TISSUE-SPECIFIC STEM CELLS

Characterization and Function of Histamine Receptors in Human

Bone Marrow Stromal Cells

KRISZTIAN NEMETH,aTODD WILSON,

bBALAZS RADA,

bALISSA PARMELEE,

aBALAZS MAYER,

aEDIT BUZAS,

c

ANDRAS FALUS,c SHARON KEY,a TAMAS MASSZI,d SAROLTA KARPATI,e EVA MEZEYa

aNational Institutes of Dental and Craniofacial Research, Craniofacial and Skeletal Diseases Branch; bLaboratory

of Host Defenses, National Institute of Allergy and Infectious Diseases, NIH, Rockville, Maryland, USA; cFaculty

of Medicine, Department of Genetics, Cell- and Immunobiology and eDepartment of Dermato-Venereology and

Dermato-Oncology, Semmelweis University, Budapest, Hungary; dSzent Laszlo Hospital, Department of

Hematology and Stem Cell Transplantation, Budapest, Hungary

Key Words. Adult stem cells • Apoptosis • Bone marrow stromal cells • IL-6R/IL-6 • Mesenchymal stem cells

ABSTRACT

There are several clinical trials worldwide using bonemarrow stromal cells (BMSCs) as a cellular therapy tomodulate immune responses in patients suffering from

various inflammatory conditions. A deeper understandingof the molecular mechanisms involved in this modulatory

effect could help us design better, more effective protocolsto treat immune mediated diseases. In this study, we dem-onstrated that human BMSCs express H1, H2, and H4 his-

tamine receptors and they respond to histaminestimulation with an increased interleukin 6 (IL-6) produc-

tion both in vitro and in vivo. Using different receptorantagonists, we pinpointed the importance of the H1 hista-mine receptor, while Western blot analysis and application

of various mitogen-activated protein kinase inhibitorshighlighted the role of p38, extracellular signal-regulatedkinase, and c-Jun N-terminal kinase kinases in the

observed effect. When BMSCs were pretreated with eitherhistamine or degranulated human mast cells, they exhib-

ited an enhanced IL-6-dependent antiapoptotic effect onneutrophil granulocytes. Based on these observations, it islikely that introduction of BMSCs into a histamine-rich

environment (such as any allergic setting) or pretreatmentof these cells with synthetic histamine could have a signifi-

cant modulatory effect on the therapeutic potential ofBMSCs. STEM CELLS 2012;30:222–231

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

INTRODUCTION

Bone marrow stromal cells (BMSCs, also called mesenchymalstem cells or MSCs) are known to generate osteogeneic, adipo-geneic, and chondrogeneic lineages and help to maintain themicroenvironment necessary for hematopoiesis. In the last 5years, it became evident that BMSCs—in addition to nursinghematopoietic stem cells—also have potent immunoregulatoryfunctions; they appear to regulate the differentiation, survival,and activation of a wide variety of immune cells [1–4]. Inrecent studies, we have shown that when BMSCs are introducedinto a pathological milieu, they can detect soluble disease-spe-cific mediators (e.g., lipopolysaccharide and tumor necrosis fac-tor a in sepsis or interleukin 4 [IL-4] and IL-13 in asthma) andrespond to them in ways that are optimal for the host [5, 6].

Histamine is a biogenic amine that plays an essential rolein several physiological and pathophysiological processes. It

acts as a neurotransmitter in the central nervous system, reg-ulates HCl synthesis in the stomach, and mediates anaphy-laxis in allergic conditions. Released from mast cells (MCs)and basophil granulocytes, it also contributes to the patholog-ical changes seen in sepsis and several autoimmune andalloimmune disorders [7, 8] and plays an important role inimmunomodulation [9] through its effect on cytokines. Hista-mine appears to stimulate the production and release of cyto-kines including IL-1a and IL-6 in different cells, both knownto be produced by BMSCs [10–12] and has proinflammatory,anti-inflammatory, and immunoregulatory functions [13, 14].As in our previously published study [15], a G-protein–coupled receptor (GPCR) array based on multiplex polyme-rase chain reaction (PCR) suggested that BMSCs mightexpress mRNAs encoding at least two of the histamine recep-tors, H1 and H2, we wondered whether BMSC-derived IL-6production might be regulated by histamine in vitro as wellas in vivo.

Author contributions: K.N.: conception and design, collection, assembly, analysis and interpretation of data, and manuscript writing;T.W., E.B., A.F., T.M., and S.K.: provision of study material or patients; B.M.: performing and evaluating of RT-PCR and in vitroexperiments and collection and/or assembly of data; B.R.: performing and evaluating superoxide assays; A.P.: collection and/orassembly of data; E.M.: conception and design, data analysis and interpretation, and illustrations and manuscript writing.

Correspondence: Krisztian Nemeth, M.D., Ph.D. Telephone: 301-451-9880; Fax: 301-496-1339; e-mail: nemethk@mail.nih.gov; or EvaMezey, M.D., Ph.D. Telephone: 301-435-5635; Fax: 301-496-1339; e-mail: mezeye@mail.nih.gov Received June 15, 2011; acceptedfor publication September 30, 2011; first published online in STEM CELLS EXPRESS November 1, 2011. VC AlphaMed Press 1066-5099/2012/$30.00/0 doi: 10.1002/stem.771

STEM CELLS 2012;30:222–231 www.StemCells.com

MATERIALS AND METHODS

BMSCs were isolated from bone marrow aspirates donated byhealthy volunteers. Cells were grown in complete minimumessential medium a (MEM-a) medium (20% fetal bovine se-rum [FBS], 1% Pen/Strep, 1% glutamine). Characterization ofthe cells showed osteogeneic and adipogeneic differentiationpotential in vitro and expression of various BMSC-specificcell surface markers (CD73, CD90, CD105, and CD146) butlack of hematopoietic markers (CD45, CD14, and CD34).

Immunohistochemistry

For immunostaining, human BMSCs in chamber slides (eight-well chambers from Lab-Tek) were used (5,000 cells per wellfixed with 4% paraformaldehyde). The slides were blockedwith 1� Universal Blocking Reagent (Biogenex, San Ramon,CA) for 10 minutes at room temperature (RT). Primary anti-bodies (rabbit anti-human H1R, H2R, H3R, H4R from AlphaDiagnostic) were applied overnight at 4�C, diluted 1:100 in1% BSA containing 0.25% Triton X100. After washing threetimes in phosphate-buffered saline (PBS), Alexa-488-conju-gated anti-rabbit secondary antibody was used at a 1:1,000dilution for 1 hour at RT. 40,6-Diamidino-2-phenylindole wasused to visualize cell nuclei. The fluorescent staining wasobserved with a Leica DMI600 inverted fluorescent micro-scope using the FITC filter set.

Reverse Transcriptase PCR

Total RNA was isolated from human BMSCs using the Stra-tagene Absolutely RNA Microprep Kit (Stratagene, La Jolla,CA) with modification of the DNase treatment step. Briefly,on-column DNA digestion was performed using QiagenDNase (RNase-free DNase Set, Qiagen, Valencia, CA) at RTfor 30 minutes. Isolated RNA (40 ng) was amplified with theone-step QuantiTect SYBR Green RT-PCR Kit (Qiagen)using previously published primers for H1R, H2R, H3R (16),and H4R (17).

The one-step reverse transcriptase PCR (RT-PCR) condi-tions were as follows: 50�C, 30-minute reverse transcription,95�C, 15-minute initial activation of the Taq polymerase anddenaturation of the reverse transcriptase, and then 40 cyclesof 94�C, 15-second denaturation, 58�C, 30-second annealing,and 72�C 30-second extension. For the H4R primers, theannealing temperature was 55�C. PCR product was run on2% agarose gel and visualized with ethidium bromide.

Enzyme-Linked Immunosorbent Assay (ELISA)

IL-6 and IL-8 levels were measured by ELISA following theDuoSet ELISA Development Kit Instructions (R&D Systems).

Western Blots

BMSCs were collected following trypsin digestion, and totalprotein concentrations were determined using the Micro BCAKit (Pierce, www.piercenet.com). Total protein lysates (20lg) were run on 4%–12% Bis-Tris gels and blotted onto nitro-cellulose membranes (Invitrogen). The membranes wereblocked with 4% nonfat dry milk in TBS (1� TBS, 0.01%Tween 20) and incubated overnight at 4�C with antibodiesagainst phosphorylated p38 mitogen-activated protein (MAP)kinase (p38), phosphorylated p44/42 MAP kinase (extracellu-lar signal-regulated kinase [ERK]), and phosphorylated c-junterminal NH2 kinase at 1:500, 1:1,000, and 1:500 dilution,respectively, or as a control with antibodies against total p38,total ERK, and total c-Jun N-terminal kinase (JNK) at 1:1,000dilution each. All antibodies were from Cell Signaling(www.cellsignal.com).

Anti-rabbit horseradish peroxidase (Jackson Immunore-search, www.jacksonimmuno.com) was used as a secondaryantibody for 1 hour at RT. The blots were visualized withWestern lightning enhanced chemiluminescence reagents (Per-kin Elmer, www.perkinelmer.com).

Cytosolic Ca21 Measurements

For cytosolic Ca2þ measurements, cells were loaded with 3lM fura-2/acetoxy methylester (45 minutes, room temperature).Ca2þ measurements were performed at room temperature in amodified Krebs-Ringer buffer containing 120 mM NaCl, 4.7mM KCl, 1.2 mM CaCl2, 0.7 mM MgSO4, 10 mM glucose,and 10 mM Na–4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid, pH 7.4. An inverted microscope (IX70; Olympus, www.olympusmicro.com/primer/techniques/fluorescence/tirf/ix70cutaway.html) equipped with an illuminator (Lambda-DG4;Sutter Instrument Co., www.sutter.com) and a digital camera(MicroMAX-1024BFT; Roper Scientific, www.roperscientific.com) and the appropriate filter sets were used for Ca2þ analy-sis. Data acquisition and processing were performed by theMetaFluor software (Molecular Devices, www.moleculardevices.com/Products/Software/.../MetaFluor.html).

Histamine Stimulation with or Without Antagonists,Inhibitors

For in vitro IL-6 measurements, BMSCs were cultured in 96-well flat bottom cell culture plates (Corning, catalog2.corning.com/Lifesciences/) at a density of 20,000 cells per well per200 microliters in complete MEM-alpha medium. Histaminewas applied at a concentration of 10�4, 10�5, 10�6, 10�7, or10�8 M, and histamine receptor antagonists were applied at aconcentration of 10�5 M (diphenhydramine hydrochloride [H1antagonist], cimetidine [H2 receptor antagonist], ciproxifanhydrochloride [H3 antagonist], JNJ7777120 [H4 antagonist]).MAP kinase pathway inhibitors (SP203580 a specific inhibitorof p38, PD98059 a specific inhibitor of ERK phosphorylation,SP600125 a specific inhibitor of JNK) and the phospholipase C(PLC) inhibitor U73122 were used at 10�5 or 10�6 M concen-trations. Antagonists or inhibitors were added 1 hour prior toaddition of histamine. IL-6 was measured from the supernatantsafter 6-, 12-, or 24-hour histamine stimulation using DuoSetELISA Kit following the manufacturer’s instructions (R&DSystems, www.rndsystems.com).

Stimulation of BMSCs with Additional MastCell-Derived Factors

To test the hypothesis of additional MC-derived factors affect-ing the BMSCs IL-6 production, we repeated the in vitroexperiments and stimulated the BMSCs with additional fac-tors released by MCs [18], such as leukotriene C4 (LTC4),platelet-activating factor (PAF), serotonin (5HT), leukotrieneB4 (LTB4), and prostaglandin D2 (PGD2). We chose thesecompounds, because based on our GPCR array [15] BMSCsbear the appropriate receptors to respond (Supporting Infor-mation Table 1). A total of 10,000 BMSCs per well wereplated in 96-well plates in MEM-alpha medium with 5%FBS. After overnight incubation, the medium was changedand the cells were stimulated with PGD2 at a concentration of10�5, 10�6, and 10�7 M for 6, 12, or 24 hours. The same vol-ume of medium and vehicle (ethanol) was added to the con-trol wells as in the wells with PDG2 stimulation. Other media-tors we used to stimulate BMSCs for 6 hours in theseexperiments were: serotonin (concentration range: 10�4–10�10

M), platelet activating factor (10�6–10�12 M), LTB4 (10�6–10�10 M), and LTC4 (10�6–10�10 M). All of the mediatorswere purchased from Cayman Chemical (Ann Arbor, MI,

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www.caymanchem.com), except serotonin that was from Toc-ris Bioscience (Ellisville, MO, www.tocris.com). Cell culturesupernatants were measured in quadruplicates using thehuman IL-6 DuoSet ELISA Kit (R&D Systems). Nonstimu-lated cells (medium alone) and histamine-stimulated cellswere included in each assay (data not shown). Experimentswere done using BMSCs derived from four different donors.

Culture and Assays Involving Human Mast Cells

Human-derived MCs (HuMC) were cultured as described. Inbrief, peripheral blood CD34þ progenitor cells were collectedfrom healthy donors after informed consent and affinity col-umn apheresis. CD34þ cells were cultured in StemPro-34 se-rum-free medium (Life Technologies, Grand Island, NY,www.lifetechnologies.com/us/en/home.html) in the presenceof recombinant IL-3 (first week only), recombinant IL-6, andrecombinant human stem cell factor (rhSCF; Peprotech,www.peprotech.com). HuMC cultures were maintained up to10 weeks at 37�C and 5% CO2.

HuMCs were sensitized overnight with 100 ng/ml biotin-ylated IgE (Sigma-Aldrich, www.sigmaaldrich.com). The nextday, 50,000 HuMCs were added to the upper chamber of a96-well transwell system (Corning) in complete MEM-alphamedium and degranulated by the addition of 100 ng/ml strep-tavidin (Sigma-Aldrich). After 6 hours, the transwell insertswere removed and the lower chamber was gently washedwith cell culture medium twice. After the last wash, 200 llfresh medium was added to each wells. After 6 hours, super-natants were collected and assayed for IL-6.

Isolation of Granulocytes from Whole Blood

Granulocytes were isolated using Lymphocyte Poly(R) den-sity gradient (Cedarlane Labs, www.cedarlanelabs.com) fol-lowing the manufacturers instructions. Briefly, anticoagulatedwhole blood is layered on top of the density solution. Aftercentrifugation, the granulocyte layer was removed, and con-taminating red blood cells were lysed using ACK lysingbuffer. Granulocyte purity was routinely more than 90% asconfirmed by FACS using CD66b-FITC antibody (BD Bio-sciences, www.bdbiosciences.com/home.jsp). For the super-oxide production experiments, granulocytes were isolatedusing a different method. In these experiments, anticoagu-lated (heparin) blood was layered on top of Histopaque 1119(Sigma, St. Louis, MO, www.sigmaaldrich.com/united-states.html) and centrifuged (800g, 30 minutes RT). Upperserum layer was aspirated; the middle phase containing whiteblood cells was collected. Pellets were resuspended in PBSand layered on top of a Percoll gradient (65%, 70%, 75%,80%, and 85%, Sigma, St. Louis, MO). After centrifugation(800g, 20 minutes RT), the 70%–75%–80% Percoll layerscontaining neutrophils were collected, washed, and resus-pended in PBS. Viability of the cells was determined by Try-pan Blue dye extrusion and resulted in >99% viable neutro-phils. The purity of the preparations was determined byWright-Giemsa staining and yielded >95% neutrophil granu-locytes (PMNs).

Granulocyte/BMSC Cocultures and Assay forApoptosis

A total of 200,000 granulocytes were added in 200 ll com-plete medium to 96-well U-bottom plates either alone or to-gether with 20,000 BMSCs. After 18 hours of culture, per-centage of apoptotic granulocytes were assessed by FACSusing Annexin-V–allophycocyanin (APC) and 7-aminoactino-mycin D (7-AAD) (both from BD Biosciences). Annexin-Vsingle-positive cells were considered early apoptotic,

whereas Annexin-V/7-AAD double-positive cells were lateapoptotic.

In case of histamine or HuMC prestimulation of stemcells, BMSCs were pretreated for 24 hours with 10�5 M hista-mine or cocultured for 6 hours with 50,000 degranulatedHuMCs. Granulocytes were given thereafter.

Granulocyte/BMSC Cocultures and Measurement ofNeutrophil Superoxide Production

Granulocytes were cultured with or without BMSCs in Ros-well Park Memorial Institute 1640 complete medium on six-well plates at a 500:1 granulocyte/BMSC ratio for 24 hours.Neutrophils were collected, washed, resuspended in Hanks’balanced salt and transferred to 96-well white opaque plates.Cells were stimulated in triplicates at a final concentration of1.2 � 106 per milliliter with 1 lM formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma, St. Louis, MO) or 10 nM phor-bol myristate acetate (Sigma, St. Louis, MO). Superoxide pro-duction was measured by the Diogenes cellular luminescenceenhancement system (National Diagnostics, Atlanta, GA,www.nationaldiagnostics.com). Luminescence was followedfor 15 minutes on a Luminoskan Ascent microplate luminom-eter (ThermoScientific, Hudson, NH, www.thermoscientific.com). Cumulative superoxide production was calculated asthe area under the measured curve and it is presented as inte-grated relative luminescence units.

In Vivo Experiments

Assaying the Specificity of Histamine in the Effect. 6–8week-old male WT C57BL/6 mice (JAX, jaxmice.jax.org),WT BALB/c, and HDC (histidine-decarboxylase) KO mice(provided by Dr. Andras Falus, Department of Genetics,Cell- and Immunobiology, Semmelweis University, Hun-gary) or MC-deficient KitW-sh/W-sh mice (provided by Dr.David M. Segal, NCI, NIH) were sensitized i.p. with 1 lgmonoclonal IgE anti–2,4-dinitrophenol (anti-DNP) (Sigma)in 200 ll PBS followed 24 hours later by i.p. challenge with0.5 mg/ml DNP–human serum albumin in 200 ll PBS. Im-mediately following challenge, 4 million human BMSCswere injected into the peritoneal cavity. After 6 hours, peri-toneal cavity was washed with 2 ml PBS. After centrifuga-tion of the lavage fluid to remove cellular components, thesupernatants were frozen and stored at �80� until assayingfor human IL-6.

Testing Histamine Prestimulation on BMSCs Effect inZymosan-Induced Peritonitis. Zymosan A (2 mg/ml; 500 ll[Sigma]) was injected intraperitoneally into 10-week-old maleC57/B6 mice (n ¼ 4 per group). Human BMSCs (0.5 millionin 200 ll PBS) were injected 30 minutes later intraperitone-ally. In some cases, BMSCs were prestimulated overnightwith 10�4 histamine or cocultured with degranulated humanMCs with transwell separation for 6 hours. After 6 hours,peritoneal cavity was washed with 5 ml PBS. Peritoneal cellswere analyzed using FACS. To rule out possible contamina-tion of human BMSCs, first we gated on mouse CD45-posi-tive cells (CD45-PerCP from BD Biosciences). PMNs wereidentified as Gr-1 (high) CD11b (high) double-positive cells(Gr-1 FITC, CD11b-PE both from BD Biosciences). Apopto-tic cells were detected using APC-conjugated Annexin-V(eBioscience). The readout of the experiment was the absolutenumber of living (functional) peritoneal neutrophils.

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RESULTS

The actions of histamine are mediated by four G-protein-coupled receptor subtypes (H1–H4), each of which has a tis-sue- and cell-specific distribution in the human body. To findout which histamine receptors are present in BMSCs, we usedRT-PCR and immunostaining. We observed a relativelystrong expression of the H1 receptor, a less pronouncedexpression of the H2 and H4 receptors, and a complete lackof H3 receptors (Fig. 1).

Next, we measured the amount of IL-6 in the media ofhuman BMSC cultures after exposing the cells to differentconcentrations of histamine, and found a time-and dose-de-pendent increase in the IL-6 output. The dose/response curvewas bell-shaped with a maximum IL-6 response (a fourfold tofivefold increase over the baseline) at 10�5 M histamine. Thistrend can be seen at all time points examined—6, 12, and 24hours after introduction of histamine into the culture medium(Fig. 2A). This effect of histamine could be completelyblocked using a specific H1 receptor antagonist but was notaffected by H2–H4 receptor antagonists (Fig. 2B).

To see if histamine released from human MCs could alsostimulate IL-6 production and secretion by BMSCs, we cocul-tured the two cell types in a transwell system. Preincubationof BMSCs with activated (degranulated) MCs resulted in asignificant increase in their IL-6 secretion. Unlike when onlyhistamine was used, this effect was blunted but not eliminatedby the addition of a histamine H1 antagonist to the culturemedium. The effect of H1 antagonist studied on IL-6 secre-tion (Fig. 3A) was incomplete suggesting that MC-derivedfactors in addition to histamine might mediate the effect oncytokine release. When we tested additional factors releasedby MCs that had the appropriate receptors present in BMSCs(Supporting Information Table 1), we found that PGD2 was

the only compound that induced BMSC-derived IL-6 release(Fig. 3B).

Next, after inducing MC degranulation, we introducedhuman BMSCs into the peritoneal cavities of control and his-tamine-deficient mice—animals lacking HDC, an enzymerequired for histamine biosynthesis [19]. After 6 hours, IL-6increased in peritoneal lavage fluid from control animals butsignificantly reduced in fluid from the histamine-deficientmice. It was not absent, however, suggesting that IL-6 pro-duction by the BMSCs in the presence of activated MCs isnot only due to histamine but also due to other factors. WhenBMSCs were introduced into MC-deficient mice, the samesensitization/challenge protocol resulted in no IL-6 increase(Fig. 3C) suggesting that the MCs are indeed responsible fordriving the release of IL-6.

Histamine mobilizes calcium in several cell types [8, 9]. Totest if it has the same effect on BMSCs, we measured calciumconcentrations in individual cells after addition of histamine at adose of 10�5 M. Immediately after the amine was added, cyto-plasmic calcium concentrations started to increase and peakedafter 30 seconds and reached a plateau thereafter (Fig. 4A).

PLC plays a critical role in mobilization of intracellularcalcium. Upon cleavage of phosphatidylinositol 4,5-bisphos-phate, phospholipase C releases inositol trisphosphate, whichtriggers calcium release from the smooth endoplasmic reticu-lum. PLC inhibitors can prevent cells from mobilizing cal-cium in response to histamine, and we used U73122, a spe-cific inhibitor of PLC, to determine whether this enzymemediated the effect of histamine on BMSCs described above.When the cells were pretreated with this agent, histaminecould no longer elicit the same level of IL-6 increase seenearlier, pointing to the importance of PLC-triggered cellularevents (Fig. 4B)

Histamine has been shown to trigger the activation ofMAP kinase pathways in different cell types [20]. To

Figure 1. Human bone marrow stromal cells (BMSCs) express histamine receptors. Immunostaining of cultured human BMSCs using specificantibodies to recognize the H1 (A), H2 (B), H3 (C), and H4 (D) receptors. The green fluorescence is due to the Alexafluor-488 secondary anti-body. Scale bar ¼ 25 lm. The upper right insets demonstrate reverse transcriptase-PCR results using receptor-specific primers. The arrows pointat the expected size of the PCR products (all around 500 bases). The H1, H2, and H4 receptors are present with both techniques, while the H3does not seem to be expressed. Abbreviations: PCR, polymerase chain reaction.

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determine whether histamine can stimulate MAP kinases inBMSCs, we performed Western blot analysis measuring theamount of phosphorylated p38, ERK, and JNK. Addition ofhistamine elicited a time-dependent activation of all of the ki-nases examined. Maximal phosphorylation of p38, ERK, andJNK occurred after 15 minutes and declined to basal level by

60 minutes (Fig. 4C). Levels of the nonphosphorylated MAPkinases studied remained unchanged at all time points.

To explore how much individual MAP kinases contributeto the histamine-induced IL-6 increase, we looked at theeffects of specific MAP kinase inhibitors. SB203580, a p38inhibitor, PD98059, an ERK inhibitor, and SP600125, a JNK

Figure 2. Histamine stimulates IL-6 and IL-8 secretion in bonemarrow stromal cells (BMSCs). The IL-6 and IL-8 concentrations ofthe BMSC supernatants are dependent on the length of the treatmentand concentration of histamine used, 10�5 M resulting in the largesteffect when IL-6 is measured (A) and 10�4 M being the strongeststimulator when IL-8 release is studied (B). Using specific antagoniststo block the effect on the individual histamine receptors demonstratesthat H1 receptor block eliminates the increase in IL-6 at all concen-trations (C). Abbreviations: IL, interleukin.

Figure 3. Mast cell-derived factors increase IL-6 production ofBMSCs in vitro. (A): IL-6 production of BMSCs is not affected bycoculturing them with human MCs at a 1:1 ratio. When the MCs arefirst degranulated (ActMC) and then cocultured with the BMSCs,there is a significant increase in the IL-6 production (p < .001). Add-ing a H1 receptor antagonist to the media significantly decreases theproduction of IL-6 by BMSCs. (B): PGD2, another major MC media-tor also induces BMSC-derived IL-6 secretion. As PGD2 was dis-solved in ethanol, the individual controls (C1–3) contain the sameEtOH concentration. In 10�6 and 10�5 M concentrations, PDG2 sig-nificantly increases the IL-6 production of BMSCs after 6 hours oftreatment. (C): Human BMSCs produce increased amounts of IL-6 invivo in a HDC- and mast cell-dependent manner. Human BMSCswere placed into the peritoneal cavity of control and histamine-defi-cient (lacking HDC, a vital enzyme in histamine biosynthesis) miceafter inducing MC degranulation. After 6 hours, the increase in IL-6content of the cell-free peritoneal wash was significantly reduced, butstill present, in the histamine-deficient mice. Once again this suggeststhat increased IL-6 production is not only due to histamine but alsodue to other MC-derived factors. When BMSCs were introduced intoMC-deficient mice the same sensitization/challenge protocol resultedin no IL-6 suggesting the MCs to be responsible for the effect. Abbre-viations: BMSCs, bone marrow stromal cells; HDC, histidine-decar-boxylase; MC, mast cell; IL, interleukin; PGD2, prostaglandin D2;WT, wild type; KO, knock-out.

226 Modulation of BMSCs by Histamine

inhibitor all significantly impaired histamine’s ability toinduce IL-6 production by the stem cells (Fig. 4D).

PMNs are professional phagocytes that rapidly engulfinvading organisms that are coated with antibodies and com-plement. PMNs are inherently short-lived cells, with a half-life of approximately 6–12 hours in the circulation. At theend of their lives, they undergo spontaneous apoptosis.Recently Pistoya’s group reported that BMSCs can signifi-cantly prolong survival of PMNs in vitro in an IL-6-depend-ent manner [12]. As histamine seemed to increase IL-6 pro-duction by BMSCs, we asked whether histamine pretreatmentcould enhance the antiapoptotic effect of BMSCs on PMNs.As expected, BMSCs suppressed spontaneous apoptosis ofneutrophils as measured by Annexin-V binding and concur-rent 7-AAD staining using FACS. Histamine prestimulationof the BMSCs increased their efficacy; the number of apopto-tic PMNs was even smaller than it had been when unstimu-lated BMSCs were cocultured with neutrophils (Fig. 5A–5C).To find out if histamine prestimulation of BMSCs can alsohelp preserve the reactive oxygen species producing activityof neutrophils, we measured superoxide production of PMNsfollowing coculture with unstimulated or histamine-pretreatedBMSCs. Although histamine prestimulation of BMSCs didnot cause a measurable effect on the spontaneous superoxideproduction of neutrophils, we were able to detect a significantincrease in fMLP-triggered superoxide synthesis (Fig. 5D). Totest if histamine pretreatment of BMSCs will result in anincrease of neutrophil survival in vivo in an infectious envi-ronment, we used a model of zymosan-induced peritonitis[21]. When the mice were injected with histamine-pretreatedBMSCs intraperitoneally, we found that the peritoneal wash

contained significantly higher numbers of live neutrophilsthan if the BMSCs were not pretreated or were cultured withactivated MCs (Fig. 6).

To explore the molecular mechanisms through whichBMSCs acted, we repeated the apoptosis experiments in thepresence of an IL-6 blocking antibody or using an H1 recep-tor inhibitor. Potentiation of the antiapoptotic effect ofBMSCs was eliminated when neutralizing anti-IL-6 antibodieswere added to the culture medium, suggesting that IL-6 medi-ates the phenomenon. In another set of experiments, insteadof adding histamine we used degranulated human MCs toprestimulate the stromal cells. BMSCs treated this way exhib-ited a significantly greater antiapoptotic effect than thosetreated with histamine alone, and the increase in PMN sur-vival in response to degranulated MCs was only partly coun-tered by a histamine H1 receptor antagonist. As stated earlier,the most likely reason for this is that factors in addition tohistamine are secreted by MCs to modulate BMSCs. Basedon our findings, these other factors are likely to contribute tothe increased production and secretion of IL-6, which appearsto be the principal mediator of the antiapoptotic effect of theBMSCs on PMNs.

DISCUSSION

Histamine was discovered a century ago by Dale and Laidlaw[22], and it soon became obvious that the amine has a varietyof biological functions. With regard to BMSCs, histamine hasbeen suggested to play a role in osteogenesis, and the H1

Figure 4. Histamine stimulation of bone marrow stromal cells (BMSCs) regulates IL-6 production via the MAP kinase pathway. (A): Wemeasured cytosolic Ca2þ concentrations with Fura2 in individual cells. Addition of histamine (10�5 M) evoked a rapid increase in cytosolic Ca2þ

peaking at 30 seconds after stimulation, followed by a sustained elevation. (B): IL-6 increase in the presence of specific PLC inhibitor. (C):Western blot analysis shows the presence of p38, ERK, and JNK. Addition of histamine elicited the activation of p38, ERK, and JNK after 15minutes and declined to basal level by 60 minutes. (D): IL-6 increase in the presence of specific MAP kinase inhibitors. SB203580, a specificp38 inhibitor, PD98059, a specific ERK inhibitor, and SP600125, a specific JNK inhibitor all significantly impaired histamine’s ability to induceBMSCs IL-6 production. Abbreviations: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated pro-tein kinase; PLC, phospholipase C; SAPK, stress-activated protein kinase.

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receptor was identified as one of the downstream targets forthe vitamin D receptor and was suggested to play a role inosteogenic differentiation of BMSCs [23]. It soon becameclear that histamine was also a very important proinflamma-tory mediator. Its role in the immediate type allergic reactionswas quickly recognized, but its other immunomodulatoryeffects were only appreciated in the last decade. Histamine isnow known to alter cytokine and chemokine production by avariety of cells including MCs, eosinophils, dendritic cells,and T cells [9, 24-26]. We wondered if and how histaminemight affect the immunomodulatory functions of humanBMSCs [8, 11, 27-30]. Histamine was described to inducemouse bone marrow cells to produce IL-1a, a proinflamma-tory cytokine that is needed in small amounts to combatinfection and initiate healing processes [31, 32] but one thatcan be harmful when its levels are excessive. A human dis-ease, mastocytosis is a condition characterized by infiltrationof MCs into the tissues of the body, including the bone

Figure 5. Histamine prestimulation increases the IL-6-driven antiapoptotic effect of BMSCs on peripherial blood-derived granulocytes. Whenplaced in coculture, BMSCs effectively suppressed spontaneous apoptosis of neutrophils as measured by Annexin-V binding and concurrent 7-AAD staining using FACS (A–C). Histamine prestimuation of BMSCs was able to even further decrease the number of apoptotic neutrophil gran-ulocytes (PMNs). This potentiation of antiapoptotic function was eliminiated in the presence of neutralizing anti-IL-6 antibodies in the media. Inanother set of experiments, instead of adding histamine itself we used degranulated human MCs as a source of histamine to prestimulate the stro-mal cells. These pretreated BMSCs exhibited a significantly greater antiapoptotic effect as compared to histamine stimulation alone. Moreover,this increased prosurvival phenotype was only partially suspended in the presence of H1 receptor blockade. IL-6 neutralization eliminated theantiapoptotic effect. PMNs increased their fMLP-induced superoxide production when they were cocultured with BMSCs. Histamine prestimula-tion of BMSCs further, significantly, increased the superoxide production of neutrophil granulocytes (D). Abbreviations: AAD, amino-actinomy-cin D; BMSCs, bone marrow stromal cells; fMLP, formyl-methionyl-leucyl-phenylalanine; IL, interleukin; MC, mast cell; RLU, relativeluminescence unit.

Figure 6. Number of live neutrophils in the peritoneal wash ofmice with zymosan-induced peritonitis. When the intraperitoneallyinjected BMSCs were pretreated with histamine, there were almostthree times more live neutrophils, than without pretreatment. Abbrevi-ations: BMSCs, bone marrow stromal cells; MCs, mast cells.

228 Modulation of BMSCs by Histamine

marrow. MCs release chemicals including large amounts ofhistamine during a variety of inflammatory and allergic condi-tions. In patients with mastocytosis, the plasma histamine lev-els are usually high and increased IL-6 levels were reported[33]. In patients with elevated IL-6 levels, a significantincrease in circulating neutrophil counts was also observed[33]. We hypothesized that human BMSCs might respond tohistamine stimulation by increasing their IL-6 production justas other immune cells do. Using cultured human BMSCs, wefound that histamine stimulation results in an increase ofBMSC-derived IL-6 secretion in a dose- and time-dependentmanner. Next, we asked which of the four histamine receptorsmight be responsible for this effect and found that in the invitro conditions it is the H1 receptor that is responsible forthe majority of the increase in BMSC IL-6 production inresponse to histamine. Histamine is a major mediator of acuteinflammatory and immediate hypersensitivity responses andMCs are known to release large amounts of histamine duringdegranulation, thus they might be a natural source of hista-mine when interacting with BMSCs in vivo. This is why weused a coculture of human BMSCs and human MCs as thesource of histamine, and found that—unlike when histaminealone was used—the H1 receptor blockade only partiallyeliminated the IL-6 increase in the medium. To determine thecontribution of MC-derived histamine to the effect seen, weperformed an in vivo experiment when we introduced humanBMSCs into the peritoneal cavity of control and histamine-de-ficient mice [19] after inducing MC degranulation. Althoughthe increase in IL-6 content of the cell-free peritoneal washwas significantly reduced in the histamine-deficient mice, theIL-6 content of the peritoneal wash was still significantlyhigher than that without inducing MC degranulation. Themost likely explanation for this result is that degranulatingMCs release a number of mediators in addition to histamine,such as monoamines, leukotrienes, proteases, and SCF [7,18]. We focused on a few MC mediators that affect receptorsthat we have found to be present in human BMSCs based onour GPCR array [17]. These included LTB4, LTC4, PAF, se-rotonin, and PGD2. We found that in our experimental condi-tions only PGD2 was able to induce IL-6 release fromBMSCs at the 6-hour time point. In fact, as PGD2 is one ofthe major lipid mediators in MCs [18], it is possible that theIL-6 increase that we could not block using H1 blockers isdue—at least in part—to released PGD2.

Using kinase inhibitors, we established that the signalingmolecules responsible for IL-6 induction belong to the MAPkinase family; p38, ERK, and JNK are all phosphorylated inBMSCs in response to histamine. This result is in agreementwith data showing that histamine receptors trigger the MAPKpathways in other cell types [20]. In addition to MAP kinases,Ca signaling has also been suggested to play a role in hista-mine receptor-induced cellular effects [8] and we indeedfound that addition of histamine induces increase in intracel-lular calcium concentrations and blocking calcium mobiliza-tion eliminates the IL-6 increase.

After we established that both histamine and MCs inducehuman BMSCs to produce and release IL-6, we wonderedabout the physiological significance of this effect. A numberof studies suggested that IL-6 can block apoptosis of a vari-ety of cell types including immune cells and neural stemcells [11, 12, 29, 34, 35]. As our previous study in septicmice [6] suggested that increasing the number of circulatingPMNs has a significant effect in fighting infection, wewanted to see whether BMSCs might release IL-6 todecrease PMN apoptosis. Our results showed that in a cocul-ture system of human PMNs and BMSCs, the BMSCs effec-tively suppressed spontaneous apoptosis of neutrophils. Pres-

timulation of BMSCs with histamine further decreased thenumber of apoptotic PMNs while increasing fMLP-inducedsuperoxide production of these phagocytic cells. Coculturingthe BMSCs with degranulated human MCs before they wereadded to PMN cultures significantly increased the antiapop-totic effect. This effect that could only be partially blockedby H1 blockade is suggesting a role for other MC-derivedfactors in the process. To examine the significance of IL-6in such effect, we also used neutralizing antibodies to IL-6in the media, and this treatment eliminated the antiapoptoticfunction. We set up an in vivo experiment using a mousemodel to test if histamine pretreatment would increase theefficiency of BMSCs in improving PMN survival. In thismodel, zymosan, a polysaccharide cell wall componentderived from Saccharomyces cerevisiae, causes an acuteinflammation, and the zymosan-induced peritonitis model isextensively used to quantify the recruitment of monocytesand neutrophils into the peritoneal cavity [21]. We foundthat the BMSCs were significantly more efficient if theywere pretreated with histamine prior to their intraperitonealinjection. In the peritoneal wash, we found a significantincrease in the number of live neutrophils if these hista-mine-pretreated BMSCs were applied. Interestingly,although, if instead of pretreatment with histamine, theBMSCs were cocultured with human MCs prior to injection,we did not find more PMNs in the peritoneal wash. Thismight be due to a variety of factors, such as using humanMCs for pretreatment (unlike our previous in vivo experi-ment, when we inject the human BMSCs and the respondingcells are naturally mouse MCs) or the likelihood that MCsrelease many additional moderators other than histamine,and some of these might have opposing effects on neutrophilsurvival.

Figure 7. A schematic drawing summarizing the hypothesizedeffect of histamine stimulation on bone marrow stromal cells(BMSCs). The histamine might derive from a variety of cellular sour-ces (or might be added as a prestimulation before delivering the cells)and effects the H1 receptor of the BMSC. In response, the BMSCswill increase their IL-8 production, a cytokine that is a strong chemo-attractant for PMNs. At the same time, the increased release of IL-6will ensure the better survival of these attracted cells by its strongantiapoptotic function. Thus the end-result is a larger number of live,functioning PMNs that will normalize an inflammatory/infectiousenvironment. Abbreviations: IL, interleukin; MCs, mast cells; MSC,mesenchymal stem cell; PMNs, neutrophil granulocytes.

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Most of the in vivo studies suggesting the immunomodu-latory actions of BMSCs are based on i.v. injection ofBMSCs. After introducing the BMSCs into the circulation,the cells will end up in small vessels (arterioles, capillaries,and postcapillary venules) by following the natural path ofblood circulation. In these vascular beds, some of the BMSCsmight participate in clot formation that attracts large numberof platelets (that might also release histamine) while othersmight engage in direct interactions with endothelial cells andeventually enter the perivascular space. During these proc-esses, the BMSCs are exposed to platelet and endothelial cell-derived factors both of which might release histamine [24,36] that will activate the BMSCs. This activation can be fur-ther enhanced when BMSCs encounter MCs in the perivascu-lar space (where most of the MCs reside), especially whenadministered in an allergic setting or in a tissue that previ-ously recruited large number of MCs (as seen in mastocytosisor in tolerated allogeneic organ grafts). In these in vivo set-tings, the BMSCs could be bathed in histamine that wouldcause them (through their histamine receptors) to make andrelease large amounts of IL-8 a potent neutrophil chemoat-tractant and a cytokine known to potentiate the oxidativeburst of neutrophils [37] as well as IL-6 a known antiapop-totic cytokine (Fig. 7). From the four receptors examined, theH1 receptor had the highest relative expression in BMSCs,and using receptor-specific antagonists, we also found thathistamine-induced IL-6 production in vitro is solely dependenton H1 binding. Our experiments using human BMSCs andhistamine-deficient mice confirmed that MC-derived histamineworks in concert with other MC-specific factors to induce theobserved IL-6 increase.

CONCLUSION

Our studies revealed another new mechanism underlying theanti-inflammatory/anti-infectious effects of human BMSCs.We established the presence of three of the histamine recep-tors on these cells and pinpointed the H1 receptor as themajor target receptor for the effect. As H1 receptor antago-nists are available and widely used, their effects on theBMSCs function could be significant and this possible sideeffect should be considered. Conversely, as histamine treat-ment seems to induce the ability of BMSCs to attract PMNsand block their apoptosis, one could also imagine an advant-age of using pretreatment of donor BMSCs before their thera-peutic use to enhance their efficacy in improving survival inseptic settings or in other inflammatory disorders in patients.

ACKNOWLEDGMENTS

We thank Dr. Dean Metcalfe for his help with the human mastcells and Dr. Tamas Balla for his guidance and help with calciummeasurements. This work was supported by the DIR, NIDCR, ofthe IRP, NIH, DHHS.

DISCLOSURE OF POTENTIAL

CONFLICTS OF INTEREST

The authors indicate no potential of interest.

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