 2) obtained from MSC isolated from different donors (range 7–8 ng/ml). These experiments are shown in Figure 7B. Neutrophils were incubated for 18 hours with a blocking anti-human-IL-6 receptor mAb or an isotype-matched mAb in the presence or absence of MSC supernatant. Figure 7C, shows that the antiapoptotic effect mediated by MSC supernatant was significantly decreased upon IL-6 receptor blockade. Similar results were obtained when neutrophils were incubated with MSC supernatant that had been pretreated with a neutralizing anti-human-IL-6 or an isotype-matched mAb (data not shown).

Human recombinant interleukin (rIL)-6 was found to inhibit spontaneous neutrophil apoptosis in a dose-dependent manner (Fig. 7D). The antiapoptotic activity of rIL-6 tested at a concentration of 10 ng/ml was equivalent to that exerted by MSC supernatants (Fig. 7D).

IL-6 signaling involves phosphorylation of the Stat-3 transcription factor [45]. We therefore investigated whether Stat-3 had a role in neutrophil rescue from apoptosis induced by MSC supernatants. To this end, in three different experiments, neutrophils were cultured in medium alone (control) or with MSC supernatant in the presence of the anti-IL-6R mAb or the isotype-matched control mAb. Cell lysates were subsequently blotted with an mAb directed to phosphorylated Stat-3. As shown in the representative experiment of Figure 7E, a band corresponding to phosphorylated Stat-3 (92-kDa band) was detected in neutrophils incubated with MSC supernatant in combination with an isotype-matched control mAb. This band was barely detectable in cells incubated with MSC and anti-IL-6R mAb (Fig. 7E). These results indicate that the antiapoptotic activity of MSC-derived IL-6 on neutrophils involved cell signaling through Stat-3.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
Previous in vitro and in vivo studies have highlighted the protean immunoregulatory activities of MSC [22], which include inhibition of T-cell, B-cell, and NK-cell proliferation and effector functions, as well as of dendritic cells maturation, activation, and antigen presentation [14]. In the present study, we have addressed for the first time the modulation of neutrophil function by MSC. We demonstrate that human MSC potently inhibit in vitro apoptosis of both resting and activated neutrophils at very low MSC:neutrophil ratios (up to 1:500). Such results may recapitulate the interactions occurring in vivo between these cell types both under physiological conditions and in the course of inflammatory responses.

The immunoregulatory activities of MSC are mediated by either cell-cell contacts or soluble factors [22]. In this study, transwell experiments, in which MSC and neutrophils were physically separated, showed unequivocally that MSC exerted their antiapoptotic effects through soluble factors without a cell-cell contact requirement. Accordingly, neutrophils were rescued from apoptosis upon incubation with MSC supernatants.

Prolongation of neutrophil survival by MSC was found to involve reciprocal modulation of two mitochondrial proteins of the Bcl-2 family, Bax and MCL-1, but not of CD95. Expression of proapoptotic Bax increases in neutrophils undergoing spontaneous apoptosis, whereas that of antiapoptotic MCL-1 is upregulated in surviving neutrophils [46, 47]. Accordingly, we demonstrated that the protection of neutrophils from apoptosis provided by MSC was paralleled by Bax downregulation and MCL-1 upregulation in the former cells. It is of note that MSC-mediated inhibition of apoptosis has also recently been reported for quiescent T cells and thymocytes [48].

The antiapopotic effects of MSC were of similar magnitude and duration as those reported by other investigators upon neutrophil incubation with various cytokines, including IL-1β, TNF, IL-6, interferon-{gamma}, granulocyte colony-stimulating factor, and granulocyte macrophage–colony-stimulating factor [49, 50]. In analogy to our findings, cytokine-driven rescue of neutrophils from apoptosis occurred through rapid modulation of Bax and MCL-1 [47].

These considerations prompted us to investigate whether a constitutively produced MSC-derived cytokine was responsible for neutrophil protection from apoptosis. IL-6 was selected as the target for these experiments since it is expressed and secreted constitutively by MSC [41, 42, 51, 52] and inhibits neutrophil apoptosis [35, 43, 53, 54], as also shown in the present study. Indeed, blocking experiments with an anti-IL-6R mAb demonstrated unambiguously that IL-6 present in MSC culture supernatants was a key molecule for neutrophil rescue from programmed cell death. This conclusion was reinforced by the results of neutralization experiments of MSC supernatants with an anti-IL-6 mAb. Finally, phosphorylated Stat-3, a transcription factor involved in IL-6 signaling [45], was detected at a high level in neutrophil incubated with MSC supernatant and was found to be dampened by mAb-mediated IL-6R blockade. In this connection, another member of the Stat family (i.e., Stat-5) was recently identified as a key transcription factor involved in nitric oxide-mediated inhibition of murine T-cell proliferation by MSC [55].

Reactive oxygen species (ROS) production is an essential step required for the elimination of invading pathogens by neutrophils. This is exemplified by chronic granulomatous disease, a genetic disorder characterized by defective production of superoxide metabolites [56], in which neutrophils display impaired intracellular killing of ingested microorganisms. Nonetheless, to the best of our knowledge, there are no published data suggesting increased infectious risk in MSC-treated patients.

On the other hand, when activation of the respiratory burst is excessive or inappropriate, ROS participate in severe host tissue injury and are involved in various pathological conditions, including ischemia-reperfusion injury, chronic obstructive pulmonary diseases, acute respiratory distress syndrome, atherosclerosis, malignancy, and rheumatoid arthritis [57]. It is therefore apparent that ROS formation is finely modulated by several stimuli through different intracellular pathways and represents a critical step in the cascade of events culminating in persistent activation of the inflammatory response [40, 58, 59]. The finely tuned balance between ROS-mediated protection from infection and role in tissue damage must be considered when treating patients with MSC.

In this study, MSC inhibited basal and f-MLP-stimulated production of ROS by neutrophils, supporting the conclusion that MSC generate in vitro a protective milieu where the respiratory burst is strongly downregulated. Interestingly, inhibition of the respiratory burst mediated by MSC was not detected in neutrophils exposed to OPZ, thus allowing the efficient phagocytosis of the opsonized particles. This latter finding suggests that MSC, while exerting a protective role from an overwhelming and potentially harmful inflammatory reaction, preserve the essential neutrophil effector functions. Accordingly, we did not observe any modulatory activity of MSC on either neutrophil expression of the CD11b and CD62L, which promote adhesion to the endothelium, or chemotaxis to C5a, IL-8, and f-MLP, three potent neutrophil chemoattractants.

In conclusion, modulation of resting neutrophil survival in vitro represents a constitutive property of MSC, perhaps reflecting a physiological function exerted by MSC in the bone marrow niche. Such a hypothesis is mitigated by the use throughout this study of in vitro-cultured MSC that likely differ from their in vivo precursors in a number of properties, such as shape, size, and adherence.

Neutrophils of the bone marrow storage pool show enhanced phagocytic and killing capacities compared with the circulating pool that are not attributable to differences in cell maturity. Therefore, a mechanism of preservation of neutrophil activity during margination in vivo has been postulated [27, 60–62]. Our present results are consistent with the hypothesis that, in the bone marrow niche, MSC protect neutrophils of the storage pool from apoptosis, preserving their effector functions and preventing the excessive or inappropriate activation of the oxidative metabolism. Similar mechanisms may operate in human lungs, where a population of resident MSC has recently been identified [63] and where a large pool of marginated neutrophils adheres to sinusoid vessels [34].

The host-damaging potential of activated neutrophils is limited by elimination of the primary triggers of inflammation and by neutrophil inactivation through tachyphylaxis to proinflammatory mediators and apoptosis [34]. The present study delineates a novel mechanism whereby the inflammatory potential of activated neutrophils is harnessed by inhibition of apoptosis and ROS production without impairing phagocytosis and chemotaxis. Support for this hypothesis comes from the ubiquitous distribution of MSC, which have been isolated from virtually all tissues [64].


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
L.R. was a recipient of a fellowship from the Italian Association of Neuroblastoma. We thank Drs. Francesco Dazzi, Chiara Boccelli, Francesco Frassoni, and Antonio Uccelli for critically reading the manuscript and providing suggestions and Dr. Anna Garuti for the help in the real-time PCR experiments. This work was supported by Ricerca Finalizzata 2005 "Cellule staminali e terapie cellulari regenerative. Caratterizzazione delle proprietà di immuno-modulazione delle cellule staminali mesenchimali e possible applicazione nel trattamento delle malattie autoimmuni" from Italian Ministry of Health and by a grant from Fondazione Carige. L.R., G.B., and M.B. contributed equally to this work as first authors. L.O. and V.P. contributed equally to this work as last authors.


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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

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