©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conditioned Media from a Cell Strain Derived from a Patient with Mastocytosis Induces Preferential Development of Cells That Possess High Affinity IgE Receptors and the Granule Protease Phenotype of Mature Cutaneous Mast Cells (*)

(Received for publication, September 12, 1994; and in revised form, November 3, 1994)

Lixin Li (1) (2) Janet J. Macpherson (1) (2) Stephen Adelstein (1) (2) Clive L. Bunn (2) Kerry Atkinson (5) Kiran Phadke (3) Steven A. Krilis (1) (2)(§)

From the  (1)University of New South Wales, School of Medicine, Kensington 2033, the (2)Departments of Immunology, Allergy and Infectious Disease, and (3)Oncology, St. George Hospital, Kogarah, New South Wales 2217, (4)Biotech Australia, Roseville 2069, and the (5)Department of Haematology, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have demonstrated for the first time that a conditioned medium from a human cell strain can induce morphologically mature mast cells that express FcRI and three mast cell-specific proteases from normal bone marrow progenitor cells. In contrast, recombinant human Kit ligand induced the differentiation of mast cells that were tryptase-positive but negative for chymase, carboxypeptidase, and FcRI. This data indicates that factors other than Kit ligand are critical for inducing the differentiation and maturation of mast cells in the human. The HBM-M cell was originally derived from a patient with mastocytosis. As mastocytosis is thought to represent a reactive hyperplasia rather than a mast cell malignancy, the factor secreted by the HBM-M cell strain could well be responsible for the mast cell hyperplasia seen in some patients with mastocytosis.


INTRODUCTION

Despite the substantial progress that has been made in deriving phenotypically distinct populations of non-transformed mouse mast cells, less progress has been made in inducing human progenitor cells to preferentially differentiate in vitro into cells that resemble mature cutaneous mast cells. Kit ligand (KL),(1, 2, 3, 4, 5, 6) , interleukin (IL) 3(7, 8) , IL-4(9, 10) , IL-9(11, 12) , IL-10(13, 14, 15) , granulocyte macrophage colony stimulating factor(16, 17) , and nerve growth factor (18) can each regulate the growth, differentiation, and/or granule maturation of mouse mast cells in vitro. While neither IL-3 nor IL-4 by themselves induces human hematopoietic progenitor cells to differentiate into mast cells(19, 20) , morphologically immature populations of human mast cells are obtained when hematopoietic progenitor cells from normal bone marrow, peripheral blood, fetal liver, or umbilical cord blood are cultured in the presence of recombinant human KL (rhKL)(21, 22, 23) . Chymase(24, 25, 26) , tryptase(27, 28, 29, 30) , and carboxypeptidase (31, 32, 33, 34, 35) are granule proteases that are specifically expressed by cutaneous human mast cells. While the majority of the KL-derived human mast cells express tryptase, they do not express chymase(21, 36) .

One of the characteristics of all known in vivo differentiated mast cells is that they bind IgE with high affinity via the FcRI receptor. This receptor also is expressed in vivo on basophils and recently has been identified on Langerhans cells(37) . It consists of an alpha, beta, and two chains. Although the alpha chain binds IgE, it requires the beta and chains for cell surface expression.

Using a coculture approach, Levi-Schaffer and co-workers found that mouse 3T3 fibroblasts can maintain the viability and phenotype of rat serosal (38) and human lung (39) mast cells ex vivo and can induce immature IL-3-dependent mast cells to mature, increasing their carboxypeptidase content 100-fold(40, 41) . Furitsu and co-workers (42) reported that mature, chymase/tryptase mast cells were obtained when human cord blood mononuclear cells were cocultured with mouse 3T3 fibroblasts. In subsequent studies, these investigators found that fibroblast conditioned media containing soluble KL could induce stem cells from human cord blood to differentiate into a population of immature mast cells that expresses tryptase but not chymase(21, 36) . Two KL transcripts have been detected that result from differential exon splicing of the KL gene (43) . One transcript encodes a protein that is retained in the plasma membrane, whereas the other encodes a protein that is constitutively secreted. Because neither recombinant nor purified KL alone will induce human or mouse progenitor cells to fully differentiate into mature mast cells, the fibroblast-induced differentiation and maturation process of cord blood progenitor cells into mast cells is dependent on either membrane-bound KL or another fibroblast-derived factor.

The HBM-M cell is a cell derived from the bone marrow of a patient with diffuse cutaneous mastocytosis(44, 45, 46) . In the present study we show that conditioned media from this cell strain will induce bone marrow progenitor cells from normal subjects to preferentially differentiate into mast cells that express tryptase, chymase, carboxypeptidase, and FcRI.


EXPERIMENTAL PROCEDURES

Preparation of HBM-M Conditioned Medium

HBM-M cells were maintained in 75-cm^2 tissue culture flasks (Corning) containing 20 ml of enriched medium (alpha minimal essential medium (Life Technologies, Inc.) supplemented with 20% heat-inactivated fetal calf serum (Flow Laboratories), 2 mML-glutamine (Life Technologies, Inc.), and 100 units/ml penicillin-streptomycin (Life Technologies, Inc.)) at 37 °C in a humidified atmosphere of 5% CO(2). Conditioned medium (HBM-M-CM) was prepared by collecting spent supernatant from growing cells every 2-3 days, centrifuging at 1500 times g for 10 min and filtering through 0.2-µm low protein binding Millex GV filters (Millipore Corp., Bedford, MA).

Cultures of Human Bone Marrow Cells

Following informed consent, 10 ± 1 ml (mean ± S.D., n = 8) of bone marrow from normal donors was collected in heparinized tubes. The cells were suspended in Dulbecco's phosphate-buffered saline (PBS, Life Technologies, Inc.) and then centrifuged at 700 times g for 10 min. Erythrocytes were lysed by hypotonic treatment in 0.5% ammonium chloride. The remaining cells were washed in enriched media, and then replicate aliquots were resuspended at 1 to 3 times 10^6 cells/ml in enriched media alone, enriched media supplemented with 100 ng/ml rhKL (Amgen Inc., Thousand Oaks, CA) alone, 50% enriched medium/50% HBM-M-CM, or 50% HBM-M-CM and 100 ng/ml rhKL. The biological activity of the rhKL was assessed in a proliferation assay using the MO7e cell line(47) . The cells were cultured at 37 °C in a 5% CO(2) humidified atmosphere with the medium being changed every 2-3 days. The adherent cell population was separated from the nonadherent cells when the adherent cells reached confluence. As the adherent cells in the cultures were negative for tryptase, chymase, and FcRI (data not shown), these cells were not examined further.

Histochemistry and Immunocytochemistry

Cytocentrifuge preparations of bone marrow cells were stained with May-Grunwald Giemsa reagent for assessment of morphology and trypan blue to assess viability. Metachromatic granule-containing cells were identified by staining with 1% toluidine blue in methanol, pH 2.5.

At different times during culture, cytocentrifuge preparations of bone marrow cells were examined for their expression of tryptase, chymase, carboxypeptidase, and FcRI by immunocytochemistry and immunofluorescence. The following reagents were used: mouse anti-human mast cell tryptase and chymase monoclonal antibodies (Chemicon International Inc., Temecula, CA), mouse monoclonal anti-human carboxypeptidase antibody (Sigma), purified human myeloma IgE, and Bsp-1 (a basophil-specific monoclonal antibody).

To analyze immunochemically for the expression of mast cell-specific proteases, cytocentrifuge preparations of the cultured cells were air-dried, placed in Carnoy's fixative for 15 min at room temperature, incubated for 30 min at room temperature with the relevant monoclonal antibody, and then incubated with alkaline phosphatase-labeled rabbit anti-mouse IgG (1:50 dilution, Dakopatts) and alkaline phosphatase anti-alkaline phosphatase complex (1:50 dilution, Dakopatts) for 30 min at room temperature. Slides were developed for 20 min in 0.1 M Tris-HCl, pH 8.2, buffer containing 0.2 mg/ml naphthol AS-MX phosphate (Sigma) and 1 mg/ml fast red.

Indirect immunofluorescence was used to detect IgE receptors and Bsp-1 epitopes. For these analyses, cells were fixed for 10 min in acetone, washed in PBS, and then incubated for 60 min at 37 °C with purified human myeloma IgE in PBS, pH 7.4, or with human IgG as a control. Fluorescein isothiocyanate-conjugated rabbit anti-human IgE (Dakopatts) was used as a second antibody to detect IgE binding. Fluorescein isothiocyanate-conjugated rabbit (Fab`) anti-mouse IgM (Caltag Laboratories, South San Francisco, CA) was used to detect anti-Bsp-1 binding.

RNA Analysis by RT-PCR

Total RNA was purified from 3 times 10^5 non-adherent cells by the method of Chomczynski (48) in order to determine by RT-PCR if any of the cultures contained the transcripts that encode the alpha-chain of FcRI, mast cell tryptase, or mast cell chymase. Since the N-Ras transcript is ubiquitous in cells, the presence or absence of this N-Ras transcript was used to assess integrity of the RNA preparations. Oligonucleotides used in the RT-PCR were as follows.

The oligonucleotide primers for analysis of the tryptase, chymase, and N-Ras transcripts were synthesized on a PCR-MATE DNA synthesizer (Applied Biosystems Inc). Oligonucleotide primers for analysis of the FcRI alpha-chain transcripts were synthesized and HPLC-purified by DNA Express (Macromolecular Resources, Fort Collins, CO). The isolated total RNA was reverse transcribed in a total volume of 20 µl in a buffer containing 50 mM potassium chloride, 3 mM magnesium, 0.01% gelatin, and 10 mM Tris, pH 8.3. 500 mM dNTPs (Pharmacia Biotech Inc.), 20 pM random hexamer, 20 units of recombinant RNasin® ribonuclease inhibitor (Promega Corp.), and 100 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). Double-stranded DNA was obtained following the addition of 1 unit of Taq DNA polymerase (Boehringer Mannheim) and the appropriate oligonucleotide primers. Denaturing, annealing, and extension reactions were carried out each at 94, 58, and 72 °C, respectively, for 30 cycles on a Hybaid thermocycler.

Transmission Electron Microscopy

The nonadherent cells in the bone marrow culture were processed for electron microscopy. Cells were fixed in 1.25% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, for 2-4 h at 4 °C. Fixed cells were washed three times in 0.1 M cacodylate buffer, postfixed in 1% osmium tetroxide at 4 °C for 1 h, dehydrated in graded ethanol, and then embedded in Spurr's resin. Sections (60-80 nm), were cut on an ultramicrotome (Reichert-Jung Ultracut E) and examined using a Hitachi 7000 electron microscope.


RESULTS

Development of Metachromatic Human Mast Cells in Vitro from Bone Marrow Cells

One ml of normal bone marrow yielded 1.7 times 10^6 ± 0.64 (mean ± S.D., n = 8) nucleated cells. The viability of the dispersed cells was greater than 96 ± 2% (mean ± S.D.). At the start of each experiment, none of the cells could be stained by toluidine blue or by anti-tryptase, chymase, or carboxypeptidase antibodies. Cells were divided into aliquots, and the effects of different culture conditions on cell numbers and viability were assessed over time (Fig. 1). rhKL maintained cell viability and increased total cell number by 41 ± 1.5% (mean ± S.D., n = 4) at day 14 (Fig. 1A). Although slightly decreased at day 30, cell number was maintained for 50 days. Total number of cells in the cultures maintained with a combination of rhKL and HBM-M-CM was similar to that obtained with rhKL alone. However, the total number of cells in those cultures maintained in media alone or HBM-M-CM alone decreased significantly over time (p < 0.005 by Student's t test). At day 21, total cell number had decreased by 10-fold.


Figure 1: Comparison of the number of cells in the culture (A) and the development of mast cells when bone marrow cells were cultured with 50% HBM-M-CM alone (), 50% HBM-M-CM and 100 ng/ml rhKL (), 100 ng/ml rhKL alone (times), or enriched media alone (box). The percentages of cells staining positive for toluidine blue (B), tryptase (C), and chymase (D) are shown. For all time points, n = 3.



With HBM-M-CM alone, the percentage of cells that could be stained by toluidine blue reached a maximum of 18 ± 3% (mean ± S.D., n = 3) at day 21 (Fig. 1B). In the presence of rhKL alone, only a few toluidine blue cells were detected in the culture at day 21 or 30, but 56 ± 6% (mean ± S.D., n = 3) of these cells were toluidine blue-positive by day 48 (Fig. 1B and Fig. 2B). In enriched media with both rhKL and HBM-M-CM, 12 ± 2.8% and 50 ± 3% (mean ± S.D., n = 3) of cells were toluidine blue-positive at day 21 and day 48, respectively ( Fig. 1and Fig. 2). Metachromatic staining cells in the cultures derived with both rhKL and HBM-M-CM appeared as large cells with prominent metachromatic granules (Fig. 3G). The intensity of granule staining was similar in cells cultured with rhKL with or without HBM-M-CM at day 48 (Fig. 3, D and G).


Figure 2: Cytochemical and immunochemical characteristics of mast cells derived by culturing human bone marrow cells from normal donors for 21 days (A) or 48 days (B) in 100% enriched media, 50% enriched media/50% HBM-M-CM, enriched media supplemented with rhKL, or 50% enriched media/50% HBM-M-CM supplemented with rhKL.




Figure 3: Cytocentrifuge preparations of bone marrow cells cultured in the presence of HBM-M-CM (50%) on day 21 (A-C) and rhKL (100 ng/ml) on day 48 (D-F) and HBM-M-CM (50%) and rhKL on day 48 (G-I) stained with toluidine blue (D and G), anti-tryptase Ig (A, E, and H), anti-chymase Ig (B, F, and I), or anti-carboxypeptidase Ig (C). Magnification, times 2000 (A-D, and G-I) and times 1000 (E and F).



Expression of Mast Cell Granule Proteases and the High Affinity IgE Receptor in Cultured Mast Cells

rhKL and HBM-M-CM alone or in combination had differential effects on the expression of mast cell-specific proteases (Fig. 1Fig. 2Fig. 3). Whereas 40 ± 11% of the cells in day 48 cultures derived with rhKL alone expressed tryptase, only 5 ± 1% of the cells expressed chymase (Fig. 1, C and D; 2B; and 3, E and F) and no cells were detected that expressed carboxypeptidase. As assessed immunohistochemically, after 21 days of culture with HBM-M-CM alone, 17 ± 5%, 8 ± 5%, and 9 ± 4% (mean ± S.D., n = 6) of the cells expressed tryptase, chymase, and carboxypeptidase, respectively (Fig. 1, C and D; 2A; and 3, A-C). However, in cultures supplemented with both HBM-M-CM and rhKL at day 21, there were fewer cells that expressed carboxypeptidase (Fig. 2A). In the day 48 cultures, 40 ± 9% of the cells in the culture derived with both HBM-M-CM and rhKL expressed tryptase, 30 ± 3% expressed chymase, and there were no cells detected that expressed carboxypeptidase (mean ± S.D., n = 3) (Fig. 2B and Fig. 3, H and I). Numerous cells in the cultures supplemented with HBM-M-CM possessed a segmented or lobulated nucleus (Fig. 3). By RT-PCR, transcripts that encode mast cell tryptase and chymase were present in cultures in which progenitor cells were cultured in the presence of HBM-M-CM or rhKL (Fig. 4).


Figure 4: Electrophoretic analysis of the products of the RT-PCR amplification from RNA obtained from mast cells derived in enriched medium supplemented with HBM-M-CM (A), rhKL (B), or both HBM-M-CM and rhKL (C) using oligonucleotide primers of N-Ras (1), FcRI (2), tryptase (3), and chymase (4). The 248-bp N-Ras, 338-bp FcRI, 440-bp tryptase, and 296-bp chymase DNA products and the molecular weight markers (M) were visualized by ethidium bromide staining.



Human mast cells, derived by culturing normal bone marrow cells in the presence of HBM-M-CM with or without rhKL, specifically bound IgE. At day 21, 27 ± 2.3% (mean ± S.D., n = 3) of the cells in the culture derived with HBM-M-CM alone specifically bound IgE (Fig. 2A). After culture for 21 and 48 days in the presence of both HBM-M-CM and rhKL, 12 ± 2% and 26 ± 2.4%, respectively, of the cells bound IgE (Fig. 2, A and B). None of the mast cells in the cultures that were derived with only rhKL bound IgE. By RT-PCR, the transcript that encodes the alpha chain of FcRI was present in only one of three cultures with rhKL-derived human mast cells (Fig. 4). However, this transcript was present in all cultures in which progenitor cells were cultured in the presence of HBM-M-CM.

Ultrastructural Analysis

At day 48, many of the cells that arose in the cultures with both HBM-M-CM and rhKL were morphologically mature mast cells. The cells had electron-dense membrane-bound granules of variable number and size (Fig. 5A). A scroll or a crystalline granule morphology was seen occasionally (Fig. 5C). Non-membrane-bound lipids were also observed but were less frequent. Approximately 50% of the mast cells in each HBM-M-CM/rhKL treated culture exhibited the morphology of the cells depicted in Fig. 5(A and C). In contrast, the mast cells that were obtained by culturing bone marrow cells for 48 days in the presence of rhKL alone contained a large nucleus with a prominent nucleolus and condensed chromatin. These cells had blunt folds on their surface and had membrane-bound cytoplasmic secretary granules that were less well filled than HBM-M-CM/rhKL-derived mast cells. Crystal or scroll-pattern granules generally were not seen (Fig. 5, B and D).


Figure 5: Ultrastructural appearance of mast cells derived by culturing bone marrow cells for day 48 in the presence of rhKL alone (B and D) or in the presence of both HBM-M-CM (50%) and rhKL (A and C). Magnification, times 9200 (A and B) and times 45,500 (C and D).




DISCUSSION

We have examined the consequences of culturing normal human bone marrow cells for up to 50 days in a conditioned medium derived from a cell strain obtained from a patient with bullous mastocytosis. Conditioned medium from the HBM-M cells promoted the development from bone marrow precursors of morphologically mature mast cells that expressed FcRI, chymase, tryptase, and carboxypeptidase.

Although HBM-M-CM could induce human cells to differentiate into a mast cell lineage after only 1 week of culture, rhKL was required to maintain the long term survival of the mast cells. HBM-M-CM-derived mast cells express tryptase, chymase, and carboxypeptidase. In addition these cells possessed multilobed nuclei. This observation has important implications for categorizing metachromatic cells as basophils or mast cells based on nuclear morphological criteria. While maintaining the viability, rhKL suppressed the ability of HBM-M-CM to induce the expression of cells that contained carboxypeptidase and FcRI. In the BALB/c mouse, IL-9 and IL-10 induce expression of certain proteases, whereas IL-3 and IL-4 each suppress the expression of these proteases (12, 14, 15) . While rhKL induces mouse bone marrow-derived mast cells to express mMCP-4, it does not suppress the steady state level of the carboxypeptidase and the FcRI transcripts. The demonstration that rhKL prevents the induction of these two transcripts has not previously been reported in any other in vitro system. As carboxypeptidase is a granule protease of mature cutaneous mast cells, this supports previous findings that in the human rhKL induced immature mast cells.

The in vitro development of mature mast cells both in humans and rodents has been achieved using hematopoietic progenitor cells from a variety of sources cocultured with fibroblasts(36, 42) . Fibroblasts (43) and stromal cells (49) express both membrane and soluble forms of KL in addition to other cytokines. Human mast cells derived from progenitors of cord blood mononuclear cells cocultured with 3T3 fibroblasts are ultrastructurally quite mature and express both tryptase and chymase(42) . In contrast, Irani et al.(50) , using the same fibroblast coculture system and dispersed human fetal liver cells as a source of progenitors, obtained human mast cells that expressed tryptase but not chymase. This implies either that the progenitors from the two sources were different or that there were other cells in the cord blood culture providing accessory mast cell regulatory cytokines which induced chymase expression. Our results suggest that rhKL induces mast cells that are tryptase-positive and that supplementation with HBM-M-CM induces chymase in the cells. The synergy between rhKL and HBM-M-CM in increasing the total number of mast cells has also been demonstrated in human and rodent systems where bone marrow is cultured with KL and IL-3(51, 52) . The factor in HBM-M-CM remains to be determined, but HBM-M-CM does not contain detectable levels of IL-3, IL-5, granulocyte/macrophage colony-stimulating factor, or KL(46) .

Mast cells derived from human bone marrow using rhKL do not bind fluoresceinated human IgE, in contrast to the cells that were grown in HBM-M-CM. The addition of rhKL to HBM-M-CM resulted in a down-regulation of IgE binding at both day 21 and day 48 of culture. In both culture systems, RT-PCR analysis revealed the presence of the transcript that encodes the alpha chain of FcRI (Fig. 4). Mast cells derived by culturing human fetal liver cells in the presence of rhKL alone also do not bind human IgE but occasionally in some cultures have the FcRI alpha transcript(53) . The expression of FcRI without IgE binding could be due to lack of transcription of the beta and genes or a failure of the alpha, beta, and transcripts to be translated or the receptor complex to be properly assembled in the endoplasmic reticulum.

Human mast cells derived from cord blood progenitors cocultured with 3T3 fibroblasts bind human IgE but with an intensity of fluorescence less than that of human lung mast cells sensitized with IgE(42) . Moreover, IgE binding was demonstrated only after 74 days of coculture of cord blood mononuclear cells with 3T3 fibroblasts, when less than 10% of cells were identified as mast cells(42, 54) .

In contrast to our study, Valent and co-workers (22) induced differentiation of human mast cells from bone marrow using rhKL in long term cultures and reported that these cells bound human IgE. This difference may be due to the fact that we have used a total bone marrow cell preparation and not a mononuclear fraction as Valent and co-workers have done. Nilsson and co-workers (53) were unable to induce the expression of FcRI in cultures of fetal liver cells with rhKL supplemented with conditioned medium from a human T cell line. Other workers have used human hematopoietic progenitor cells from a variety of sources, co-cultured with a range of human stromal/fibroblast cell lines, but have been unable to induce the development of cells that exhibit the phenotype of cutaneous mast cells(36) .


FOOTNOTES

*
This study was supported by a grant from the National Health and Medical Research Council of Australia and the St. George Hospital Cancer Research Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Immunology, Allergy and Infectious Disease, St. George Hospital, Kogarah, New South Wales 2217, Australia. Tel.: 61-2-350-2955; Fax: 61-2-350-3981.

(^1)
The abbreviations used are: KL, kit ligand; rhKL, recombinant human KL; IL, interleukin; PBS, phosphate-buffered saline; RT-PCR, reverse transcription polymerase chain reaction; HBM-M-CM, HBM-M cell conditioned medium; bp, base pair(s).

(^2)
KL is also known as kit ligand, stem cell factor, mast cell growth factor, and steel factor.


ACKNOWLEDGEMENTS

We thank A. Coleman for excellent secretarial assistance; Dr. G. Layton, Medical Innovations, Queensland, Australia for kindly providing purified myeloma IgE; and Dr. M. Bodger Christchurch Hospital, Christchurch, New Zealand, for gift of Bsp-1 monoclonal antibody.


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