Development of human mast cells from their progenitors

Development

of human mast cells from their progenitors

Teruko Ishizaka, Hideki Mitsui, Makoto Yanagida, Toru Miura and Ann M Dvorak La Jolla Institute

for Allergy

and Immunology,

Harvard

Medical

La Jolla and Beth Israel Hospital

School,

Boston,

Two types of human mast cells, which are morphologically

similar to skin mast

cells and lung mast cells, respectively, can be developed from pluripotent cells under different development

culture conditions.

is c-kit ligand, which

this cytokine is not sufficient

Current

Opinion

and

USA

stem

The major growth factor for mast-cell

induces mastocytosis for full maturation

in immunology

1993,

in’vivo.

However,

of the cells.

5:937-943

Introduction

Development

cells and basophils play a primary role in the clinical manifestation of allergic inflammations and atopic diseases. In immediate hypersensitivity reactions, IgE antibodies bind to mast cells and basophils through their high affinity IgE receptors (FcERI), and crosslinking of membrane-bound IgE by multivalent antigen induces the initial triggering signals for the release of chemical mediators, e.g. histamine, leukotrienes and prostaglandins 111. Mast cells and basophils are also activated by various IgE-independent stimuli, such as complement components C5a, C3a, some neuropeptides and a variety of other agents, resulting in the release of mediators 121.More recently, evidence has accumulated that mast cells may play an important role in immediate hypersensitivity reactions as a source of various cytokines (reviewed in 131). Crosslinking of FcERI on normal mouse mast cells results in an increase in the expression of mRNA for several cytokines and the release of the cytokines 141.Bradding et al. 15’1 demonstrated mRNA for interleukin (IL)-4 in human mast cells from the skin and respiratory tissues of atopic patients, and IgE-mediated release of IL-4 from these cells.

In the murine system, recombinant IL-3 promotes the proliferation of mouse mast-cell lines 161and facilitates the formation of mast-cell colonies in semisolid cultures of mouse bone marrow (BM) cells 171. Suspension culture of murine BM cells with recombinant IL-3 results in the development of a pure mast-cell population 181. These IL-3-dependent, BM-derived mast cells appear to be analogous to mast cells in the gastrointestinal mucosa, but different from those in connective tissues 191. However, LeviSchaffer et al. 1101 demonstrated that co-culture of the BM-derived, IL+dependent mast cells with 3T3 fibroblasts in the presence of IL-3-containing conditioned medium resulted in the mast cells undergoing a phenotypic change so that they resembled those of connective tissue.

Mast

In order to analyze the biochemical mechanisms involved in the activation of mast cells for their various functions, a sufficient number of morphologically and functionally mature human mast cells of high purity is required. It is anticipated that the development of mast cells in vitro will facilitate the opportunity to establish a human mast cell line that may eventually be used for analysis of the biochemical processes of mast-cell activation and allergic reactions in humans.

of human mast cells in vitro

In contrast with the murine mast-cell system, in vitro development of human mast cells proved quite difficult. Several groups of investigators have succeeded in the development of human basophils from progenitors in BM, umbilical cord blood, and fetal liver in vitro [ll-141. Recombinant human IL-3 promoted the differentiation of human basophils from progenitors in BM and cord blood cells; however, neither IL-3 or IL-4 on their own, nor various combinations of cytokines facilitated the development of human mast cells 115-171. In vitro development of human mast cells from progenitors in umbilical cord blood was achieved by co-culture of cord blood mononuclear cells with 3T3 fibroblasts derived from Swiss albino mouse skin (Swiss/3T3 fibroblasts) [181. After 7-8 weeks of coculture, mononuclear cells containing metachromatic granules appeared in the culture. After 9-10 weeks,

Abbreviations BM-bone

marrow; IL-interleukin; 0 Current

mAb--monoclonal

Biology

Ltd ISSN

antibody; MCF-mast

0952-7915

cell growth factor.

937

938

Atopic allergy and other hypenensitivities

when the number of the other hematopoietic cells declined, cells that contained metachromatic granules comprised up to 20% of the total cells in the culture. It appears that co-culture of mononuclear cells from cord blood 1181, or CD34+ pluripotent progenitor cells from BM 1191, with Swiss/3T3 fibroblasts promoted the differentiation of mast-cell progenitors in an early stage of the culture, and then selectively supported the survival of the cells. Irani et al. 1201 demonstrated that co-culture of dispersed human fetal liver cells with Swiss/3T3 fibroblasts resulted in the development of human mast cells within 4 weeks of culture. Whereas, co-culture of cord blood cells with human fibroblast cell line cells, i.e. IMR-90, MRC-9 and Detroit 550 (American Type Culture Collection) did not lead to the development of human mast cells 1181. After establishing reproducible development of human mast cells when cord blood cells are co-cultured with 3T3 fibroblasts, attempts were made to identify growth factor(s) for human mast cells. It became clear that the concentrated culture supernatant of 3T3 fibroblasts contained growth factors for mast cells, which promoted the development of human mast cells in the absence of fibroblast feeder layers 1211. The human mast cell growth-promoting activity could be enriched by fractionation of the culture supernatant of Balb/3T3 libroblasts using ammonium sulfate precipitation and by ion-exchange chromatography. The molecular size of the factor, estimated by gel filtration, was between 70 and 1OOkDa 1211. While the purification of the facto&> was in progress, a novel cytokine, the ligand for the proto-oncogene receptor encoded by c-kit was characterized, and molecular cloning of the cytokine gene was accomplished by several groups of investigators [22-261 The c-kit ligand was called stem cell factor 121,221, kit ligand 124,251 and mast cell growth factor (MGF) 1261by different investigators. As the murine c-kit ligand was characterized as a mouse MGF [26l,several groups of investigators explored the possibility that human c-kit ligand promotes the development of human mast cells from their progenitors in bone marrow 127,281, fetal liver 1291, peripheral blood 1271, cord blood 130’1 and highly purified CD34+ cells from BM 1281. The results showed that both human and mouse recombinant c-kit ligand induced differentiation and proliferation of mastcell progenitors in the early stages of culture. Affinity chromatography using Affigel coupled with polyclonal antibody against mouse c-kit ligand, the major human mast cell growth factor in the culture supernatant of 3T3 fibroblasts was identified as mouse c-kit ligand 130’1. However, the apparently selective growth of mast cells induced by c-kit ligand in a long-term culture of cord blood cells is due mainly to the cytokine maintaining the survival of immature mast cells in the culture.

Functional and morphological properties of cultured mast cells Human mast cells developed in co-culture of cord blood mononuclear cells and 3T3 fibroblasts, and those matured in a long-term suspension culture of cord blood cells in the presence of human or murine c-kit ligand express approximately 105 FcERI per cell, and contain histamine (1.6-2.8 pg/cell) 130’1. The cultured mast cells sensitized with human IgE released histamine and arachidonic acid, and generated prostaglandin D2 and leukotriene C4 upon challenge with antigen that binds IgE. Mast cells obtained by both types of culture bear the human mast cell surface marker recognized by the monoclonal antibody (mAb) YB5.B8 1311, but are not stained by the mAb VlM12, which is specific for human basophils [321. After the human c-kit ligand was identified, it became clear that YB5.B8 is specific for the product of the human c-kit proto-oncogene 1331. Thus, cultured human mast cells bear c-kit on their cell surface, whereas human basophils do not. Ultrastructural analysis of human mast cells that developed in the co-culture of cord blood mononuclear cells and Swiss/3T3 fibroblasts revealed that the cells recovered after 8 weeks of culture were mature human mast cells 1181. As shown in Figure 1, they were mononuclear cells and their nuclei displayed partially condensed chromatin. Narrow surface folds were generally distributed over the cell surface. In the granules, previously reported substructural granule patterns, such as crystalline arrays of several periodicites, spirals, scrolls and particles were evident, but the cells rarely contained or did not contain lipid bodies. Most of the mast cells in the culture expressed some granules with regular crystalline arrays resembling those frequently seen in human skin mast cells 118,341. Ultrastructural analysis of the cells unveiled a remarkable difference between mast cells that developed in the co-culture with 3T3 fibroblasts and those that developed in suspension culture in the presence of c-kit ligand. As shown in Figure 2, mast cells that developed in suspension culture in the presence of c-kit ligand did not reach full maturity even after I4 weeks of culture. The cells contain an immature nucleus and granules, many lipid bodies but no crystal granules [30*1. One of the characteristics of human mast cells is the presence of unique neutral proteases in their granules. Granules of human mast cells can be visualized using immunofluorescent mAbs against tryptase and/or chymase, whereas these antibodies do not bind to granules in human basophils [35,361.In the course of cell development, the tryptase in mast-cell granules can be detected earlier than metachromasia. An important feature of human mast cells is that they can be classified into two types based on granule-associated proteases [36-401. The majority of mast cells in alveolar walls of

Development of human mast cells lshizaka et al.

Fig. 1 (a) Human mast cell that developed in a co-culture of cord blood cells with 3T3 fibroblasts for 12 weeks. The photograph shows that the mast cell has a monolobed nucleus with partially condensed peripheral chromatin, narrow surface folds, intracytoplasmic canaliculi (arrow), a lipid body and numerous membranebound granules. Substructure of granules delineated by boxes in (a) are seen at higher magnification in (b) and (c). (b) A crystal granule is seen in cross-section adjacent to a mixed granule with homogeneous dense contents and peripheral scrolls or lamellae. (c) A mixed granule parallel crystalline array and with homogeneous dense contents adjacent to a dense lipid body with focal peripheral lucency. (a) x 9 160; (b) x 50 000; (c) x 51 000.

the lung and those in the small intestinal mucosa contain tryptase but no chymase (MCT cells). In contrast, the predominant type of mast cells in the skin and submucosa of the small intestine contains not only tryptase but also chymase, a cathepsin G-like protease 1411, and carboxypeptidase [421 (MCT~ cells). Interesting observations concerning these proteases have been made in vitro more than 90% of mast cells that developed in a co-culture of cord blood cells with 3T3 fibroblasts for 7-10 weeks, were stained by both anti-tryptase and anti-chymase antibodies, i.e. were MCTc cells [IS], whereas the majority of mast cells that developed in suspension cultures in the presence of c-kit ligand contained only tryptase (MCT cells) [30*1. Thus, mast cells that developed in the co-cultures were similar to mature skin mast cells in both ultrastructural criteria and the type of granule-associated neutral proteases present, whereas those that developed in suspension culture in the presence of c-kit ligand were more like those in the lung and intestinal mucosa. It is possible that a maturation or differentiation factor, other than c-kit ligand, is associated with the plasma membrane of fibroblasts. However, it is also possible that the two subsets of mast cells have separate progenitors, and that differentiation of MC,, progenitors requires a membrane-associated cytokine or direct contact with

Fig. 2. Immature mast cell that developed in a suspension culture of human cord blood mononuclear cells supplemented with murine ckit ligand for 14 weeks. The cell has a large immature nucleus lyith partially dispersed chromatin and a round, central nucleolus. The cell surface has a large number of short narrow surface folds. Many membrane-bound cytoplasmic secretory granules with heterogeneous contents are present. Some granules contain scrolls; most contain mixtures of membranous and dense materials. Nearly half of the secretory granules are immature with incomplete deposition and condensation of dense granule materials. Other cytoplasmic organelles include elongated mitochondria, free ribosomes and a cluster of small dense lipid bodies adjacent to the nucleus. (x 7 100) An interesting observation in this respect is that 94% of mast cells that developed after co-culture of fetal liver cells and 3T3 fibroblasts for 30 days contained only tryptase (MCT cells) 1201.However, both the histamine content and tryptase content were comparable in the MTc type mast cells derived from cord blood and MCT type mast cells derived from fetal liver. For mast cells derived from both cord blood and fetal liver, no evidence has been obtained supporting the possible conversion of MCT cells to MC-rc cells. One might speculate that mast-cell progenitors are committed to develop into either MCT cells or MC-rc cells at a stage of differentiation earlier than granulation [201.

fibroblasts.

Ontogeny

of human mast cells

The co-culture system for the development of mature mast cells from cord blood mononuclear cells has proved useful in the analysis of the ontogeny of human mast cells. Kirshenbaum et al. 1431 have shown that co-culture of highly purified CD34+ pluripotent bone marrow stem cells with 3T3 fibroblasts, or culture of the cells over agarose surface, resulted in the development of both mast cells and basophils, indicating that both these cell types are derived from the CD34+ agranular progenitors [19,431. Dvorak et al. L44.1 co-cultured aliquots of cord blood cells from the same individuals with 3T3 fibroblasts, and recovered the cells after

939

Atopic allergy and other hypersensitivities

cuM for different times, ranging between 2 weeks e 3.5 mod. The cells were examined by light lecron microscopy and by immunofluthe presence of tryptase. At times as 3 weeks of culture, Dvorak and COGes -Id detect vyptase positive Cells and Ultraly identified mast-cell progenitors. The identification was based on the presence of at least one and possibly several qtoplasmic granules that contained dense materials and variable numbers of granule-sized, membrane-bound containers, which initially clustered around an enlarged Golgi area (Fig. 3). The cells also contained lipid bodies. This ultrastructure was shared by immature mast cells, which appeared during the later period of culture. After co-culture for periods between 25 days and 4 weeks, mast-cell progenitors were found to display a large number of immature granules, many of which contained particulate contents. In some cells, typical mast cell granule scroll patterns superimposed upon the particulate material. The mast-cell progenitor had a single lobular nucleus, large nucleoli and a surface structure which varied from smooth to narrow folds. The morphological features of mast cells observed with an electron microscope are distinct from cells of the basophil lineage. Indeed, in the co-cultures sampled between 2 and 5 weeks OS-1.5% of the cells were basophils, which could be easily distinguished from mast-cell progenitors. A recent report actually indicated that co-culture of human basophils with Swiss/3T3 fibroblasts did not support the survival of basophils and did not alter the phenotype of the cells to that of mast cells (AR Kirshenbaum, abstract 459, J Allergy Clin Immunol1993, 91:255).

Effect of IL-3 and IL-4 on the development

of

human mast cells It is clear that IL-3 cannot promote the development of human mast cells in either BM or cord blood cell cultures, but the addition of IL-~ to the cultures together with c-kit ligand generally increased the total number of mast cells l30.1. However, the proportion of mast cells in the IL-3-containing cultures was comparable with that obtained in the cultures that received c-kit ligand alone. Thus, the effect of IL-~ is not selective for mast cells [30*1. Kirshenbaum et al. [43] reported that mast cells developed within 3 weeks of culture of highly purified CD34+ bone marrow cells in the presence of both c-kit ligand and IL-3. The cells contained tryptase and were similar ultrastructurally to those that developed after co-culture of the CD34+ cells with 3T3 fibroblasts for 10 weeks. The findings suggest that IL-~ may play a role in the differentiation of human mast cells. However, IL-3 receptors could not be detected either on the immature mast cells that differentiated in the suspension culture containing c-kit ligand or on mature mast cells that differentiated in the co-culture of cord blood cells and 3T3 fibroblasts (A Miyajima, personal communication).

Fig. 3. A mast cell progenitor from a 3 week old co-culture of cord blood cells and 3T3 fibroblasts. The cell is distinguished by variable numbers of granule-sized, membrane-bound containers that cluster around the Golgi (G) area. Some of these immature granules have accumulated dense materials (arrow). The cytoplasm is filled with ribosomes, small amounts of rough endoplasmic reticula, which do not have dilated cisternae, mitochondria and lipid bodies (arrowhead). The large lobular nucleus often appears separated into several lobes in thin sections. (x9 140)

Sillaber et al. 1451 tested possible effects of various cytokines, namely IL-I-IL-9, granuloqte macrophage colony stimulating factor, interferon and tumor necrosis factor, on the expression of the product of the c-kit proto-oncogene by immunofluorescence with the mAb YB5.B8 and by northern blot analysis. Among the cytokines tested, only IL-4 downregulated the expression of YB5J38 antigen on the human mast cell line HMC-1 [461 and progenitor cells in normal bone marrow, and downregulated the transcription of mRNA for c-kit in leukemic myeloid cells and HMC-1 cells. In agreement with these observations, Nilsson and colleagues (G Nilsson, personal communication) found that IL-4 significantly suppressed development of mast cells in fetal liver cell cultures in the presence of c-kit ligand. It appears that IL-4 regulates differentiation of human hematopoietic cells, including human mast cells, mediated by c-kit ligand through the inhibition of the expression of c-kit. As murine IL-4 is one of the growth factors for mouse mast cells 171, and it enhances IgE synthesis through the induction of isotype switching [471, this cytokine is believed to play a key role in allergic diseases. It should be noted, however, that human IL-4 inhibits, rather than promotes, the differentiation of human mast cells.

Development

of human mast cells in vivo

In order to elucidate the function of c-kit ligand in viuo, Galli et al. [481 administered human recombi-

Development of human mast cells lshizaka et al.

nant c-kit ligand to monkeys. Two daily subcutaneous injections of either 0.1 mg or bmgkg-Idayof c-kit ligand to Macaca fascicularis for 21 days resulted in a remarkable expansion of the mast-cell population in many tissues and organs, and discontinuation of the treatment with c-kit ligand resulted in a rapid decline in the number of mast cells to nearly baseline levels. This observation in monkeys was reproduced in their phase I trial of recombinant human c-kit ligand in patients with breast cancer (JI Costa, abstract 97, Proc Subcutaneous adminAm Assoc Can Res 1993,34:210. istration of 10 mg or 50 mgkg-‘day-1 of c-kit ligand for 7 or 14 days resulted in a doubling in the number of dermal mast cells and a slight increase in the level of circulating histamine. The results showed that human c-kit ligand promotes differentiation and development of human mast cells in vivo. Langley et al. 149’1 studied the expression of the gene encoding c-kit ligand in the skin of patients with cutaneous mastocytosis using immunohistochemcal methods and the polymerase chain reaction. Their studies showed that immunoreactive c-kit ligand was associated with keratinocytes and some scattered dermal cells in normal subjects. In skin biopsies from mastocytosis patients with or without skin lesions, however, c-kit ligand was detected not only on keratinocytes but also within extracellular spaces in the dermis. As the altered distribution of c-kit ligand in the cutaneous lesions of the patients is consistent with abnormal production of the soluble form of the ligand, these authors speculated that an increase in levels of the soluble form of c-kit ligand may cause accumulation of mast cells and hyperpigmentation in the patient’s skin.

obtained in this system are similar to those present in the lung and intestinal mucosa. As 3T3 fibroblasts produce c-kit ligand, this cytokine is considered to be involved in the development of MCTC cells. However, the failure of soluble c-kit ligand to develop MCT~ cells indicates that additional differentiation factor(s) provided by the fibroblasts or the interaction of mast-cell progenitors with fibroblasts is required for the development of MC& cells. Both MCT cells and MCT~ cells are derived from CD34+ pluripotent stem cells; however, mast-cell progenitors committed to develop into MCT~ cells may be distinct from those committed towards MCT cells. Unlike murine mast cell systems, no evidence is available so far that human MCT cells could develop into MCT~ cells in the presence of fibroblasts. In vitro culture systems for the development of human mast cells provide a useful tool for identification of mast-cell progenitors. They are distinct from myeloid cells and the findings provide definitive evidence that the mast-cell lineage is distinct from the basophil lineage even at the progenitor cell stage. It should be noted that, in the human system, IL-3 is a growth factor for basophils but not for mast cells, whereas c-kit ligand promotes the differentiation and maturation of mast cells but not basophils. Indeed, evidence has been provided that c-kit ligand induces proliferation of mast cells not only in in vitro cultures, but also in vivo, and may be involved in the development of mastocytosis.

Acknowledgements The authors

Conclusion Human mast cells have proved to be one of the most difficult cells to differentiate in culture in vitro. In the past few years, however, differentiation of mast cells in vitro has been achieved by co-culture of umbilical cord blood mononuclear cells with 3T3 fibroblasts or by suspension culture of the mononuclear cells with c-kit ligand. Human mast cells obtained by the two culture systems are functionally mature cells. Sensitization of the cells with human IgE, followed by challenge with anti-IgE, resulted in the release of histamine, leukotrienes and prostaglandins. However, mast cells obtained in the two culture systems have quite different morphological characteristics. The cells obtained in a long-term co-culture with 3T3 fibroblasts were mature human mast cells in ultrastructural criteria, contained both tryptase and chymase in their granules (MC-rc cells), and were morphologically similar to mast cells present in the skin. In contrast, mast cells obtained in the suspension culture in the presence of c-kit ligand were immature in ultrastructural criteria and contained tryptase but no chymase (MCT cells). Thus, mast cells

express their special gratitude to Drs Anne-Marie A Irdni and Lawrence 1) Schwartz, Medical College of Virginia, Virginia Commonwealth University, Richmond, ViiInia for their collaboration and contribution. This is publication number 85 from La Jolla Institute for Allergy and Immunology, supported by Health and Human Service grants AI-loo60 and AI-33372, and a research contract with Gemini Science, Inc.

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T lshizaka, H Mitsui, M Yanagida and T Miura, la Jolla Institute for Allergy and Immunology, 11149 North Torrey Pines Road, la Jolla, California 92037, USA. AM Dvorak, Beth Israel Hospital and Harvard Medical School, 300 Brookline Avenue, Boston, Massachusetts 02210, USA.