Selective Growth of Human Mast Cells Induced by Steel Factor, 11-6, and Prostaglandin E, from Cord Blood Mononuclear Cells'

Hirohisa Saito,** Motohiro Ebisawa,* Hiroshi Tachimoto,* Michitaka Shichijo,* Kazumi Fukagawa,* Kenji Matsumoto,* Yoji likura,* Takeo Awaji,+ Gozo Tsujimoto,+ Makoto Yanagida,* Hiroya Uzumaki,* Gen Takahashi,§ Koichiro Tsuji,' and Tatsutoshi Nakahata'

To establish the method for generating a large number of mature human mast cells, we cultured cord blood mononuclear cells (CBMC) in several conditions in the presence of Steel factor (SF). Among several cytokines tested, 11-6 enhanced SF-dependent mast cell growth from purified CD34+ cells for more than 8 wk in culture. When CBMC were cultured instead of CD34+ cells, IL-6 enhanced the mast cell development in the presence but not in the absence of PGE,. PGE, enhanced the SF- and IL-6- dependent development of mast cells from CBMC probably by blocking granulocyte-macrophage CSF (GM-CSF) secretion from accessory cells, because 1 ) PGE, or anti-GM-CSF enhanced the mast cell development induced by SF and 11-6 from CBMC, but not from CD34+ cells; 2) CM-CSF inhibited the enhancing effect of 11-6 on the mast cell development from CD34+ cells; and 3) PGE, inhibited CM-CSF secretion from CBMC. The mast cells cultured in the presence of SF, 11-6, and PGE, for >10 wk were 99% pure, and seemed to be functionally mature, because 1) they contained 5.62 p g of histamine and 3.46 p g of tryptase per lo6 cells; and 2) when sensitized with human IgE and then challenged with anti-human IgE, the cells released a variety of mediators such as histamine, and an increase in intracellular Caz+ was found in advance of the activation of membrane movement by using a confocal laser-scanning microscope. Electron-microscopic analysis revealed that some of the cultured mast cells are morphologically mature since they filled with scroll granules and contained crystal granules. The journal of Immunology, 1996,157: 343-350.

M ast cells play a central role in allergic inflammation by releasing various kinds of cytokines as well as vaso- active mediators (1, 2). Since mast cell heterogeneity has been found in different species and tissues (3,4), it is expected to use human cells for investigating the role of mast cells in al- lergic disorders. And, since the number of cells that can be ob- tained from human tissues is limited, numerous attempts to estab- lish human mast cell culture had been made for a decade after discovery of the method for culturing a large number of mouse mast cells in the presence of mouse IL-3 (5). The development of human mast cells was found in the coculture system of hemopoi- etic cells with 3T3 fibroblast cell line (6).

One of the major factors supporting the development of human mast cells has later been found to be stem cell factor (7-lo), which

*Division of Allergy and 'Department of Pediatric Pharmacology, National Chil- dren's Medical Research Center, Tokyo, Japan; *Pharmaceutical Development Laboratory, Kirin Brewery Co., Maebashi, Gunrna, Japan; §Department of Anat- omy, Hirosaki University School of Medicine, Hirosaki, Aomori, Japan; and ¶Department of Clinical Oncology, The Institute of Medical Science, University of Tokyo, Tokyo, Japan

Received for publication November 15, 1995. Accepted for publication April 22, 1996.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This work was supported in part by Pediatric Research Grant 6-04 from the ences Foundation, 1995. Ministry of Health and Welfare, and by Grant 51 14 from the Japan Health Sci-

Allergy, National Children's Medical Research Center, 3-35-31, Taishido, Seta- ' Address correspondence and reprint requests to Dr. Hirohisa Saito, Division of gaya-ku, Tokyo 154, Japan.

Copyright 0 1996 by The American Association of Immunologists

is derived from the Steel locus gene and has a ligand activity for the c-kit proto-oncogene product (1 1-13). Stem cell factor, which is also called c-kit ligand or Steel factor (SF),3 is known to promote colony growth of crude hemopoietic cells (14-16). SF alone, how- ever, induces only minimum proliferation of hemopoietic colonies from purified CD34" cells. In combination with other cytokines, SF strongly stimulates hemopoietic colonies from CD34+ cells (1 7-1 9). The synergistic effect of SF with other cytokines is hardly detected when crude cell populations are cultured, since accessory cells spontaneously secrete a variety of cytokines (20, 21). Simi- larly, SF induces development of a substantial number of mast cells from crude hemopoietic cell preparations (8-10). Although human mast cells have been proven to be derived from CD34' cells, SF alone induces only a minimum proliferation of the cells from purified CD34+ cells (7). These reports suggest that addi- tional growth factors for mast cell development may be released from accessory cells.

We have recently found in a preliminary experiment (22) that IL-6 enhances the SF-induced development of human mast cells from purified CD34+ cells. We have also previously reported that PGE enhances the growth of mast cell colonies and inhibits the growth of granulocyte-macrophage (GM) colonies in the murine system (23). In the present study, therefore, we tried to confirm the effect of IL-6 in several culture conditions, and tested the effect of PGE on the SF- and IL-6-dependent development of human mast cells from crude cord blood mononuclear cells (CBMC) to establish a simple method for generating a large number of human mast cells.

' Abbreviations used in this paper: SF, Steel factor; CBMC, cord blood mono- nuclear cells: CM, granulocyte-macrophage.

0022-1 767/96/$02.00

344 CULTURED HUMAN MAST CELLS

Materials and Methods Cell preparation

Heparin-treated umbilical cord blood was obtained under informed consent based on guidance from the hospital, diluted, and layered over lymphocyte separation medium (LSM; Organon Teknika Corp., Durham, NC) at room temperature within 12 h after delivery. Mononuclear cell fractions, which contained lymphocytes (60-80%), monocytes (20-40%), and other cell types (

The Journal of Immunology 345

FIGURE 1. Effect of cytokines on SF-induced proliferation of tryptase-positive or -negative cells. Purified CD34+ cells were cultured at 10’ to 104/ml in the presence of cytokines, as indicated. After 4 wk in culture, the number of cultured cells was counted, the smears were stained for tryptase, and the percentages of cells having tryptase-pos- itive granules (B) or tryptase-negative cells (0) were calculated. Each column and bar represents the mean and SE of four separate experi- ments, and p values between the number of tryptase-positive cells in cultures with SF and that in cultures with SF plus cytokines were 95% of the tryptase- negative cells were judged to be macrophages by their prominent phagosomes) developed in the presence of SF. IL-6 enhanced the SF-induced development of tryptase-positive mast cells with the highest proportion. Further addition of GM-CSF at 100 pg/ml to the cultures containing SF and IL-6 enhanced the proliferation of tryptase-negative cells, and inhibited the enhancing effect of IL-6 on the SF-induced growth of tryptase-positive mast cells (Fig. 1). In the absence of SF, the growth of tryptase-positive cells was not detected even in the presence of these three cytokines (data not shown). Other than the experiments shown in Figure 1 , we have obtained similar results that IL-6, IL-3, and GM-CSF in combina- tion with SF stimulated the growth of tryptase-positive cells in three separate experiments, although the cell number was not suf- ficient enough for counting tryptase-positive cells in the cultures

FIGURE 2. Effect of PGE, on mast cell development from crude CBMC induced by SF and IL-6. CBMC were cultured at 105/ml in the presence of SF at 80 ng/ml and IL-6 at 50 ng/rnl for 4 wk. After 4 wk in culture, the number of cultured cells was counted, the smears were stained for tryptase, and the percentages of tryptase-positive cells were calculated. Each column and bar represents the mean and SE of eight separate experiments, and p values were

346 CULTURED HUMAN MAST CELLS

Table II. Effect of PGE, on the increase in cellular histamine and cellular tryptase concentrations in mast cells cultured in the presence of SF and IL-6 for 4 wk

Control PCE, lo-’ M PGE, 10-7 M PCE, 1 0-6 M

Histamine (nglwell)” 51.4 t 35.2‘ 75.2 t 49.9‘ 11 9.6 t 75.2‘ 1 10.0 ? 84.8 Tryptase (nglwell)” 81.2 t 53.2 220.6 ? 189.2 286.4 t 182.8‘ 242.2 t 105.1‘

a Each value indicates the concentration of mediators in the total cultured cells per each well where 1 X lo5 mononuclear cord blood cells were plated.

c p < 0.05 by a double-tailed paired Student’s t test. Each value represents an average 2 SEM of five separate experiments.

FIGURE 3. Effect of PGE, on the SF- and IL-6-induced mast cell de- velopment from purified CD34+ cells. Cord blood-derived CD34+ cells were cultured at 1 O3 to 104/ml in a well in the presence of SF at 80 nglml and IL-6 at 50 nglml for 4 wk. After cell count, the smears were stained for tryptase, and then percentages of tryptase-positive cells were calculated. Each column represents the mean of three sep- arate experiments. Tryptase-positive cells were developed at 2.98 t 1, 3.05 t 0.82,2.62 t 0.8, and 2.89 t 0. A quantity amounting to 98 X 1 Os per well for 4 wk in culture was in the presence of PGE, at 0 , l O-’ M, 10” M, and M, respectively (not significant).

lo4 tryptase-positive cells from lo5 mononuclear cord blood cells after 4 wk in the medium supplemented with IL-6 at 0, 2, 10, and 50 ng/ml, respectively (mean ? SEM; n = 3; p < 0.05 by Stu- dent’s paired t test between groups cultured with IL-6 at 10-50 ng/ml and 0 ng/ml). In the absence of PGE, and the presence of SF, the addition of IL-6 did not affect the number of tryptase- positive cells that arose from 10’ mononuclear cells (3.99 X io4 cells for IL-6 at 50 ng/ml and 3.82 X lo4 cells for control in three separate experiments).

When lo5 CBMC were cultured for 4 wk in 1 ml of medium provided with SF and IL-6, but not PGE,, the addition of anti- GM-CSF (10 pg/ml) enhanced the growth of tryptase-positive cells ((8.03 ? 2.01) X lo4 for anti-GM-CSF and (1.54 ? 2.01) X io4 for control, in four separate experiments. The value represents the mean ? SEM; p < 0.05 by paired t test.).

Morphologic features

In the presence of SF at 80 ng/ml, IL-6 at 50 ng/ml, and 300 nM of PGE,, the cultured mast cells increased in size as well as in number until day 100 in culture (Fig. 4). Ultrastructural analysis revealed that most granules of the cultured mast cells were filled with scroll structures, although irregular periodicity was seen in the structures (Fig. 5). We were also able to demonstrate a typical crystal granule in the mast cells (Fig. 6). We used IL-6 at 50 ng/ml, because the cytokine at 100 ng/ml induced atypical characteristics, i.e., the cells had >10 pg of cellular histamine per cell and mul- tilobed nuclei. As has been reported elsewhere (28), the cultured mast cells were stained for tryptase at 98.6 ? 0.4%, and chymase for 18.4 ? 4.4% (n = 7).

Functional features

When CBMC were cultured in the presence of SF at 80 ng/ml, IL-6 at 50 ng/ml, and 300 nM of PGE, for >10 wk, the mast cells

FIGURE 4. Typical mast cells developed in the presence of SF, IL-6, and PGE, for 12 wk. May-Grunwald Giemsa stain. Note the prominent microvilli-like processes. X1099.

contained 5.62 ? 1.88 pg of histamine (n = 6) and 3.46 ? 0.89 pg of tryptase (n = 6; each value represents mean ? SEM) per lo6 cells. Cells sensitized with 1 pglml of human IgE released 52.9% histamine at 30 min, and 1.76 ng of TNF-a per lo6 cells at 6 h after challenge with 1.5 pg/ml of anti-human IgE, whereas the control cells spontaneously released 3.7% histamine and 0.18 ng of TNF-a (Fig. 7). Release of TNF-a reached a plateau at 6 h (data not shown), while the increase in intracellular Ca2+ (Fig. 8) and his- tamine release (data not shown) reached a plateau before 10 min. An increase in intracellular Ca” could be detected in advance of partial swelling of the plasma membrane by using a confocal laser- scanning microscope (Fig. 9). Some parts of the membrane had ballooned outward and were soon shrinking. The ballooning was repeated from 196 to 476 s after anti-IgE challenge. We could not observe the dynamic movement of the plasma membrane after 8 min. Mast cells also released 3, 85, 98, and 58 pg of IL-5 per lo6 cells at 6 h after challenge with anti-human IgE at 0,0.15, 1.5, and 15 pg/ml, respectively (n = 2). The protein levels of IL-3 and IL-4 were

The Journal of Immunology 347

FIGURE 5. Mast cell developed in the presence of SF, IL-6, and PGE, for 16 wk. The photograph shows that the cell filled with dense scroll-type granules (A) . Higher magnification of the granules was seen in 6. A, X4,200. 6, X21,OOO.

absence of accessory cells. Several cytokines such as IL-3, IL-6, and GM-CSF have been shown to synergize with SF for prolifer- ation of hemopoietic cells (17-19, 27). Therefore, it was not sur- prising that these cytokines enhanced the SF-induced proliferation of mast cells probably by stimulating the expansion of primitive hemopoietic cells in the early stage of cell proliferation, as shown in this study. Among these cytokines, only IL-6 stimulated the expansion of hemopoietic cells without inducing marked prolifer- ation of macrophages, which are known to secrete a variety of cytokines such as GM-CSF (20, 21, 30). Subsequent secretion of GM-CSF, which may occur in the presence of exogenous GM-CSF or IL-3, would therefore accelerate the differentiation of primitive hemopoietic cells toward the GM lineage, thereby reducing the differentiation capacity of the cells toward the mast cell lineage in a prolonged period. IL-6 was also effective for proliferation of differentiated mast cells after 4 wk in culture, as well as for the expansion of undifferentiated progenitors. Indeed, we have ob- served recently that IL-3, IL-4, and IL-5 as well as IL-6 prevent apoptosis of the mast cells cultured for > I O wk (31).

FIGURE 6. Mast cell developed in the presence of SF. IL-6, and PGE, for 14 wk, showing immature granules and microvilli-like mem- brane processes (A). A crystal granule was seen in 6. A, x5,400. 6, X83,OOO.

anti-lgE ( Fglml)

FIGURE 7. IgEdependent release of histamine and TNF-a from CUI- tured mast cells. The mast cells cultured >10 wk were sensitized with 1 pg of myeloma IgE, and were challenged with either control buffer or 0.1 5 to 15 pg of anti-human IgE for 30 min (0) (histamine release) and for 6 h (0) (TNF-a release). Each point represents mean t SEM of five (TNF-o! release) or six (histamine release) separate experiments. The purity of mast cells used in this experiment was 98.6% 2 0.4% (mean 2 SEM).

It has been reported by Metcalfe et al. (7,32) that IL-3 markedly enhanced the SF-dependent mast cell growth from bone marrow- derived CD34+ cells in the early culture period. We had similar

348 CULTURED HUMAN MAST CELLS

t - 1 min

Anti-lgE (1.5 ,ug/ml)

FIGURE 8. Increase in the intracellular concentrations of Ca2+ in the cultured mast cells. The mast cells with 99.5% purity at 1 2 wk in culture were sensitized with 1 pg of myeloma IgE, and were labeled with 1 p M of fura-2 AM and suspended at 1 X 1 Ob per 1 mi of Tyrode‘s solution in a cuvette of a Ca2+ analyzer, CAF-100. The cells were then challenged with either 1.5 pg (left side) or 15 pg (right side) of anti- human IgE in the analyzer. The two fluorescent intensities were mea- sured at excitation wavelengths of 340 nm and 380 nm, and their ratio was calculated. Similar results were obtained in two other separate experiments.

results in the present study, especially in three of the seven exper- iments showing SF alone failed to induce a substantial amount of mast cells from purified CD34+ cells. In those three experiments, E - 3 seemed to be a powerful synergistic factor for the SF-induced proliferation of mast cells. In one of the seven purified CD34+ samples and in two of ten crude samples with PGE, addition, we failed to obtain a substantial number (>lo6 per sample) of mast cells even in the presence of SF and IL-6, whereas IL-3 alone ‘always induced a substantial amount of basophils (33) from the same cord blood samples (data not shown). It may be related to the number of cytokine receptors present on cord blood-derived CD34+ cells, which may be individually different. In most cases, however, the combination of L - 6 and SF would be the best way for generating highly purified mast cells in a prolonged period.

We have reported previously that PGE enhances the colony for- mation of mast cells and inhibits GM colony formation in the presence of IL-3-containing medium in murine system (23). In the present study, PGE, inhibited proliferation of adherent macro- phages by blocking GM-CSF secretion, as has been reported pre-

FIGURE 9. Membrane ballooning of the cultured mast cells following an increase in intracellular Ca*+. The mast cells at 12 wk in culture were sen- sitized with 1 pg of myeloma IgE overnight on a fi- broblast layer to which the mast cells were fixed, and weie labeled with 1 p M of fluo-3, and sus- pended in a temperature-controlled dish. The cells were then challenged with 15 pg of anti-human IgE. A single cell was scanned every 7 s by using a con- focal laser microscope, Olympus GB-200. The mast cell at 0 s ( A ) , 21 s (61, and 399 s (C) after challenge with anti-lgE was respectively shown. Relative Ca’+ concentrations were shown in the upperpartby col- oring the fluorescent intensities in the following rank order, i.e., red > orange > yellow > green > blue. In the lower part, the same cell was examined by using a phase-contrast technique. Similar results were obtained in three separate experiments, al- l though intracellular Ca*+ started to increase at var- ious intervals, from 35 s to >1 min after anti-lgE challenge. The fading of fluo-3 fluorescence was clearly found in the control cells within 5 min.

267 bp. I p-actln IL-3 IL-4 IL-5

FIGURE 10. IgE-dependent mRNA expression of cytokines. A half- million mast cells at day 150 in culture with >99.9% purity were challenged with 1.5 pgml of anti-lgE. After 60 min of incubation at 37C, the cells were treated with RNAzol B, as shown in Materials and Methods.

viously by others (29), and it enhanced the development of human mast cells. Since PGE, failed to affect mast cell development from purified CD34+ cells, and GM-CSF was never detected until day 4 in culture with CD34+ cells in the presence of SF and E-6, the enhancing effect of the lipid mediator is probably brought on by modulating cytokine secretion, especially by blocking GM-CSF secretion from accessory cells. Indeed, exogenous addition of anti- GM-CSF Ab enhanced the proliferation of mast cells in the pres- ence of SF and IL-6. IL-6 and granulocyte CSF were released from the 2 X 10’ mononuclear cord blood cells at 181 pg and 150 pg in the present study (data not shown in the results) and previous re- ports (20, 21, 29), and the addition of PGE, slightly enhanced the secretion of the cytokines. However, their concentrations were not sufficient for modulating the proliferation of mast cells.

Thus, we established a method for obtaining a large number 01 human mast cells. We were able to generate >lo7 mast cells in 2 of IO samples and 10‘ to IO’ mast cells in 6 of 10 samples with >95% purity when 3 to 8 X lo7 crude CBMC were cultured in the presence of SF at 80 ng/ml, IL-6 at 50 ng/ml, and 300 nM of PGE, for > I O wk. Similar inconstant results were obtained even by cul- turing purified CD34+ cells depending on samples. Since the num- bers of CD34’ cells, GM colony-forming cells, monocytes, and lymphocytes present in the CBMC were almost constant depend- ing on samples (data not shown), we may have to examine the

A C

The Journal of Immunology

expression of c-kit and ILdR on the CD34+ cells to define dif- ferentiation capacity toward the mast cell lineage of the cells. Hu- man mast cells developed in the presence of SF alone often exert somewhat immature functional properties (9, 10). The lung mast cells are known to possess 2 to 3.9 p g of histamine and 11 p g of tryptase per 10" cells (34, 35). The mast cells cultured in the present method seemed to be functionally mature, because 1) they contained 5.62 pg of histamine and 3.46 pg of tryptase per lo6 cells; 2) the cultured mast cells had IgE receptors (36), and the cells sensitized with 1 pglml of human IgE released 52.9% of the histamine content, 693 ng LTC, (28), 1.76 ng TNF-a, and 98 pg IL-5 per 10" cells when challenged with 1.5 pg/ml anti-human IgE, whereas the control cells spontaneously released 3.7% hista- mine, 9 ng LTC,, 0.18 ng TNF-a, and 3 pg IL-5; and 3) the re- actions were accompanied by an increase in intracellular Ca2+. By using a confocal laser-scanning microscope, some parts of the plasma membrane were found to have ballooned outward follow- ing the IgE-dependent increase in intracellular Ca2+. The mem- brane ballooning was started from 3 min and finished until 8 min after anti-IgE challenge, and its time course was similar with that of histamine release (34), suggesting that the partial swelling of the membrane reflects IgE-dependent degranulation of human mast cells.

The cultured mast cells were positively stained for anti-tryptase and anti-chymase Abs at 99 and 18% (28), respectively. In an electron-microscopic analysis, some of the cells nearly filled with scroll-type granules that are seen frequently in lung mast cells (37). Crystal granules, which are seen frequently in human skin mast cells and in some of the cultured mast cells developed in coculture system with 3T3 fibroblasts (6), were also detectable. These results suggest that the cultured mast cells developed in SF plus IL-6 are morphologically more mature than those developed in SF alone, which are reported to have only incomplete condensation of gran- ule materials (lo), and are a mixture of tryptase- and chymase- positive skin-type mast cells and tryptase-positive lung-type mast cells (35).

Since it is hard to obtain lo7 purified mast cells from human tissues, analyses requiring a large number of pure cells, such as intracellular mechanisms or cytokine production, have not been intensively examined. Contaminated monocytes may produce a va- riety of cytokines when they adhere to plastic flasks (21, 29), and basophils may produce IL-4, as has been reported (38). In the present experiment for cytokine production, cells consisting of >99.9% mast cells and 1 ng of TNF-a per lo6 cells, which amount is enough for eosinophil transendothelial migration (41). A substantial amount of IL-5 was also detected. We were able to show that the cultured mast cells expressed IL-5 mRNA after challenge with anti-IgE. In the same samples, however, mRNA expression of IL-3 and IL-4 was not detected. As has been reported elsewhere (28), the cultured mast cells expressed a series of surface CD molecules in keeping with human mast cells in vivo (42,43), except CD13, CD14, and CD38 present on the cultured cells. Taken together, the present method for generating human mast cells should greatly facilitate investi- gation of the role of mast cells in human allergic disorders, espe- cially when such studies require large numbers of pure mast cells.

349

Acknowledgments We thank Drs. Kimishige Ishizaka and Teruko Ishizaka (La Jolla Institute for Allergy and Immunology, L a Jolla, CA) for their critical reviewing of the manuscript. We are grateful to Dr. Shigenobu Shoda (Department of Obstetrics, Gyoda Chuo Hospital, Saitama, Japan) for his continuous sup- port by generously providing the umbilical cord blood. We also thank Dr. Naoya Sakaguchi, Dr. Katsushi Miura. Dr. Akira Akasawa, Mr. Takashi Numazaki, and Mr. Masahiro Kimata (Division of Allergy, National Chil- dren's Medical Research Center, Tokyo, Japan) for their advice on the manuscript and for excellent technical suppon for the experiments.

References 1.

2.

3.

4.

5.

6.

7.

8

9

10.

1 I .

12.

13.

14.

15.

16.

17.

18.

Ishizaka, T., and K. Ishizaka. 1984. Activation of mast cells for mediator release through IgE receptors. In Progress in Allergy, Vol. 34. P. Kallos, ed. Karger AG, Basel, pp. 188-235. Plaut, M., J. H. Pierce, C. J. Watson, J. Hanley-Hyde, R. P. Nordan, and W. E. Paul. 1989. Mast cell lines produce lymphokines in response to cross-linkage of FcERI or to calcium ionophores. Nature 339:64. Katz, H. R., R. L. Stevens, and K. F. Austen. 1984. Heterogeneity of mammalian mast cells differentiates in vivo and in vitro. J. Allergy Clin. lmmunol. 78:250. Bienenstock, J., D. Befus, J. Denburg. T. Goto, T. Lee, H. Otsuka, and

species and sites. lnt. Arch. Allergy Appl. fmmunot. 77.126. F. Shanahan. 1985. Comparative aspects of mast cell heterogeneity in different

Ihle, J. N., J. Keller, S. Oronszlan, L. E. Henderson, T. D. Copeland, R. Fitch, M. B. Prystowsky, E. Goldwasser, J. W. Schrader, E. Paranszynski, M. Dy, and B. Lehel. 1983. Biologic properties of homogeneous interleukin 3.:1. Demon- stration of WEHI-3 growth factor activity, mast cell growth factor activity, P cell-stimulating factor activity, colony-stimulating factor activity and histamine- producing cell-stimulating factor activity. J. lmmunol. 131:282. Furitsu, T., H. Saito, A. M. Dvorak, L. B. Schwartz, A.M. A. Irani, J. F. Burdick, K. Ishizaka, and T. Ishizaka. 1989. Development of human mast cells in vitro. Proc. Nutl. Acad. Sci. USA 86:10039. Kirshenbaum, A. S., J. P. Goff, S . W. Kessler, J. M. Mican, K. M. Zsebo, and D. D. Metcalfe. 1992. Effect of IL-3 and stem cell factor on the appearance of human basophils and mast cells from CD34+ pluripotent progenitor cells. J. lm- munol. 148:772. Valent. P., E. Spanblochl, W. R. Sperr, C. Sillaber, K. M. Zsebo. H. Agis,

entiation of human mast cells from bone marrow and peripheral blood mononu- H. Strobl, K. Geissler, P. Bettelheim, and K. Lechner. 1992. Induction of differ-

clear cells by recombinant human stem cell factorlkit-ligand in long-term culture. Blood 110.2237. Irani, A. M. A., G. Nilsson, U. Miettinen. S . S. Craig, L. K. Ashman, T. Ishizaka, K. M. Zsebo, and L. B. Schwartz. 1992. Recombinant human stem cell factor

Blood 80:3009. stimulates differentiation of mast cells from dispersed human fetal liver cells.

Mitsui, H., T. Furitsu, A. M. Dvorak, A.M. A. Irani, L. B. Schwartz, N. Inagaki, M. Takei, K. Ishizaka, K. M. Zsebo, S. Gills, and T. Ishizaka. 1993. Development of human mast cells from umbilical cord blood cells by recombinant human and murine c-kit ligand. P roc. Nutl. Acud. Sci. USA 90.735. Zsebo, K. M., D. A. Williams, E. N. Geissler, V. C. Broudy, F. H. Martin, H. L. Atkins, R. Y. Hsu, N. C. Birkett, K. H. Okino, D. C. Murdock, F. W. Jacobsen,

Suggs. 1990. Stem cell factor is encoded at the SI locus of the mouse and is the K. E. Langley, K. A. Smith, T. Takeishi, B. M. Cattanach, S. J. Galli, and S. V.

ligand for the c-kit tyrosine kinase receptor. Cell 63:213. Copeland, N. G., D. J. Gilbert, B. C. Cho, P. J. Donovan, N. A. Jenkins.

growth factor maps near the steel locus on mouse chromosome 10 and is deleted D. Cosman, D. Anderson, S. D. Lyman, and D. E. Williams. 1990. Mast cell

in a number of steel alleles. Cell 63:175. Nocka, K., J. Buck, E. Levi. and P. Besmer. 1990. Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors. EMBO J. 9.3287. Broxmeyer, H. E., L. Lu. G. Hangoc, S. Cooper, P. C. Hendrie, J. A. Ledbetter, M. Xiao, D. E. Williams, and F. W. Shen. 1991. CD45 cell surface antigens are linked to stimulation of early human myeloid progenitor cells by interleukin 3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), a GM- CSFiIL-3 fusion protein, and mast cell growth factor (a c-kit ligand). J. Exp. Med. 1741447. Tsuji, K., K. M. Zsebo, and M. Ogawa. 1991. Enhancement of murine blast cell colony formation in culture by recombinant rat stem cell factor, a ligand for c-kit. Blood 78,1223. Migliaccio, G., A. R. Migliaccio, M. L. Druzin, P. J. Giardina, K. M. Zseho. and J. W. Adamson. 1991. Effect of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro. J. Cell. Physiol. 148:503. Bernstein, I. D., R. G. Andrew, and K. M. Zseho. 1991. Recombinant human stem cell factor enhances the formation of colonies by CD34+ and CD34+ lin- cells cultured with interleukin-3, granulocyte colony-stimulating factor, or gran- ulocyte-macrophage colony-stimulating factor. Blood 77.2316. Brandt, J., R. A. Briddell, E. F. Srour, T. B. Leemhuis. and R. Hoffman. 1992. Role of c-kit ligand in the expansion of human hematopoietic progenitor cells. Blood 79.634.

350 CULTURED H U M A N MAST CELLS

19.

20.

21.

22.

23.

24.

25.

26.

21.

28.

29.

30.

31.

Brugger, W., W. Mocklin, S. Heimfeld, R. J. Berenson, R. Mertelsmann, and L. Kanz. 1993. Ex vivo expansion of enriched peripheral blood CD34+ progen- itor cells by stem cell factor, interleukin-lp ( L I P ) , IL-6, IL-3, interferon-y, and erythropoietin. Blood 81.2579. Guba, S . C., C. I. Sartre, L. R. Gottschalk, Y. H. Jing, T. Mulligan, and S. G. Emerson. 1992. Bone marrow stromal fibroblasts secrete interleukin-6 and gran- ulocyte-macrophage colony-stimulating factor in the absence of inflammatory stimulation: demonstration by serum-free bioassay, enzyme-linked immunosor- bent assay, and reverse transcriptase polymerase chain reaction. Blood 80:llYO. Ishiguro, A,, T. Nakahata. K. Koike, H. Yoshida, T. Shimbo, and A. Komiyama.

factors from human monocytes stimulated by Fc fragments of human IgG. 1991. Induction of granulocyte and granulocyte-macrophage colony-stimulating

Br. J. Haematol. 79114. Nakahata, T., K. Tsuji, R. Tanaka. K. Muraoka, N. Okumura, N. Sawai, M. Takagi, S. Itoh, C. Ra, and H. Saito. 1995. Synergy of stem cell factor and other cytokines in mast cell development. In Biological and Molecular Aspects of Mast Cell and Basophil Dlferentiation and Function. Y. Kitamura, S. Yamamoto, and S. J. Galli, eds. Elsevier Science Publishers, New York, pp. 13-24. Saito, H., Y. Sanai, and Y. Nagai. 1985. Cholera toxin enhances factor-dependent colony growth of murine mast cell progenitors. Exp. Hematol. 13.261. Craig, S. S., G. DeBlois, and L. B. Schwartz. 1986. Mast cells in human keloid, small intestine, and lung by an immunoperoxidase technique using a murine monoclonal antibody against tryptase. Am. J . Pathol. 124r427. Saito. H., N. Sakaguchi, M. Ebisawa, K. Matsumoto. A. Akasawa, and Y. Iikura.

cells. 11. Mechanisms involved in histamine release induced by extracellular ATP 1991. The stimuli releasing histamine from murine bone marrow-derived mast

and its metabolites. Jpn. J. Allergol. 401680. Takahashi, G. 1990. Tannin-ferrocyanide-OsO, method for scanning electron microscopy with use of microwave irradiation. Elecrron Microsc. 3126. Miura, N., S . Okada, K. M. Zsebo, Y. Miura, and T. Suda. 1993. Rat stem cell factor and IL-6 preferentially support the proliferation of c-kit-positive murine hemopoietic cells rather than their differentiation. Exp. Hematol. 21.143. Saito, H.. M. Ebisawa, N. Sakaguchi, T. Onda, Y. Iikura, M. Yanagida, H. Uzumaki, and T. Nakahata. 1995. Characterization of human mast cells cul- tured in the presence of sreel factor and interleukin 6. in?. Arch. Allergy Appl. Immunol. 107.63. Saito, H., N. Sakaguchi, K. Matsumoto, T. Tsubaki, T. Numazaki, M. Ebisawa, M. Kobayashi, R. Shoji, H. Yanagi, A. Akasawa, and Y. Iikura. 1994. Growth in methylcellulose of human mast cells in hematopoietic colonies stimulated by steel factor, a c-kit ligand. int. Arch. Allergy Appl. immunol. 1031143. Lee, M. T., K. Kaushansky. P. Ralph, and M. B. Ladner. 1990. Differential expression of M-CSF, G-CSF, and GM-CSF by human monocytes. J. Leukocyte B id . 471275. Yanagida, M., H. Fukamachi, K. Ohgami, T. Kuwaki, H. Ishii, H. Uzumaki, K. Amano, T. Tokiwa, H. Mitsui, H. Saito, Y. Iikura, T. Ishizaka, and

T. Nakahata. 1995. Effect of T helper 2-type cytokines, interleukin (IL)-3, IL-4. IL-5, and IL-6 on the surv~val of cultured human mast cells. Blood 8613705.

32. Rottem, M., T. Okada, J. P. Goff, and D. D. Metcalfe. 1994. Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis orig- inate from a CD34+/FceRI-cell population. Blood 84.2489.

33. Ebisawa, M., H. Saito, D. C. Reason, N. Sakaguchi, T. Katsunuma, and Y. Iikura.

histamine release from IL-3-dependent cultured basophils. In [ . Arch. AllerRv 1991. Changes in filament actin accompanying IgE-dependent and -independent

Appl. lmmunol. 94: 71. 34. Lavens, S. E., D. Proud, and 1. A. Warner. 1993. A sensitive colorimetric asaoy

for the release of tryptase from human lung mast cells in vitro. J. lmmunol. Methods 166193.

35. Schwartz, B. 1990. Human mast cell neutral protease,: markers of mast cell heterogeneity and function. In Progress in Allergy and Clinical Immunology. W. J. Pichler, B. M. Stadler, C. A. Dahinden, A. R. Pecoud, P. Frei, C. H. Schneider, and A. L. de Weck, eds. Hogrefe & Huber Publishers, Tront, pp. 1-5.

36. Igarashi, Y.. M. Kurosawa, 0. Ishikawa, Y. Miyachi, H. Saito, M. Ebisawa, Y. Iikura, M. Yanagida, H. Uzumaki, and T. Nakahata. 1995. Characteristics of histamine release from human cultured mast cells. Ctin. Exp. Allergy. In press.

37. Dvorak, A. M., T. Furitsu, S. Kissell-Rainville, and T. Ishizaka. 1992. Ultra- structural identification of human mast cells resembling skin mast cells stimulated to develop in long-term human cord blood mononuclear cells cultured with 3T3 murine skin fibroblasts. J. Leukocyte B id . 51.557.

38. Brunner, T., C. H. Heusser, and C. A. Dahinden. 1993. Human peripheral blood

globulin E receptor stimulation. J. Exp. Med. 177:605. basophils primed by interleukin 3 (IL-3) produce IL-4 in response to immuno-

39. Bradding, P., 1. H. Feather. P. H. Howarth, R. Mueller. J. A. Roberts, K. Britten. J. P. A. Bews, T. C. Hunt, Y. Okayama, C. H. Heusser, G. R. Bullock, M. K. Church, and S. T. Holgate. 1992. Interleukin 4 is localized to and released by human mast cells. J. Exp. Med. 176.1381.

40. Bradding, P., Y. Okayama, P. H. Howarth, M. K. Church, and S. T. Holgate. 1995. Heterogeneity of human mast cells based on cytokine content. J. Immunol. 1551297.

41. Ebisawa, M., B. S. Bochner, S. N. Georas, and R. P. Schleimer. 1992. Eosinophil transendothelial migration induced by cytokines. 1. Role of endothelial and eo-

munol. 149.4021. sinophil adhesion molecules in IL-lp-induced transendothelial migration. J. Im-

42. Guo, C. B., A. Kagey-Sobotka, L. M. Lichtenstein, and B. S. Bochner. 1992. Immuno-phenotyping and functional analysis of purified human uterine mast cells. Blood 79.708.

43. Valent, P., 0. Majdic, D. Maurer, M. Bodger, M. Muhm, and P. Bettelheim. 1990. Further characterization of surface membrane structures expressed on hu- man basophils and mast cells. lnt. Arch. Allergy Appl. lmmunol. 911198.