International Immunopharmacology 8 (2008) 1737–1743

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International Immunopharmacology

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Accelerated differentiation of bone marrow-derived dendritic cellsin atopic prone mice

Eiko Koike ⁎, Hirohisa Takano ⁎, Ken-ichiro Inoue, Rie YanagisawaEnvironmental Health Sciences Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

⁎ Corresponding authors. Tel./fax: +81 29 850 2336, 2E-mail addresses: ekoike@nies.go.jp (E. Koike), htaka

1567-5769/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.intimp.2008.08.006

a b s t r a c t

a r t i c l e i n f o

Article history:

NC/Ngamice are atopic pron Received 18 February 2008Received in revised form 11 August 2008Accepted 13 August 2008

Keywords:Allergic/atopic diseaseNC/Nga miceDendritic cellsGM-CSF

emice that can be an animalmodel for human atopic dermatitis (AD). Dendritic cells(DC) as professional antigen-presenting cells (APC) are the most capable inducers of immune responses. Thepresent study usingBALB/c, C57BL/6J, andNC/Ngamalemice investigatedwhether differentiation and function ofDC were associated with atopic prone. Bone marrow-derived DC (BMDC) were differentiated by culture withgranulocyte macrophage colony stimulating factor (GM-CSF). At days 0, 6, and 8 of culture with GM-CSF, theexpression of MHC class II, co-stimulatory molecules (CD80, CD86), and of DC markers (CD11c, DEC205) wasmeasured by flow cytometry. Antigen-presenting activity of BMDC and cytokine production were measured byELISA. The cell numbers and the expressionofMHCclass II, co-stimulatorymolecules, andofDCmarkers onBMDCfrom NC/Nga mice were significantly larger than those from BALB/c and C57BL/6J mice. Antigen-presentingactivity of BMDC was significantly greater in NC/Nga and C57BL/6J mice than in BALB/c mice. BMDC-stimulatedIFN-γ production from T-cells was significantly lower in NC/Nga or BALB/c mice than in C57BL/6J mice,whereas IL-4 production was significantly greater in NC/Nga and C57BL/6J mice than in BALB/c mice. Takentogether, GM-CSF-stimulated differentiation of BMDCwasmore accelerated in atopic prone NC/Ngamice than inthe other strains of mice. The enhancement of differentiation and function of DC caused by genetic backgroundmay be related, at least partly, to the induction or aggravation of allergic/atopic diseases.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skindisease in individuals often having a family history of atopy [1,2] and isfrequently associated with elevated serum level of total and/or specificIgE antibodies [3,4]. It has been reported that lymphocytes, monocytes/macrophages, mast cells [5], eosinophils [6], dermal dendritic cells (DC)and epidermal langerhans cells are observed in the skin lesions of AD[7,8]. However, the pathogenesis of AD and the mechanisms of hyperproduction of IgE have not been fully clarified.

NC/Ngamice, an inbred strain, were established from Japanese fancymice by K. Kondo in 1957 [9]. NC/Nga mice spontaneously develop aneczematous AD-like skin lesion when kept under air-uncontrolledconventional conditions, but not under SPF conditions [10]. The skinlesions with increased number of mast cells, eosinophils, CD4+ T-cells,and macrophages and elevation of serum level of IgE are clinically andhistologically similar to humanAD. Therefore,NC/Ngamice are availableas an animal model for human AD.

In general, enhancement of antigen-presenting activity can lead toan increase in the antigen-related immunoglobulin (Ig) productionand allergic inflammation through the proliferation and activation of

334.no@nies.go.jp (H. Takano).

l rights reserved.

lymphocytes and eosinophils [11,12]. Therefore, antigen-presentingcells (APC) play an important role in the aggravation of allergic/atopicdiseases and/or responses.

DC, asprofessionalAPC, are themost capable inducers in the initiationof primary immune responses and in the enhancement of secondaryimmune responses. MHC class II molecules [13] and co-stimulatorymolecules such as CD80 and CD86 [14,15] are essential for antigenpresentation; thus, DC differentiation/activation is typically defined byenhanced expression of MHC class II and co-stimulatory molecules.

We hypothesize that the differentiation/maturation of APC such asDC may be accelerated in genetic atopic prone subjects, which is, atleast partly, responsible for the aggravation of allergic/atopic diseases.Therefore, we investigated the differences in the differentiation andfunction of bone marrow-derived DC in response to granulocytemacrophage colony stimulating factor (GM-CSF) using atopic proneNC/Nga mice and the other strains of mice.

2. Materials and methods

2.1. Animals

Seven-week-old SPF NC/NgaTndCrlj male mice were purchasedfromCharles River Japan (Osaka, Japan) andwere used at 12–15-wkold(weighing 24 to 27 g). Seven-week-old SPF BALB/cAJcl and C57BL/6JJclmale mice were purchased from CLEA Japan Inc. (Tokyo, Japan) and

Fig. 1. Numbers and phenotypes of isolated bone marrow cells from different strains of mice. Cell numbers (a) and phenotypes (b) of bone marrow cells isolated from NC/Nga micewere compared with those from BALB/c or C57BL/6J mice. Cell counts and cell viability were determined by the trypan blue exclusion method. The expression of MHC class II and co-stimulatory molecules (CD80 and CD86) and DC markers (CD11c and DEC205) was analyzed by flow cytometry. Values are presented as mean ± SEM of 8 animals in each strain.*Pb0.05, **Pb0.01 versus BALB/c; †Pb0.05, ††Pb0.01 versus C57BL/6J; #Pb0.05, ##Pb0.01 versus NC/Nga.

Fig. 2.Numbers of BMDC differentiated by culturewith GM-CSF from different strains ofmice. The numbers of BMDC from NC/Nga mice were compared with those from BALB/cor C57BL/6J mice. BMDCwere differentiated from bone marrow cells (4.8×105) culturedin 1.2 ml R10 medium containing GM-CSF (20 ng/ml). At day 8 of culture, non-adherentand loosely adherent cells were harvested and cell numbers were counted. Cell countsand cell viability were determined by the trypan blue exclusion method. Values arepresented as mean ± SEM of 9 animals in each strain. **Pb0.01 versus BALB/c; ††Pb0.01versus C57BL/6J; ##Pb0.01 versus NC/Nga.

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were used at 12–15-wk old (weighing 27 to 31 g for BALB/c, 21 to 28 gfor C57BL/6J). Micewere given sterile distilledwater and a commercialdiet (CE-2; CLEA Japan Inc.) ad libitum. They were housed in an animalfacility that was maintained at 24–26 °C with 55–75% humidity and a12-h light/dark cycle under conventional conditions.

2.2. Preparation of bone marrow cells

Mice were anesthetized with sodium pentobarbital (DainipponPharmaceutical Co., Osaka, Japan) given intraperitoneally (50 mg/kg),and exsanguinated from the cut abdominal aorta and vein. Afterremoving the surrounding muscle tissue by rubbing with kleenextissues, the bones were left in 60 mm dish with 70% ethanol for 3 minand washed with Dulbecco's calcium and magnesium-free, phos-phate-buffered saline (PBS; TAKARA BIO INC. Shiga, Japan). Both endsof the bones were cut and then the marrow was flushed with PBSusing a syringe with 25G needle. The marrow suspension was passedthrough nylon mesh to remove small pieces of bone and debris andred blood cells were lysed with ammonium chloride. After washingtwice with PBS, the cells were suspended in culture medium R10,which was RPMI 1640 medium (GIBCO BRL, Eggenstein, Germany)

Fig. 3. BMDC phenotypes at day 6 of culture with GM-CSF from different strains of mice. At day 6 of culture, BMDC were harvested and the expression of MHC class II and co-stimulatory molecules (CD80 and CD86) and DC markers (CD11c and DEC205) was analyzed by flow cytometry. Histograms are representative of BMDC from each stain of mice. Thefine solid, dotted, and bold lines reveal the staining pattern of BMDC from BALB/c, C57BL/6J, and NC/Nga mice, respectively. Values of bar graph are presented as mean ± SEM of 8animals in each strain. *Pb0.05, **Pb0.01 versus BALB/c; †Pb0.05, ††Pb0.01 versus C57BL/6J; #Pb0.05, ##Pb0.01 versus NC/Nga.

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Fig. 4. BMDCphenotypes at day 8 of culturewithGM-CSF fromdifferent strains ofmice. At day 8 of culture, BMDCwere harvested and the expression ofMHC class II and co-stimulatorymolecules (CD80 and CD86) and DC markers (CD11c and DEC205) was analyzed by flow cytometry. Histograms are representative of BMDC from each stain of mice. The fine solid,dotted, and bold lines reveal the staining pattern of BMDC fromBALB/c, C57BL/6J, and NC/Ngamice, respectively. Values of bar graph are presented asmean ± SEM of 8 animals in eachstrain. *Pb0.05, **Pb0.01 versus BALB/c; †Pb0.05, ††Pb0.01 versus C57BL/6J; #Pb0.05, ##Pb0.01 versus NC/Nga.

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Fig. 5. Antigen-presenting activity of BMDC from different strains of mice. At day 8 ofculture, BMDCwere harvested. T-cells (2×105) from Dp-sensitized syngeneic mice wereco-cultured with 1.25% or 2.5% BMDC (2.5×103 or 5×103 respectively) in the presence of2 μg of Dp in 200 μl of R10 medium for 4 days and pulsed with BrdU for the last 20 h.Antigen-presenting activity of BMDC was performed in triplicate and was evaluated byBrdU incorporation into proliferating T-cells. Data represent themean±SEMof 6 animals.*Pb0.05, **Pb0.01 versus BALB/c; †Pb0.05, ††Pb0.01 versus C57BL/6J; ##Pb0.01 versusNC/Nga.

Fig. 6. Cytokine production stimulated by BMDC from different strains of mice. Dp-sensitized T-cells were co-cultured with BMDC for 4 days as Fig. 5 and then culturesupernatant was collected. The levels of IFN-γ (a) and IL-4 (b) in the culture supernatantwere measured by ELISA. Data represent the mean±SEM of 9 animals. *Pb0.05,**Pb0.01 versus BALB/c; ††Pb0.01 versus C57BL/6J; #Pb0.05, ##Pb0.01 versus NC/Nga.

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supplemented with 10% heat-inactivated fetal bovine serum (FBS; MPBiomedicals Inc., Eschwege, Germany), 100 U/ml penicillin, 100 μg/mlstreptomycin (Sigma, St. Louis, MO), and 50 μM 2-mercaptoethanol(GIBCO BRL). The numbers of viable cells were determined by thetrypan blue (Gibco Laboratories, Grand Island, NY) exclusion method.

2.3. Generation of bone marrow-derived DC (BMDC)

BMDC were differentiated using a modified protocol of Lutz et al.[16]. Briefly, 4×105 cells/ml of bone marrow cells were cultured in1.2 ml R10 medium containing 20 ng/ml recombinant mouse GM-CSF(Sigma) in 6-well plate in duplicate. At day 3, another 1.2 ml R10medium containing 20 ng/ml GM-CSF were added to the well. At day6, half of the culture medium was turned into fresh medium in thecase of 8 days culture. At days 6 and 8, non-adherent and looselyadherent cells were collected by gentle pipetting, and centrifuged at400×g for 5 min at 20 °C. These cells (immature BMDC) weresuspended in fresh R10 medium. The numbers of viable cells weredetermined by the trypan blue exclusion method.

2.4. FACS analysis

For FACS analysis, the following monoclonal antibodies were used:MHC class II molecules: I-A/I-E (2G9, FITC-conjugated, BD BiosciencesPharmingen, CA); co-stimulatory molecules: CD80 (16-10A1, PE-con-jugated, BD Biosciences Pharmingen), CD86 (GL1, PE-conjugated, BDBiosciences Pharmingen); DC markers: CD11c (HL3, PE-conjugated, BDBiosciences Pharmingen), DEC205 (NLDC-145, PE-conjugated, MiltenyiBiotec GmbH, Gladbach Germany). Cells (3–5×105) were suspended in100 μl PBS with 0.3% BSA and 0.05% sodium azide (Wako Pure ChemicalIndustries, Osaka, Japan) and were stained with antibodies at 1 μg for30 min on ice. After incubation, the cells were washed, and thefluorescence was measured by a FACSCalibur (Becton, Dickinson andCompany, NJ). For each sample, fluorescence data from10,000 cells werecollected and positive cells were expressed % total events.

2.5. Measurement of antigen-presenting activity and cytokine production

Antigen-presenting activity of BMDC was assessed by stimulatingcapacity of antigen-specific syngeneic T-cell proliferation. Mite extract[Dermatophagoides pteronyssinus crude extract (Dp); Cosmo Bio LSL,Tokyo, Japan]-sensitized T-cells were derived from a pool of spleno-

cytes from Dp-sensitized syngeneic mice (three mice/each strain).Mice were immunized with 50 μg Dp and 1 mg Al(OH)3 in 0.1 ml ofsaline and splenocytes were harvested at day 14. T-cells were isolatedfrom the splenocytes using a nylon fiber column (Wako Pure ChemicalIndustries). BMDC at day 8 of culture were treated with 50 μg/mlmitomycin C (Kyowa Hakko Kogyo, Tokyo, Japan) for 20 min in awaterbath at 37 °C. Thereafter, Dp-sensitized T-cells (2×105) were co-culturedwith BMDC (2.5×103 or 5.0×103) in the presence of 2 μg of Dpin 200 μl of R10medium in 96-well flat-bottom plates. Co-culture of T-cells and BMDC was performed in triplicate for 4 days at 37 °C in a 5%CO2/95% air atmosphere. Proliferation of T-cells was measured with aCell-Proliferation-ELISA Kit (Roche Molecular Biochemicals, Man-nheim, Germany) according to the manufacturer's instructions. Levelsof IFN-γ (Endogen, Cambridge, MA) and IL-4 (Amersham, Buckin-ghamshire, UK) in the culture supernatant were measured by ELISAaccording to the manufacturer's instructions. The detection limit ofIFN-γ or IL-4 was 10 pg/ml or 5 pg/ml, respectively.

2.6. Statistical analysis

Datawere represented as themean ± SEMof six to nine animals eachstrain. Differences between groups were determined using ANOVA byFisher's LSD (StatView J-4.5; Abacus Concepts, Inc., Berkeley, CA). A pvalue of b0.05 was considered to indicate a significant differencebetween pairs of groups.

3. Results

3.1. Numbers and phenotypes of isolated bone marrow cells from BALB/c,C57BL/6J, and NC/Nga mice

Cell numbers and phenotypes of bone marrow cells isolated fromNC/Nga mice were compared with those from BALB/c or C57BL/6J

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mice. The numbers of freshly isolated bone marrow cells weresignificantly larger in NC/Nga and C57BL/6J mice than in BALB/c mice.There was no difference between NC/Nga and C57BL/6J mice. Themean value of the cell numbers in BALB/c, C57BL/6J, and NC/Nga micewas 3.61×107, 5.12×107, and 4.51×107, respectively (Fig. 1a).

In contrast, the expression ofMHC class II on the bonemarrow cellswas lower in NC/Nga and C57BL/6J mice than in BALB/c mice. Thesecells expressed few co-stimulatory molecules (CD80 and CD86) andDC markers (CD11c and DEC205) in any strains of mice (Fig. 1b).

3.2. Generation of BMDC from BALB/c, C57BL/6J, and NC/Nga mice

The numbers of BMDC fromNC/Ngamicewere comparedwith thatfrom BALB/c and C57BL/6J mice. Bone marrow cells (4.8×105) fromeach strainwere cultured in 1.2 ml R10 medium containing GM-CSF in6-well plate. At day 8 of culture, the numbers of differentiated BMDCwere significantly larger in NC/Nga mice than in the other strains ofmice (Fig. 2).

3.3. Phenotypes of BMDC from BALB/c, C57BL/6J, and NC/Nga mice

BMDC phenotypes from NC/Nga mice were compared with thosefromBALB/c or C57BL/6Jmice. At day 6 of culture, the expression ofMHCclass II, CD80, CD86, and CD11c of BMDCwas significantly greater in NC/Nga mice than in the other strains of mice (Fig. 3). Percentages of MHCclass II+CD80+,MHCclass II+CD86+,MHCclass II+CD11c+, andMHCclass II+

DEC205+ cells in BMDC were also significantly greater in NC/Nga micethan in the other strains of mice (Fig. 3). The expression of DEC205 onBMDC tended to larger in NC/Nga mice than in the other strains of mice,but there was no statistical significant differences (Fig. 3). At day 8 ofculture, the expression of MHC class II and CD11c of BMDC wassignificantly greater in NC/Nga mice than in the other strains of mice(Fig. 4). Percentage ofMHC class II+CD11c+ of BMDCwas also significantlygreater in NC/Nga mice than in the other strains of mice (Fig. 4).

3.4. Function of BMDC from BALB/c, C57BL/6J, and NC/Nga mice

Antigen-presenting activity and stimulating capacity of cytokineproduction of BMDC fromNC/Ngamicewere comparedwith those fromBALB/c or C57BL/6J mice. Antigen-presenting activity of BMDC wassignificantly greater in NC/Nga and C57BL/6J mice than in BALB/c mice(Fig. 5). Therewasnodifference in the activity of BMDCbetweenNC/Ngaand C57BL/6J mice. On the other hand, BMDC-stimulated IFN-γ pro-duction from T-cells was greatly higher in C57BL/6J mice than in BALB/cor NC/Ngamice (Fig. 6a). BMDC-stimulated IL-4 production from T-cellswas significantly greater in NC/Nga and C57BL/6J mice than in BALB/cmice (Fig. 6b). There was no significant difference between NC/Nga andC57BL/6J mice.

4. Discussion

In the present study, we demonstrated the differences in thedifferentiation and function of BMDC from NC/Nga mice as comparedto those from BALB/c or C57BL/6J mice. GM-CSF-stimulated differ-entiation of BMDC was more accelerated in atopic prone NC/Nga micethan in the other strains of mice.

NC/Nga mice, an inbred strain, develop AD-like skin lesions withincreased number of mast cells, eosinophils, CD4+ T-cells, and macro-phages [10], and hyper production of Th2-specific cytokines [17] andIgE [10], which may be triggered by environmental factors. Epide-miological studies have suggested that induction and/or aggravationof AD are influenced by environmental factors [18,19]. Atopic proneNC/Nga mice may exhibit higher susceptibility to the development ofenvironmental factor-induced allergic/atopic diseases. Previous stu-dies have suggested potency of NC/Nga mice as a model of humanallergic/atopic diseases including atopic dermatitis and bronchial

asthma [20]. NC/Nga mice with experimentally induced asthma showsevere eosinophilia as compared with BALB/c mice [21]. Our previousstudy has also shown that weekly intratracheal administration ofdiesel exhaust particles lead airway inflammation such as infiltrationof neutrophils and mononuclear cells in NC/Nga mice [20], however,same experimental protocol does not influence lung inflammation inICR mice [22]. These results indicate that NC/Nga mice may be moresusceptible to airway inflammation induced by environmental factorsthan the other strains of mice.

Our present study demonstrated that the expression of MHC classII on the bone marrow cells isolated from NC/Nga and C57BL/6J micewas slightly lower than that from BALB/c mice (Fig. 1). However, theexpression of MHC class II, co-stimulatory molecules, and DC markerson BMDC induced by GM-CSF was significantly larger in NC/Nga micethan in the other strains of mice (Figs. 3 and 4). The number of BMDCwas also significantly larger in NC/Ngamice than in the other strains ofmice (Fig. 2). These results suggest that NC/Nga mice may have highersusceptibility to differentiation of BMDC in response to GM-CSF butnot have innate high expression of the molecules associated withantigen presentation on bone marrow cells.

It has been reported that GM-CSF plays an important role in thedevelopment of allergic inflammatory responses through induction ofTh2-associated cytokine production [23,24] and an increase in localantigen-presentingcapacity [25]. Therefore, atopicprone inNC/Ngamicemay be determined by genetic background-mediated high susceptibilitytoGM-CSF.Ourpresent studyalsodemonstrated that antigen-presentingactivity of BMDC was significantly greater in NC/Nga and C57BL/6J micethan in BALB/c mice (Fig. 5), implicating greater potential of antigen-specific CD4 polarization in the two strains. Interestingly, BMDC-stimulated Th1 response was significantly lower in NC/Nga and BALB/cmice than in C57BL/6J mice, whereas BMDC-stimulated Th2 responsewas significantly greater in NC/Nga and C57BL/6J mice than in BALB/cmice (Fig. 6). Th1/Th2 balance might be important rather thanproduction levels of the cytokine. In fact, the ratio of IL-4/IFN-γ in theculture of T-cells with 2.5% BMDC of BALB/c, C57BL/6J, and NC/Nga was7.1×10−2, 2.5×10−2, and 5.2×10−2, respectively (data not shown). Thus,T-cells from both BALB/c and NC/Nga mice can be easily differentiatedinto the Th2 compared to C57BL/6J mice. Furthermore, it is likely thatantigen-specific T-cell polarization with Th2 skew is most prominent inthe cell system from NC/Nga mice among that from these strainsexamined.

To explore themechanismof severe allergic inflammation inNC/Ngamice as an asthmaticmodel, cytokine profiles in bronchoalveolar lavagefluid (BALF) and productivity of eosinophils have been examined in aprevious study [26]. In this study, NC/Nga mice show lower productionof IFN-γ in BALF and a large number of eosinophils derived from bonemarrow cells stimulated with IL-3, GM-CSF, and IL-5 compared withBALB/c mice [26]. These results may imply the possibility that severeallergic inflammation in NC/Nga mice are attributable to lower produc-tion of IFN-γ andhigher eosinophil productivity frombonemarrowcellson stimulationwith cytokines such as GM-CSF. Previous reports and ourpresent study suggest that bone marrow cells in NC/Nga mice aresusceptible to the stimulation by specific cytokines and induce largenumber of eosinophils and DC. NC/Nga mice could be a suitable modelfor both of in vivo and in vitro studies to investigate the effects ofenvironmental stimulation on allergic/atopic diseases and their under-lying mechanisms. We are now investigating the effects of environ-mental chemicals on differentiation/activation of BMDC from NC/Ngamice to elucidate whether environmental chemicals can be involved inthe recent increase in allergic/atopic diseases.

In conclusion, the present study demonstrated that atopicprone NC/Nga mice showed accelerated differentiation of BMDC inresponse to GM-CSF as compared with the other strains of mice.The acceleration caused by genetic background may be related, atleast partly, to the development and/or aggravation of allergic/atopicdiseases.

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Acknowledgments

The authors thank toMiho Sakurai and Satomi Abe for her technicalassistance.

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