Comparison of short term in vitro cultured human mast cells from different progenitors — Peripheral blood-derived progenitors generate highly mature and functional mast cells

Journal of Immunological Methods 336 (2008) 166–174

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Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m

Research paper

Comparison of short term in vitro cultured human mast cells from different progenitors — Peripheral blood-derived progenitors generate highly mature and functional mast cells Hanne Busk Andersen a, Mette Holm a,b, Thea Eline Hetland a,b, Christine Dahl a, Steffen Junker b, Peter Oluf Schiøtz a, Hans Jürgen Hoffmann c,⁎ a b c

Department of Paediatrics, Aarhus University Hospital, DK-8200 Aarhus N, Denmark Department of Human Genetics, University of Aarhus, DK-8000 Aarhus C, Denmark Department of Respiratory Diseases, Clinical Institute, Aarhus University & Hospital, Building 2b, Nørrebrogade 44, DK-8000 Aarhus C, Denmark

a r t i c l e

i n f o

Article history: Received 21 December 2007 Received in revised form 8 April 2008 Accepted 15 April 2008 Available online 16 May 2008 Keywords: Human mast cells FcεRI c-kit/CD117 IgE IL-4 PGD2

a b s t r a c t During the last two decades different scientific groups have investigated the phenotype and function of in vitro generated human mast cells (MC). The cells have been shown to display variable surface markers and functional characteristics. The phenotypic differences may reflect different culture conditions, protocols or the use of different progenitors. To investigate the significance of different progenitors, we have compared MC generated from CD133+ progenitor cells from cord blood (CBMC) or peripheral blood (PBMC). The progenitors were cultured for 7 weeks in the presence of IL-6 and SCF, with addition of IL-3 the first 3 weeks, and FCS during week 7. The phenotype of the established MC was characterized by surface marker expression levels, metachromasia, histamine and tryptase contents and their function was evaluated by receptor-mediated release of histamine and PGD2. The generated metachromatic (b 99%) MC were 75% tryptase+, regardless of the source of progenitor cell. Expression of c-kit/CD117, CD203c, and FcεRI was comparable. The density of ckit/CD117 receptors on CBMC was higher that of PBMC (p b 0.001). The density of CD203c and FcεRI was higher on PBMC (p b 0.001). PBMC contained more histamine (p b 0.001), expressed more FcεRI (p b 0.001) and released more histamine (p b 0.001) and PGD2 (p b 0.001) upon ligation of FcεRI, than CBMC. Culture with IL-4 increased expression of tryptase, FcεRI, CD117 and CD203c, secretion of histamine and PGD2 of PBMC, and histamine secretion of CBMC. Cord and peripheral blood may give rise to different types of MC. The question addressed should determine the progenitor cell and protocol to be used. © 2008 Elsevier B.V. All rights reserved.

1. Introduction

Abbreviations: PBS, phosphate buffered saline; BSA, bovine serum albumin; SCF, stem cell factor; FCS, fetal calf serum; APAAP, alkaline phosphatase anti-alkaline phosphatase; FACS, fluorescence-activated cell sorter; IL-4, interleukin-4; IgE, Myeloma IgE; CBMC, Cord blood-derived mast cells; PBMC, Peripheral blood-derived mast cells. ⁎ Corresponding author. Tel.: +45 89492107; fax: +45 89492110. E-mail addresses: [email protected], [email protected] (H.J. Hoffmann). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.04.016

Mast cells are morphologically and histochemically distinct cells of hematopoietic origin and they are widely and uniquely distributed throughout mammalian tissue (Church and Clough, 1999). Since the identification of mast cells, the knowledge about precursors, growth regulation and differentiation has increased considerably. In addition to contributing to the pathophysiology of allergy, mast cells also play important roles in other physiological, pathological and immunological processes, such as tissue

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Table 1 Comparison of mast cell differentiation protocols Protocol

Yield

Receptor

Histamine

Protease

Source

Prog.

Ref.

Cytokines added

Period weeks

Progenitor total

Mast cells total

CD 117

FcɛRI

Total pg/cell

HR %

Tryptase+ %

Chymase+ %

PB PB PB PB CB CB CB CB CB BM FLC FLC

CD 34+ CD 133+ Mnc CD 34 CD 133+ CD 34+ CD 133+ Mnc h-TERT_ST CD 34+ Mnc Mnc

Wang et al. (2006) Holm et al., 2008, here Iida et al. (2001) Lappalainen et al. (2007) Dahl et al. (2002) Saito et al. (1996) This work Iida et al. (2001) Yamaguchi et al. (2007) Shimizu et al. (2002) Irani et al. (1992) Kambe et al. (2000)

IL-3, IL-6, SCF IL-3, IL-6, SCF IL-3, IL-6, SCF IL-3, IL-9, IL-6, SCF IL-3, IL-6, SCF IL-3, IL-6, SCF IL-3, IL-6, SCF IL-3, IL-6, SCF IL-6, SCF, IL-6, SCF Fibroblast co culture SCF + fibroblast co culture

12 7 12 9 12 10 7 12 8 12 4 6

5.2 × 104 2.5 × 106 ND 5 × 105 1.0 × 105 1.0 × 103 1.7 × 106 ND 104 1.0 × 105 2.0 × 107 1.0 × 106

5.0 × 106 8.2 × 106 15 × 103 20 × 106 8.0 × 106 1.0 × 103 174 × 106 0.8 × 103 6 × 106 6.8 × 105 1.2 × 106 6.9 × 106

ND + ND +/− + ND + ND + + ND +

ND + + + + + + + + + ND ND

5.5 23.3 9.3 7.6 3.8 3.6 5.9 8.6 9.7 2.4 0.9 ND

48 53 16 35 N 30% 59 17.5 2 46 ND ND ND

100 72 + + 82.7 99 75 + + 100 94 96

10 0 + + 35.7 18 ND + +/− 100 0 12

Cultured human mast cells derived from CD34+, CD133+ or mononuclear cell (mnc) precursors are compared. The culture methods differ, as do the experiments of interest — selected results are listed for comparison. ND = not determined. Bone marrow progenitors are obtained after spinal puncture; cord-blood and foetal liver cells (obtained from induced abortions) require proximity of an obstetric department. Peripheral blood may be obtained from healthy volunteers or from a blood bank.

remodelling (Garbuzenko et al., 2002) cystic fibrosis (Kulka et al., 2005), cancer and inflammatory responses (Marone et al., 2002). The involvement of mast cells in these highly different processes is made possible through their expression of Fc receptors imposing an antibody specificity in the mast cells, the expression of different Toll like receptors making the cells able to respond to bacterial and viral components (Okayama, 2005) and their release of various immune mediators making them central in the initiation of immune responses (Hart, 2001). In addition, their release of proteases such as tryptase and chymase is directly involved in tissue remodelling (Garbuzenko et al., 2002; Hart, 2001). The characteristics of mature human mast cells are related to the environment in which they reside (Hart, 2001). Mast cells in various anatomical compartments or at different stages of maturation can vary considerably in morphology, histochemistry and mediator content. Various leukocyte sub-populations can be isolated from the peripheral blood to investigate their phenotype and function and their interaction with other cell types. Similarly, in vitro culture of mast cells isolated from tissue can be used to investigate the role of mast cells in complex cellular reactions and to characterize their phenotype and function in detail (Saito et al., 1995). However, isolation of live mast cells from the tissue where they reside is a major challenge, because the cells will be strongly influenced by the isolation techniques and the cell yields from these procedures are extremely low (Sellge and Bischoff, 2006; Patella et al., 1995). As an alternative to this, many scientific groups have developed protocols for in vitro differentiation and culture of human mast cells from different progenitors to establish a method that easily could provide mature, functional mast cells in high numbers. Considerable differences in mast cell yields and phenotypic and functional characteristics are apparent from these studies. A summary of studies investigating different culture techniques is presented in Table 1 (Wang et al., 2006; Iida et al., 2001; Shimizu et al., 2002; Irani et al., 1992; Kambe et al., 2000; Dahl et al., 2002; Saito et al., 1996). These variations

complicate scientific discussion of mast cell biology and make it difficult to draw general conclusions on the exact involvement of mast cells in the processes mentioned above. To investigate how the source of progenitor cell influences the characteristics of in vitro cultured mast cells, we have compared the morphology, surface marker expression, and functional characteristics when progenitor cells from cord blood or adult blood (buffy coats) were cultured under identical condition with and without IL-4 (Toru et al., 1998) using a well defined culture protocol. In this study functionally mature human mast cells were generated from CD133+ cells derived by in vitro culture for 7 weeks under identical culture conditions. 2. Materials and methods 2.1. Antibodies and reagents Ficoll-Paque cell separation solution was purchased from Amersham Pharmacia Biotech, Uppsala, Sweden. AC133 cell isolation kit and LS+ cell separation columns were purchased from Miltenyi Biotech (Bergisch-Gladbach, Germany). Stemspan culture medium was from Stem Cell Technologies (Vancouver, Canada). Foetal calf serum (FCS) and penicillin/streptomycin were purchased from GIBCO BRL (Grand Island, NY, USA). Human recombinant stem cell factor (rhSCF), human rhIL-6, and human rhIL-3 were purchased from R&D Systems (Abingdon, UK). Recombinant interleukin-4 was from Peprotech EC Ltd., London, UK. IgG1 antibodies against tryptase were purchased from Chemicon (Termecula, CA, USA). IgG1 PE conjugated monoclonal antibody (mAb) against c-kit/CD117 (clone 104D2), and its isotype control were from Serotec (Kidlington, UK). FITC conjugated antibody against CD23 (clone MHM6), and RPEconjugated CD14 (R0864) and their isotype controls were purchased from Dako Corp. (Glostrup, Denmark). CD203c/PE (CLONE 97A6) and its isotype were from Immunotech (Marseille, France). Anti-FcεRIα mAb, CRA-1 was purchased from Kyokuto Pharmaceuticals, Tokyo, Japan. Myeloma IgE was produced by U-266, a continuous human cell line producing monoclonal IgE. The alkaline phosphatase

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anti-alkaline phosphatase (APAAP) kit used was from Dako Corp. (Glostrup, Denmark.

30 min at room temperature. As negative controls the T-cell line, “Jurkat” and an isotype-matched IgG1 antibody were used.

2.2. Cell preparation

2.6. Flow cytometry

Human cultured mast cells were obtained from cord blood as described by Dahl et al. (2002) and from buffy coats from the local blood bank. Approximately 100 ml of heparinised cord blood was obtained within 30 min after delivery after informed consent from the mothers. From approximately 470 ml peripheral blood (from healthy donors) 50 ml of buffy coat (only leukocytes) was collected within 24 h after being drawn. The following procedure was applied to both cord blood and buffy coat. The material was diluted in three volumes of PBS and overlaid on a Ficoll-Paque gradient and centrifuged at room temperature for 30 min at 450 G. The interface layer containing mononuclear cells was harvested and washed three times in PBS at 4 °C for 5 min, 450 G.

Surface expression of c-kit/CD117, CD203c, CD23, CD14 and FcεRI was assessed by flow cytometry using a FACS Calibur. In some experiments, 7 weeks old MC were sensitized for 24 h with increasing concentration of myeloma IgE (0–16 µg/ml) to determine optimal IgE-concentration. For optimal sensitization, 2 µg myeloma IgE/ml was used in all experiments. Up to 106 mature mast cells/100 µl PBS with 0.1% FCS were collected and washed twice in PBS containing 0.1% FCS, and then incubated for 30 min at 4° with appropriately titrated antibodies. To determine FcεRI-expression the cells were incubated 30 min at 4° with CRA1 (0.1 µg/ml) or an isotype control, washed twice, and then incubated for another 30 min at 4° with conjugated secondary antibody. The labelling was stopped by adding ice-cold PBS containing 0.1% human serum albumin (HSA). Cell viability was evaluated by adding propidium iodine (PI) to the cells just prior to FACS analysis. The software FCS-Express was used to analyse the results, and Overton Subtraction was used to calculate the net percentage positive cells by comparison to cells stained with an isotypematched IgG (Overton, 1988).

2.3. Purification of CD133+ cells CD133+ cells were separated using AC133 cell isolation kit and a LS+ separation column according to manufacturers' instructions. Briefly, the mononuclear cells were resuspended in 300 µl of MACS buffer (PBS containing BSA, EDTA, pH 7.2) and incubated with FcR blocking reagent and AC133 micro beads for 30 min at 4 °C. After being washed once in MAC buffer, the cells were applied to the LS+ separation column where AC133+ cells were retained in the magnetic field. Subsequently, the column was kept on the magnet and washed with 12 ml MACS buffer. Finally, the column was removed from the magnet and the AC133+ cells were eluted from the column and counted in Trypan blue (0.5% in saline) to determine cell viability. 2.4. Cell cultures Purified CD133+ cells derived from cord blood or buffy coats were resuspended in Stem Span medium with 50 ng/ml IL-6, 100 ng/ml SCF, and penicillin/streptomycin 100 µg/ml. IL-3 (1 ng/ml) was added for the first 3 weeks. To induce final differentiation 10% FCS (15) was added at week 6 and mature human mast cells were harvested and analysed after 7 weeks of culture. Medium was renewed weekly to a cell-density of 5 × 105 cell/ml. Weekly, dead cells were identified by staining with Trypan blue. Alcian blue was used to stain metachromatic cells determining the numbers of Mast cells throughout the culture period. IL-4 (10 ng/ml) was added to selected 7 weeks old mast cell cultures for an additional 4 days to examine the effect of this cytokine on mast cell phenotype. 2.5. Immunostaining Immunostaining for tryptase was performed on cytospins (Craig et al., 1988). The slides were air dried for 24 h, fixed in methanol/acetone and incubated overnight at 4 °C with primary antibody against tryptase. The slides were then washed and incubated as described by manufacturers for 30 min with secondary antibody and stained using the alkaline phosphatase anti-alkaline phosphatase (APAAP method) for another

2.7. Mediator release and secretion from cultured human mast cells Seven weeks old MC were sensitized for 24 h with increasing concentrations of myeloma IgE (0–16 µg/ml) to determine optimal IgE-concentration. For optimal sensitization, 2 µg myeloma IgE/ml was used to sensitize the MC in all experiments. Seven weeks old mast cells were incubated with human myeloma IgE (2 µg/ml) for 24 h at 37 °C. The sensitized mast cells were washed and resuspended (105 cells/ml) in pipes buffer (10 mM pipes, 150 mM Na-acetate, 5 mM K-acetate, 0.6 mM CaCl2, 1.1 mM MgCl2, glucose 1 mg/ml, human serum albumin 0.3 mg/ml, and heparin 15 IE/ml, pH 7.4) containing 100 ng/ml rhSCF and 50 ng/ml rhIL-6. Cells were activated by incubation with anti-IgE 5 µg/ml for 30 min at 37 °C. Histamine release (HR) was blocked by the addition of icecold pipes buffer followed immediately by centrifugation (450 ×g) for 5 min at 4 °C. The cell pellets and supernatants were then collected separately for the histamine assay. For determination of HR, a microfiber-based method was used. The method is based on chemical extraction of histamine to ground glass microfibers and measures histamine by fluorescence of a histamine-o-phtaldehyde complex (Skov et al., 1985). Prostaglandin D2 (PGD2) from anti-IgE stimulated mast cells was measured by a PGD2-MOX-EIA kit (Cayman Chemicals, Ann Arbour, MI). The kit is based on the conversion of PGD2 to a stable MOX derivate by the treatment with methoxylamine hydrochloride. The detection limit of the assay was 6 pg/ml. 2.8. Total content of histamine per cell 50,000 mature mast cells were centrifuged for 5 min at 1500 rpm, 4 °C and washed once in PBS before pellets were frozen at −20 °C. The pellets were then incubated for 5 min in

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HR was expressed as a percentage of total cellular histamine content. FACS analysis revealed positive difference and mean fluorescence intensity (MFI). Statistical significance was analysed using Students t-test, and p b 0.05 was considered significant. 3. Results 3.1. Cell proliferation Buffy coats yielded 2.56 × 106 ± 0.17 CD133+ cells that could be cultured to 8.2 ± 1.2 × 106 PBMC (3.2 ± 1 PBMC/progenitor cell, Fig. 1). Cord blood yielded 1.71 × 106 ± 0.11 CD133+ cells cord blood that could be cultured to 174.1 ± 32.7 × 106 CBMC (101 ± 20 CBMC/progenitor cell). Fig. 1. Mast cell proliferation. Proliferation of cells cultured from CD133+ progenitors from cord blood (n = 23) and buffy coats (n = 56).

40 µl 7% HCLO4 at room temperature, before neutralizing the reaction with 400 µl pipes buffer. Total histamine content was determined as above. 2.9. Statistics All results are expressed as mean ± standard error of the mean values for n independent preparations of mast cells derived from individual cord blood samples or buffy coats.

3.2. Phenotypic analysis of cultured cells 3.2.1. Cell histochemistry Mast cells were monitored weekly by metachromatic staining with Alcian blue (Henwood, 2002), expression of tryptase and determination of histamine content. Irrespective of culture protocol and source of progenitor, more than 99% of the cells were metachromatic (examples of cells in Fig. 2), and the fraction of tryptase positive cells was 80 ± 5.4% after 7 weeks of culture (Fig. 3). After 7 weeks, PBMC contained 23.3 ± 3.3 pg histamine/cell and CBMC significantly less; 5.9 ± 1.0 pg histamine/cell (p b 0.001, Fig. 4).

Fig. 2. Mature CBMC (a, b) and PBMC (c, d) stained with Alcian blue and photographed in a Bürkner-Türk counting chamber.

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Fig. 3. Protease expression in mature mast cells. Immunostaining with tryptase antibody of CBMC (n = 5) and PBMC (n = 27) was similar. Addition of IL-4 for 4 days of culture increased tryptase staining of PBMC (n = 10, p b 0.001) but had no influence on CBMC (n = 5, n = 8 respectively).

3.2.2. Receptor expression c-kit/CD117, the receptor for the essential mast cell growth factor stem cell factor (SCF), was expressed on both cell populations, but significantly higher receptor density was found on CBMC (p b 0.001, Table 2). In the absence of human IgE, the density of FcεRI was significantly higher (p b 0.001) on PBMC than CBMC, but the fraction of cells expressing the receptor were almost identical (Table 2). As surface expression of FcεRI is stabilised by IgE (MacGlashan et al., 1998), 7 week old PBMC were sensitized for 24 h with 0–16 μg/ml of myeloma IgE. FcεRI expression was increased up to 8 µg IgE/ml, with markedly increased expression by addition of 0.5–1 µg IgE/ml, and a further slight increase from 2 to 8 µg IgE/ml (Fig. 7a). CD14 (monocyte linage marker) and CD23 (monocytes, eosinophil granulocytes and activated B-cells) were not expressed on the cultured cells (data not shown). 3.2.3. Mediator release and secretion FcεRI-mediated HR and PDG2 secretion were compared as aspects central to mast cell function. During the 7 week maturation period the cultures had not been exposed to human IgE. The cells were sensitized with myeloma IgE for 24 h before anti-IgE mediated activation was performed. To determine the IgE-concentration for optimal mediator release, CBMC and PBMC were incubated with increasing concentration of IgE (0– 16 µg/ml). HR did not increase markedly above concentrations

Fig. 4. Histamine content of mast cells. PBMC (n = 35) had a higher histamine content than CBMC (n = 12, p b 0.001).

of 1 µg/ml myeloma IgE (Fig. 7b). The spontaneous HR from sensitized PBMC was 1.3% ± 0.1 and 2.9% ± 0.7 from sensitized CBMC. Anti-IgE mediated HR from sensitized mature PBMC was significantly (p b 0.001) higher at 52.9% ± 2.5 than the release of 17.5% ± 2.1 from CBMC (Fig. 6a). The spontaneous secretion of PGD2 was 310.6 ± 129.9 pg PGD2/104cells/ml from PBMC and 169.8 ± 44.8 pg PGD2/104cells/ml from CBMC. On anti-IgE activation PBMC secreted 8066.3± 650.1 pg PGD2/104cells/ml, which was significantly more than CBMC secreted; 2433.7 ± 315.8 pg PGD2/104cells/ml (p b 0.001). Thus, PBMC released significantly more histamine and PGD2 on α-IgE activation than CBMC. 3.3. Effect of IL-4 3.3.1. Cell histochemistry Seven week old cultures were cultured for an additional 4 days ± 10 ng/ml IL-4 (Toru et al., 1996) to determine whether this would enhance the IgE-mediated response. The fraction of cells expressing tryptase was significantly increased in PBMC cultured with IL-4 (from 75.1% ± 17.6 to 94.3% ± 2.6, p b 0.001, Fig. 3), but did not change in CBMC. 3.3.2. Receptor expression The fraction of PBMC expressing FcεRI increased when cultured with IL-4 (p =0.001), but the density (MFI) was not changed (Fig. 5, Table 2). The amount (p= 0.01) and density (p b 0.001) of PBMC expressing c-kit/CD117 were significantly increased by

Table 2 Expression of c-kit/CD117, FcεRI and CD203c on CBMC and PBMC and influence of IL-4 Mast cells

CBMC (n≥20) CBMC+IL-4 (n≥17) PBMC (n≥36) PBMC+IL-4 (n≥15)

Positive difference

MFI

c-kit

FcεRI

CD203c

c-kit

FcεRI

CD203c

91.5 ± 1.1 88.6 ± 1.8 88.3 ± 2.2 97.2 ± 0.5

33.9 ± 6.6 34.8 ± 7.5 31.4 ± 3.3 41.6 ± 7.0

74.4 ± 3.8 77.1 ± 2.4 72.6 ± 3.2 89.6 ± 2.4

5537.4 ± 689.8 1184.9 ± 334.1 621.2 ± 76.4⁎ 1283.8 ± 205.0

2.3 ± 0.4 2.9 ± 0.2 64.1 ± 9.1⁎ 85.5 ± 18.2

56.4 ± 22.1 105.3 ± 34.8 200.1 ± 24.1⁎ 162.6 ± 34.5

Receptor expression was determined by flow cytometry and expressed as positive difference to the left and as MFI to the right. ⁎ indicates p b 0.001 for difference between PBMC and CBMC. IL-4-cultured PBMC (n N 15) expressed significantly more FcεRI (p = 0.007 for positive difference and 0.08 for density) and CD203c (p = 0.03 for positive difference and 0.017 for density), while c-kit/CD117 was unchanged. IL-4-cultured CBMC (n N 9) expression of FcεRI (p = 0.13 for positive difference and p = 0.11 for density) and CD203c (p = 0.14 for positive difference and p = 0.07 for density) was not significantly changed. However, the expression of ckit/CD117 was significantly decreased (p = 0.0506 for positive difference and p = 0.0004 for density) on IL-4-cultured CBMC. Number of batches examined; CBMC; N = 20 for positive difference, and 12 for MFI. PBMC; N N 36 for positive difference, and N28 for MFI.

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Fig. 5. Mature mast cells express FcεRI, c-kit/CD117 and CD203c. Representative dot plots and histograms of CBMC (top row) and PBMC (bottom row) cultures are shown. The histograms show expression of FcεRI, CD203c, and c-kit/CD117 (red), compared with an isotype (black). Positive difference is in blue. (For interpretation of the reference to color in this figure legend, the reader is referred to the web version of this article.)

IL-4. The fraction of PBMC expressing CD203c increased significantly from 72.6 ±3.2 to 89.6 ±2.4 when cultured with IL-4 (p= 0.03) whereas the density (MFI) of CD203c decreased from 200.1±24.1 to 162.6 ±34.5 (p =0.017). The fraction of c-kit/CD117 positive CBMC did not decrease, but the density (MFI) of c-kit/ CD117 was significantly reduced from 5537.4 ±689.8 to 1184.9± 334.1 (p b 0.001), when CBMC were cultured with IL-4. IL-4 had no effect on expression of FcεRI or CD203c on CBMC. 3.3.3. Mediator release and secretion The release of histamine and synthesis of PGD2 from mast cells cultured with IL-4 was investigated to determine whether the changes in mast cell phenotype described above were accompanied by functional alterations. Culture with IL-4 increased the spontaneous release of histamine from nonsensitized PBMC from 1.3 ± 0.1% to 2.7 ± 0.5% (p b 0.001, data not shown). The anti-IgE mediated degranulation was similar with (52.9 ± 2.5%) and without IL-4 (53.3 ± 3.4%). Culturing CBMC with IL-4 did not affect the spontaneous release of histamine from non-sensitized CBMC (2.9% ± 0.7 vs. 2.0% ± 0.4). The anti-IgE mediated degranulation from CBMC increased significantly from 17.5% ± 2.1 to 24.8% ± 3.0 after culture with IL-4 (p = 0.03). The spontaneous HR from CBMC was unchanged by IL-4, and the anti-IgE mediated released was significantly increased by IL-4. The spontaneous PGD2 secretion from non-sensitized PBMC was significantly reduced by culture with IL-4 from 310.6 ± 129.9 pg/104 PBMC/ml to 24.4 ± 12.6 pg/104cells/ml (p b 0.001). Anti-IgE mediated release was increased from 8066.3 ± 650.1 pg/ 104cells/ml to 10,818.3 ± 1025.0 pg/104cells/ml (p = 0.02, Fig. 6b). Spontaneous PGD2 secretion of CBMC decreased significantly from 169.8 ± 44.8 pg/104cells/ml (N = 14) to 46.1 ± 16.0 pg/104cells/ml (p b 0.001). Anti-IgE mediated secretion of PGD2 was not affected by IL-4 (2433.7 ± 315.8 pg/104cells/ml vs. 2358.1 ± 423.3 pg/104cells/ml). 4. Discussion In the last decade a number of in vitro protocols for generating human mast cells have been described (Saito et al.,

Fig. 6. Anti-IgE mediated release of histamine and PGD2-secretion by mature mast cells. Mast cell function was evaluated after sensitization with IgE and cross-linking with FcεRI. Baselines reflect the spontaneous release/secretion from sensitized, non-activated mast cells. a: PBMC (n = 57) released significantly more histamine than CBMC (n = 21, p b 0.001) on cross-linking of IgE with antiIgE. Addition of IL-4 did not significantly increase HR of either culture. b: PBMC (n = 9) secreted significantly (p b 0.001) more PGD2 than CBMC (n = 14). The release of PGD2 from PB-MCs (n = 8) was significantly (p = 0.04) increased after culture with IL-4. CBMC did not release more PGD2 (n =14) after addition of IL-4.

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1996; Irani et al., 1992; Kambe et al., 2000; Dahl et al., 2002; Shimizu et al., 2002; Wang et al., 2006; Lappalainen et al., 2007; Yamaguchi et al., 2007) and Holm et al. (2008). Mast cell cultures have been established from progenitors derived from various sources and it is striking how different methods yield mast cells with variable characteristics. In a recent review this is ascribed to the plasticity of human mast cells resulting in the development of phenotypically distinct populations of human mast cells in different anatomical sites — also described as mast cell heterogeneity (Galli et al., 2005). Mast cells are thought to be derived from CD34+ progenitor cells, which are the predominant source of precursor cells used when differentiating mast cells in vitro (Table 1). As seen from Table 1 the yield of mature mast cells varies with CD34+ progenitor origin and culture method, yet cord blood-derived methods give the highest number of mast cell/progenitor cell, and foetal liver cell derived progenitors the lowest number. Generation of mast cells from other progenitors isolated from different sources has been described and we (Dahl et al., 2002) obtained high yields of mature mast cells when culturing CD133+ progenitor cells. In more recent protocols, both PBMC (Lappalainen et al., 2007) and culture of CBMC in serum-free medium with IL-9 and irradiated stromal cells (Yamaguchi et al., 2007) have been described. In the present study we generated mast cells from CD133+ progenitor cells isolated from cord blood and peripheral blood, using identical culture conditions in a seven week protocol.1 The proliferation of MC progenitors was stimulated by IL-3 during the first 3 weeks (Fig. 1) as described by other investigators (Gebhardt et al., 2002; Tsuji et al., 1990) and as shown in Fig. 1 the potential of CB progenitors for cell division is markedly greater than for PB progenitors. Whether this is due to a higher degree of IL-3-sensitivity in highly immature cord blood-derived progenitors, or has to do with the ontogeneity of the cells has yet to be determined. Tjusi et al. has shown that IL-3 increases proliferation of isolated rodent peritoneal mast cells (Tsuji et al., 1990), which is in line with our findings. Kirshenbaum et al. (1992) find IL-3 and SCF to enhance growth of bone marrow derived mast cells from CD34+ progenitor cells, while IL-3 alone does not. They speculate that IL3 stimulates growth and SCF differentiation but firm conclusions on the exact effect of these ligands remains to be established. After 2–3 weeks of culture, proliferation declines and appears to attenuate at the time when IL-3 is withdrawn, as the number of maturing cells remains fairly constant for the rest of the culture period. As Fig.1 illustrates, the number of PBMC increased to a much lesser degree, but similar to the number of CBMC the number of cells stabilizes from culture week three to seven. This could suggest that the culture time may be even further reduced as described by Irani et al. (1992), as there was no increase in cell number during the final 4 weeks of the culture period. The ability of CD133+ progenitor cells to differentiate into mast cells was similar in buffy coats and cord blood, as the percentage of Alcian blue positive cells was comparable. Tryptase is a defining characteristic for mast cells. Tryptase positive cells (MCT) are found predominantly in lung alveolar wall and in intestinal mucosa. Tryptase and chymase positive cells (MCTC) predominate in skin and intestinal submucosa. The fractions of tryptase+ cells in cultured CBMC and PBMC were

comparable (Fig. 2). Shimizu et al. (2002) compare cord bloodderived MC with bone marrow-derived mast cells using CD34+ cells cultured for 12 weeks, and found that the tryptase level in bone marrow derived mast cells and CBMC was similar. Human mast cells contain 2–5 pg histamine per cell (Prussin and Metcalfe, 2003) — and this is in accordance with our findings in cultured CBMC (5.9 ± 1.0 pg/cell) and what has been reported by others for mast cell cultures (Henwood, 2002; Kinoshita et al., 1999). However, there is a considerable increase in intracellular histamine when MC are generated from progenitors in peripheral blood (23.3 ± 3.3 pg/cell). Kikuchi et al. (2002) generated mast cells using a 15 week protocol and report that peripheral blood-derived mast cells contain 3.5 times the histamine content of cord blood-derived mast cells (20 and 70 pg/5 × 104 MC, respectively). Kinoshita et al. (1999) cultured CBMC with SCF and IL-6 at concentrations similar to those used in the current study and found the total histamine content to be 15.5 pg/cell. Differences in specific culture conditions make it difficult to compare the results directly and variations in the methods for histamine quantitation may also account for the discrepancies in the absolute histamine

Fig. 7. Mast cells sensitized with increasing concentration of myeloma IgE. a: FcεRI expression on PBMC increased up to 8 µg/ml, with marked increase from 0 to 2 µg/ml. b: Sensitizing CBMC and PBMC for 24 h with increasing levels of myeloma IgE showed optimal HR at approximately 2 µg/ml.

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concentrations. However, it seems to be a reproducible finding that PBMC contain more histamine than CBMC. PBMC released more histamine (52.9 ± 2.5% vs. 17.5 ± 2.1%) and PGD2 (8066.3 ± 650.1 pg PGD2/104 PBMC/ml and 2433.7 ± 315.8 pg PGD2/104 CBMC/ml) than CBMC (Fig. 6). Thus, in addition to high mediator content, the PBMC were also found to be more sensitive to stimulation with anti-IgE. This results in a highly up-regulated mediator release upon stimulation (Fig. 6) and indicates that progenitors from peripheral blood should be preferred if mast cell function is investigated. The increased mediator release from PBMC could be directly related to the higher FcεRI expression found for these cells, as increasing receptor molecules per cells has been shown to increase mediator release from mast cells (MacGlashan et al., 1998). The study revealed that the turnover of the receptor is prevented and thereby increasing the net expression of the receptor. Asai et al. studied in vitro cultured murine mast cells and found IgE to increase FcεRI expression (Asai et al., 2001). We found FcεRI expression to increase slightly with increasing IgE concentrations during a 24 h-sensitisation of the mast cells (Fig. 7a).The expression may also be influenced by the length of the sensitisation period. These IgE induced effects on receptor expression which may result in changes in the activation potential of the mast cells stress that also strict assay conditions have to be applied when mast cell functions are investigated. FcεRI is known to be expressed on mast cells, dendritic cells, basophils and activated eosinophils. The c-kit/ CD117 receptor is expressed on hematopoietic progenitors and mast cells, and the high expression of this receptor on the CBMC may indicate that these cells are more immature. In humans, the activation marker CD203c has been found to be expressed selectively on blood basophils and tissue mast cells, as well as CD34+ progenitor cells (Buhring et al., 2004). CD203c is also a sensitive marker for FcεRI-mediated cell activation (Hoffmann et al., 2005), and CD203c was significantly higher expressed on PBMC than on CBMC, suggesting that PBMC are more activated cells. Culturing the different progenitor cells with IL-4 increased the expression of CD203c on CBMC whereas PBMC were not as sensitive to this cytokine. The expression of CD203c on IL-4 incubated CBMC did not increase to a level comparable to PBMC cultured without IL-4, which may have to do with the origin of the progenitors. IL-4 inhibits proliferation (Sillaber et al., 1994; Kulka and Metcalfe, 2005) and stimulates maturation of human mast cells (Toru et al., 1996; Toru et al., 1998). The addition of IL-4 for 4 days did not significantly alter the tryptase content of either MC culture even though there was a trend toward increased tryptase content induced by IL-4 in PBMC. Toru et al. found that culturing mast cells with IL-4 during weeks 10–12 promoted mast cell maturation (Toru et al.,1998). In the present study IL-4 was only added for 4 days. This may explain why the increase in tryptase content was not significant (Fig. 3). The effect of IL-4 on the expression of the SCF-receptor c-kit/ CD117, the activation marker CD203c and the high affinity receptor FcεRI was markedly different in CB and PB derived MC (Table 3). Moreover, the different responses to IL-4 were also reflected in the effect of this cytokine on histamine content and receptor-mediated HR (Fig. 6). This supports the notion that IL-4 sensitivity of MC may depend on their level of maturation, and/ or the source of progenitor cell. Previously IL-4 has been described to enhance FcεRI expression (Stahl et al., 1999) and

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down-regulate expression of c-kit/CD117 (Thienemann et al., 2004) and these two findings could be confirmed for PBMC and CBMC, respectively in the present study. In conclusion, these results indicate that cells with mast cell characteristics can be generated in vitro from CD133+ precursors from different sources. Clear differences were found with respect to mast cell yields, receptor expression, activation potential, and response to IL-4 when mast cells generated from CD133+ progenitors of different origin. In general, mature and functionally active MC are primarily generated from the peripheral blood in these short term cultures while CBMC seem to be more immature and less dedicated for IgE-mediated activation. PBMC seem to express higher and more uniform levels of all three receptors (c-kit/ CD117, FcεRI and CD203c) resulting in discrete peaks. These results highlight the need for highly standardized MC culture protocols if results on mast cell phenotype and function from different laboratories are to be compared. Acknowledgements We thank technician Ellen Margrethe Raaby for her persistent work in cell culturing. Lars K. Poulsen, National University Hospital, Allergy Unit kindly supplied the myeloma IgE and Keld Kaltoft, University of Aarhus, Denmark, kindly supplied IL-4. This work was supported by the Danish Lung Association, the Foundation of 1870, Denmark; The Institute for Experimental Clinical Research, Denmark; Dagmar Marshall Foundation; Helga and Peter Kornings Foundation; King Christian X.'s Foundation; ALK-Abello Denmark, the Velux and Lundbeck foundations and the Danish Paediatric Asthma Center.

References Asai, K., Kitaura, J., Kawakami, Y., Yamagata, N., Tsai, M., Carbone, D.P., Liu, F.T., Galli, S.J., Kawakami, T., 2001. Regulation of mast cell survival by IgE. Immunity 14, 791. Buhring, H.J., Streble, A., Valent, P., 2004. The basophil-specific ectoenzyme ENPP3 (CD203c) as a marker for cell activation and allergy diagnosis. Int. Arch. Allergy Immunol 133, 317. Church, M.K., Clough, G.F., 1999. Human skin mast cells: in vitro and in vivo studies. Ann. Allergy Asthma Immunol. 83, 471. Craig, S.S., Schechter, N.M., Schwartz, L.B., 1988. Ultrastructural analysis of human T and TC mast cells identified by immunoelectron microscopy. Lab. Invest. 58, 682. Dahl, C., Saito, H., Nielsen, H.V., Schiotz, P.O., 2002. The establishment of a combined serum-free and serum-supplemented culture method of obtaining functional cord blood-derived human mast cells. J. Immunol. Methods 262, 137. Galli, S.J., Kalesnikoff, J., Grimbaldeston, M.A., Piliponsky, A.M., Williams, C.M., Tsai, M., 2005. Mast cells as “tunable” effector and immunoregulatory cells: recent advances. Annu. Rev. Immunol. 23, 749. Garbuzenko, E., Nagler, A., Pickholtz, D., Gillery, P., Reich, R., Maquart, F.X., Levi-Schaffer, F., 2002. Human mast cells stimulate fibroblast proliferation, collagen synthesis and lattice contraction: a direct role for mast cells in skin fibrosis. Clin. Exp. Allergy 32, 23. Gebhardt, T., Sellge, G., Lorentz, A., Raab, R., Manns, M.P., Bischoff, S.C., 2002. Cultured human intestinal mast cells express functional IL-3 receptors and respond to IL-3 by enhancing growth and IgE receptor-dependent mediator release. Eur. J. Immunol. 32, 2308. Hart, P.H., 2001. Regulation of the inflammatory response in asthma by mast cell products. Immunol. Cell Biol. 79, 149. Henwood, A., 2002. Improved demonstration of mast cells using alcian blue tetrakis (methylpyridium) chloride. Biotech. Histochem. 77, 93. Hoffmann, H.J., Bogebjerg, M., Nielsen, L.P., Dahl, R., 2005. Lysis with Saponin improves detection of the response through CD203c and CD63 in the basophil activation test after crosslinking of the high affinity IgE receptor FcepsilonRI. Clin. Mol. Allergy 3, 10.

174

H.B. Andersen et al. / Journal of Immunological Methods 336 (2008) 166–174

Holm, M., Andersen, H.B., Hetland, T.E., Dahl, C., Hoffmann, H.J., Junker, S., Schiøtz, P.O., 2008. Seven week culture of functional human mast cells from buffy coat preparations. J. Immunol. Methods 336, 213 (Submitted). Iida, M., Matsumoto, K., Tomita, H., Nakajima, T., Akasawa, A., Ohtani, N.Y., Yoshida, N.L., Matsui, K., Nakada, A., Sugita, Y., Shimizu, Y., Wakahara, S., Nakao, T., Fujii, Y., Ra, C., Saito, H., 2001. Selective down-regulation of high-affinity IgE receptor (FcepsilonRI) alpha-chain messenger RNA among transcriptome in cord blood-derived versus adult peripheral blood-derived cultured human mast cells. Blood 97, 1016. Irani, A.A., Craig, S.S., Nilsson, G., Ishizaka, T., Schwartz, L.B., 1992. Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts. Immunology 77, 136. Kambe, N., Kambe, M., Chang, H.W., Matsui, A., Min, H.K., Hussein, M., Oskerizian, C.A., Kochan, J., Irani, A.A., Schwartz, L.B., 2000. An improved procedure for the development of human mast cells from dispersed fetal liver cells in serum-free culture medium. J. Immunol. Methods 240, 101. Kikuchi, T., Ishida, S., Kinoshita, T., Sakuma, S., Sugawara, N., Yamashita, T., Koike, K., 2002. IL-6 enhances IgE-dependent histamine release from human peripheral blood-derived cultured mast cells. Cytokine 20, 200. Kinoshita, T., Sawai, N., Hidaka, E., Yamashita, T., Koike, K., 1999. Interleukin-6 directly modulates stem cell factor-dependent development of human mast cells derived from CD34(+) cord blood cells. Blood 94, 496. Kirshenbaum, A.S., Goff, J.P., Kessler, S.W., Mican, J.M., Zsebo, K.M., Metcalfe, D.D., 1992. Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells. J. Immunol. 148, 772. Kulka, M., Dery, R., Nahirney, D., Duszyk, M., Befus, A.D., 2005. Differential regulation of cystic fibrosis transmembrane conductance regulator by interferon gamma in mast cells and epithelial cells. J. Pharmacol. Exp. Ther. 315, 563. Kulka, M., Metcalfe, D.D., 2005. High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCFdependent human mast cell production in vitro: correlation with apoptosis and Kit expression. Blood 105, 592. Lappalainen, J., Lindstedt, K.A., Kovanen, P.T., 2007. A protocol for generating high numbers of mature and functional human mast cells from peripheral blood. Clin. Exp. Allergy 37, 1404. MacGlashan, D., Kenzie-White, J., Chichester, K., Bochner, B.S., Davis, F.M., Schroeder, J.T., Lichtenstein, L.M., 1998. In vitro regulation of FcepsilonRIalpha expression on human basophils by IgE antibody. Blood 91, 1633. Marone, G., Galli, S.J., Kitamura, Y., 2002. Probing the roles of mast cells and basophils in natural and acquired immunity, physiology and disease. Trends Immunol. 23, 425. Okayama, Y., 2005. Mast cell-derived cytokine expression induced via Fc receptors and Toll-like receptors. Chem. Immunol. Allergy 87, 101. Overton, W.R., 1988. Modified histogram subtraction technique for analysis of flow cytometry data. Cytometry 9, 619. Patella, V., Marino, I., Lamparter, B., Arbustini, E., Adt, M., Marone, G., 1995. Human heart mast cells. Isolation, purification, ultrastructure, and immunologic characterization. J. Immunol. 154, 2855. Prussin, C., Metcalfe, D.D., 2003. 4. IgE, mast cells, basophils, and eosinophils. J Allergy Clin Immunol 111, S486–S494.

Saito, H., Ebisawa, M., Sakaguchi, N., Onda, T., Iikura, Y., Yanagida, M., Uzumaki, H., Nakahata, T., 1995. Characterization of cord-blood-derived human mast cells cultured in the presence of Steel factor and interleukin6. Int. Arch. Allergy Immunol. 107, 63. Saito, H., Ebisawa, M., Tachimoto, H., Shichijo, M., Fukagawa, K., Matsumoto, K., Iikura, Y., Awaji, T., Tsujimoto, G., Yanagida, M., Uzumaki, H., Takahashi, G., Tsuji, K., Nakahata, T., 1996. Selective growth of human mast cells induced by Steel factor, IL-6, and prostaglandin E2 from cord blood mononuclear cells. J. Immunol. 157, 343. Sellge, G., Bischoff, S.C., 2006. Isolation, culture, and characterization of intestinal mast cells. Methods Mol. Biol. 315, 123. Shimizu, Y., Sakai, K., Miura, T., Narita, T., Tsukagoshi, H., Satoh, Y., Ishikawa, S., Morishita, Y., Takai, S., Miyazaki, M., Mori, M., Saito, H., Xia, H., Schwartz, L.B., 2002. Characterization of ‘adult-type’ mast cells derived from human bone marrow CD34(+) cells cultured in the presence of stem cell factor and interleukin-6. Interleukin-4 is not required for constitutive expression of CD54, Fc epsilon RI alpha and chymase, and CD13 expression is reduced during differentiation. Clin. Exp. Allergy 32, 872. Sillaber, C., Sperr, W.R., Agis, H., Spanblochl, E., Lechner, K., Valent, P., 1994. Inhibition of stem cell factor dependent formation of human mast cells by interleukin-3 and interleukin-4. Int. Arch. Allergy Immunol. 105, 264. Skov, P.S., Mosbech, H., Norn, S., Weeke, B., 1985. Sensitive glass microfibrebased histamine analysis for allergy testing in washed blood cells. Results compared with conventional leukocyte histamine release assay. Allergy 40, 213. Stahl, J.L., Cook, E.B., Graziano, F.M., Barney, N.P., 1999. Human conjunctival mast cells: expression of Fc epsilonRI, c-kit, ICAM-1, and IgE. Arch. Ophthalmol. 117, 493. Thienemann, F., Henz, B.M., Babina, M., 2004. Regulation of mast cell characteristics by cytokines: divergent effects of interleukin-4 on immature mast cell lines versus mature human skin mast cells. Arch Dermatol Res 296, 134. Toru, H., Eguchi, M., Matsumoto, R., Yanagida, M., Yata, J., Nakahata, T., 1998. Interleukin-4 promotes the development of tryptase and chymase double-positive human mast cells accompanied by cell maturation. Blood 91, 187. Toru, H., Ra, C., Nonoyama, S., Suzuki, K., Yata, J., Nakahata, T., 1996. Induction of the high-affinity IgE receptor (Fc epsilon RI) on human mast cells by IL4. Int. Immunol. 8, 1367. Tsuji, K., Nakahata, T., Takagi, M., Kobayashi, T., Ishiguro, A., Kikuchi, T., Naganuma, K., Koike, K., Miyajima, A., Arai, K., 1990. Effects of interleukin3 and interleukin-4 on the development of “connective tissue-type” mast cells: interleukin-3 supports their survival and interleukin-4 triggers and supports their proliferation synergistically with interleukin-3. Blood 75, 421. Wang, X.S., Sze, W.S., Kwok, H.Y., Lau, H.Y., 2006. Functional characterization of human mast cells cultured from adult peripheral blood. Int. Immunopharmacol. 6, 839. Yamaguchi, M., Azuma, H., Fujihara, M., Hamada, H., Ikeda, H., 2007. Generation of a considerable number of functional mast cells with a high basal level of FcepsilonRI expression from cord blood CD34+ cells by co-culturing them with bone marrow stromal cell line under serum-free conditions. Scand. J. Immunol. 65, 581.