Proc. Natl. Acad. Sci. USAVol. 88, pp. 6382-6386, July
1991Medical Sciences
Induction of mast cell proliferation, maturation, and
heparinsynthesis by the rat c-kit ligand, stem cell factor
(developmental biology/W and Si loci/cell-cell
interaction/histamine/interleukin 3)
MINDY TSAI*, TAKASHI TAKEISHI*, HELEN THOMPSONt, KEITH E.
LANGLEY*, KRISZTINA M. ZSEBOt,DEAN D. METCALFEt, EDWIN N.
GEISSLER*, AND STEPHEN J. GALLI*§*Departments of Pathology, Beth
Israel Hospital and Harvard Medical School, and Division of
Experimental Pathology, Beth Israel Hospital, Boston, MA02215;
tMast Cell Physiology Section, Laboratory of Clinical
Investigation, National Institute of Allergy and Infectious
Diseases, National Institutes ofHealth, Bethesda, MD 20892; and
tAmgen, Inc., Amgen Center, Thousand Oaks, CA 91320
Communicated by Elizabeth S. Russell, April 22, 1991
ABSTRACT We investigated the effects of a newly recog-nized
multifunctional growth factor, the c-kit ligand stem cellfactor
(SCF), on mouse mast cell proliferation and phenotype.Recombinant
rat SCF'64 (rrSCF"64) induced the developmentof large numbers of
dermal mast cells in normal mice in vivo.Many ofthese mast cells
had features of "connective tissue-typemast cells" (CTMC), in that
they were reactive both with theheparin-binding fluorescent dye
berberine sulfate and withsafranin. In vitro, rrSCFI64 induced the
proliferation of clonedinterleukin 3 (IL-3)-dependent mouse mast
cells and primarypopulations of IL-3-dependent, bone marrow-derived
culturedmast cells (BMCMC), which represent immature mast cells,and
purified peritoneal mast cells, which represent a type ofmature
CTMC. BMCMC maintained in rrSCF164 not onlyproliferated but also
matured. Prior to exposure to rrSCF'"4,the BMCMC were alcian blue
positive, safranin negative, andberberine sulfate negative; had a
histamine content of 0.1)8 ±0.02 pg per cell; and incorporated
[35S~sulfate into chondroitinsulfates. After 4 wk in rrSCF'64, the
BMCMC were predom-inantly safranin positive and berberine sulfate
positive, had ahistamine content of 2.23 ± 0.39 pg per cell, and
synthesized35S-labeled proteoglycans that included substantial
amounts(41-70%) of [35S]heparin. These rmdings identify SCF as
asingle cytokine that can induce immature, IL-3-dependent mastcells
to mature and to acquire multiple characteristics ofCTMC. These
findings also directly demonstrate that SCF canregulate the
development of a cellular lineage expressing c-kitthrough effects
on both proliferation and maturation.
Mast cell development is a complex process that results in
theappearance of phenotypically distinct populations of mastcells
in different anatomical sites (reviewed in ref. 1). Con-nective
tissue-type mast cells (CTMC), such as those presentin the skin and
peritoneal cavity, represent a major mast cellpopulation in the
mouse (1). Mouse CTMC exhibit cytoplas-mic reactivity with safranin
and with the fluorescent heparin-binding dye berberine sulfate,
have a relatively high hista-mine content, and incorporate
[35S]sulfate into [35S]heparin(reviewed in refs. 1-4). By contrast,
immature mast cells,such as interleukin 3 (IL-3)-dependent bone
marrow-derivedcultured mast cells (BMCMC), do not exhibit
cytoplasmicreactivity with safranin or berberine sulfate, have a
lowhistamine content, and incorporate [35S]sulfate predomi-nantly
into chondroitin sulfates (reviewed in refs. 1-4). It hasbeen
demonstrated that BMCMC can mature and acquiremultiple phenotypic
characteristics of CTMC either in vivo(5) or when cocultured with
3T3 cells in vitro (6). Neverthe-less, the signals that regulate
the maturation of immaturemast cells into CTMC are not fully
understood.
Several lines ofevidence indicate that interactions
betweenproducts of the W and SI loci on mouse chromosomes 5 and10
importantly influence CTMC development. Mice homozy-gous for
mutations at these loci exhibit several strikingphenotypic
abnormalities including a macrocytic anemia (7,8) and a virtual
absence of tissue mast cells (9, 10). Both invivo and in vitro
studies indicate that W mutations producedefects intrinsic to the
erythroid and mast cell lineages, whileSI mutations impair the
tissue microenvironment required fornormal erythroid and mast cell
development (reviewed inrefs. 8-10). For example, IL-3-dependent
BMCMC of(WBB6)F1 +/+ mouse origin developed into CTMC
whenadoptively transferred to appropriate anatomical sites, suchas
the skin or peritoneal cavity, of genetically mast cell-deficient
(WBB6)F1 WIW' mice (5). By contrast, normalBMCMC failed to survive
or develop into CTMC in the skinof genetically mast cell-deficient
(WCB6)F1 S1S1d mice (11).In vitro studies showed that fibroblasts
derived from normalor (WBB6)F1 WIWv mice supported the survival and
prolif-eration of normal BMCMC in vitro, whereas fibroblastsderived
from (WCB6)F1 S1S1d mice did not (reviewed in ref.9). Moreover,
mast cells developed on membranes that hadbeen covered with
(WCB6)F1 +/+ 3T3 fibroblasts, but noton membranes covered with
(WCB6)F1 S1/S"d 3T3 fibro-blasts, after introduction of these
membranes into the peri-toneal cavity of SIIS1d mice (9,
12).Products of the W or SI loci that influence mast cell
development have recently been identified. The W locusencodes
the c-kit tyrosine kinase receptor (13, 14), whereasSl encodes a
c-kit ligand (15-23), which we have designatedstem cell factor
(SCF) (18-20, 23). Three groups demon-strated independently that
recombinant SCF (18, 19) or otherexamples of this c-kit ligand (15,
21, 22, 24) can induceproliferation of certain populations of mouse
mast cells invitro, and we showed that subcutaneous injection of
therecombinant rat factor, rrSCF'1, permits mast cells to de-velop
in genetically mast cell-deficient (WCB6)F1 S1S1d micein vivo (20).
However, it was not determined whether SCFcould induce the
development of dermal mast cells in normalmice. Nor was it known
whether SCF influenced mast cellmaturation as well as
proliferation.
MATERIALS AND METHODSCells. Mouse mast cell populations were
derived, main-
tained, or purified (peritoneal mast cells, PMC) as
described
Abbreviations: SCF, stem cell factor; rrSCFlM, recombinant
ratSCF164; CTMC, connective tissue-type mast cells; IL-3,
interleukin3; BMCMC, bone marrow-derived cultured mast cells; PMC,
peri-toneal mast cells; Hct, hematocrit.§To whom reprint requests
should be addressed at: Division ofExperimental Pathology,
Department of Pathology, Research East,Beth Israel Hospital, 330
Brookline Avenue, Boston, MA 02215.
6382
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 88 (1991)
phenotypic characteristics that are different than those
typ-ical of the resting populations (reviewed in refs. 1 and
35).The mechanisms by which SCF influences mast cell pro-
liferation and maturation remain to be fully elucidated.
Ourexperiment with (WBB6)F1 WIW" BMCMC confirms a pre-vious report
(24) indicating that the effects of SCF on mastcell proliferation
in vitro require that the c-kit ligand interactswith a functionally
competent c-kit receptor. However, thesubsequent events leading to
mast cell proliferation and/ormaturation are not known. By itself,
SCF has relativelymodest effects on the proliferation of
hematopoietic or lym-phoid cells in vitro, but it acts in potent
synergy with othercytokines to influence the development of
specific erythroid,myeloid, or lymphoid lineages (18-20, 22). We
have obtainedpreliminary evidence that rrSCF164 can activate
certainmouse mast cell populations to release their mediators
(37).Although the concentrations of rrSCFlM required to
observethese effects in vitro are substantially higher than
thoserequired to induce proliferation of the same cells,
activationof dermal mast cells in vivo is observed at rrSCF164
doseseven lower than those we used to induce expansion of
thispopulation. Activation of mast cells generated in vitro,
eithervia the FceRI or by other mechanisms, induces these cells
todevelop increased levels of mRNA for several cytokinesand/or to
secrete the products (25, 38-40). Some of thesecytokines-e.g., IL-3
and IL-4-can promote or augmentmast cell proliferation (reviewed in
refs. 38-42). In light ofthese findings, it will be of interest to
determine whether oneof the actions of SCF is to induce mast cells
to generate othercytokines with autocrine effects on mast cell
proliferationand/or maturation.
We thank Dr. Li Sun Shih and Ms. Lisa Fox for help. This workwas
supported in part by Public Health Service Grants AI22674,A123990,
CA28834, and GM45311, and by Amgen, Inc.
1. Galli, S. J. (1990) Lab. Invest. 62, 5-33.2. Galli, S. J.,
Dvorak, A. M., Marcum, J. A., Ishizaka, T.,
Nabel, G., Der Simonian, H., Pyne, K., Goldin, J. M.,
Rosen-berg, R. D., Cantor, H. & Dvorak, H. F. (1982) J. Cell
Biol. 95,435-444.
3. Bland, C. E., Ginsburg, H., Silbert, J. E. & Metcalfe, D.
D.(1982) J. Biol. Chem. 257, 8661-8666.
4. Razin, E., Stevens, R. L., Akiyama, F., Schmid, K. &
Austen,K. F. (1982) J. Biol. Chem. 257, 7229-7236.
5. Nakano, T., Sonoda, T., Hayashi, C., Yamatodani, A.,Kanayama,
Y., Yamamura, T., Asai, H., Yonezawa, Y.,Kitamura, Y. & Galli,
S. J. (1985) J. Exp. Med. 162, 1025-1043.
6. Levi-Schaffer, F., Austen, K. F., Gravallese, P. M. &
Stevens,R. L. (1986) Proc. Nati. Acad. Sci. USA 83, 6485-6488.
7. Sarvella, P. A. & Russell, E. S. (1956) J. Hered. 47,
123-128.8. Russell, E. S. (1979) Adv. Genet. 20, 357-459.9.
Kitamura, Y., Nakayama, H. & Fujita, J. (1989) in Mast Cell
and Basophil Differentiation and Function in Health and
Dis-ease, eds. Galli, S. J. & Austen, K. F. (Raven, New York),
pp.229-246.
10. Galli, S. J., Geissler, E. N., Wershil, B. K., Gordon, J. R.
&Tsai, M. (1991) in The Role of the Mast Cell in Health
andDisease, eds. Kaliner, M. A. & Metcalfe, D. D. (Dekker,
NewYork), in press.
11. Gordon, J. R. & Galli, S. J. (1990) Blood 75,
1637-1645.12. Fujita, J., Onoue, H., Ebi, Y., Nakayama, H. &
Kanakura, Y.
(1989) Proc. Natl. Acad. Sci. USA 86, 2888-2891.13. Chabot, B.,
Stephenson, D. A., Chapman, V. M., Besmer, P.
& Bernstein, A. (1988) Nature (London) 335, 88-89.14.
Geissler, E. N., Ryan, M. A. & Housman, D. E. (1988) Cell
55,
185-192.15. Williams, D. E., Eisenman, J., Baird, A., Rauch, C.,
Van Ness,
K., March, C. J., Park, L. S., Martin, U., Mochizuki, D.
Y.,Boswell, H. S., Burgess, G. S., Cosman, D. & Lyman, S.
D.(1990) Cell 63,X167-174.
16. Copeland, N. G., Gilbert, D. J., Cho, B. C., Donovan, P.
J.,Jenkins, N. A., Cosman, D., Anderson, D., Lyman, S. D.
&Williams, D. E. (1990) Cell 63, 175-183.
17. Flanagan, J. G. & Leder, P. (1990) Cell 63, 185-194.18.
Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith,
K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V.
N.,Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung,
W.,Bosselman, R. A., Mendiaz, E. A. & Langley, K. E. (1990)Cell
63, 195-201.
19. Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S.,
Ting,J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen,F.
W., Mendiaz, E. A., Birkett, N. C., Smith, K. A., Johnson,M. J.,
Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F.,Edjavec,
H. O., Herrera, C. J., Wypych, J., Sachdev, R. K.,Pope, J. A.,
Leslie, I., Wen, D., Lin, C.-H., Cupples, R. L. &Zsebo, K. M.
(1990) Cell 63, 203-211.
20. Zsebo, K. M., Williams, D. A., Geissler, E. N., Broudy,V.
C., Martin, F. H., Atkins, H. L., Hsu, R.-Y., Birkett,N. C., Okino,
K. H., Murdock, D. C., Jacobsen, F. W., Lang-ley, K. E., Smith, K.
A., Takeishi, T., Cattanach, B. M., Galli,S. J. & Suggs, S. V.
(1990) Cell 63, 213-224.
21. Huang, E., Nocka, K., Beier, D. R., Chu, T.-Y., Buck,
J.,Lahm, H.-W., Wellner, D., Leder, P. & Besmer, P. (1990)
Cell63, 225-233.
22. Anderson, D. M., Lyman, S. D., Baird, A., Wignall, J.
M.,Eisenman, J., Rauch, C., March, C. J., Boswell, H. S., Gim-pel,
S. D., Cosman, D. & Williams, D. E. (1990) Cell 63,235-243.
23. Matsui, Y., Zsebo, K. M. & Hogan, B. L. M. (1990)
Nature(London) 347, 667-669.
24. Nocka, K., Buck, J., Levi, J. & Besmer, P. (1990) EMBO
J. 9,3287-3294.
25. Gordon, J. R. & Galli, S. J. (1990) Nature (London)
346,274-276.
26. Pluznik, D. H., Tarem, N. S., Zatz, M. M. & Goldstein,
A. L.(1982) Exp. Hematol. 10 (Suppl. 12), 211-216.
27. Galli, S. J., Arizono, N., Murakami, T., Dvorak, A. M. &
Fox,J. G. (1987) Blood 69, 1661-1666.
28. Enerback, L. (1974) Histochemistry 42, 301-313.29. Arizono,
N., Koreto, O., Nakao, S., Iwai, Y., Kushima, R. &
Takeoka, 0. (1987) Virchows Arch. B. 54, 1-7.30. Shore, P. A.
(1971) in Methods ofBiochemical Analysis: Anal-
ysis ofBiogenicAmines and TheirRelatedEnzymes, eds. Glick,D.
(Wiley, New York), pp. 89-110.
31. Kanakura, Y., Thompson, H., Nakano, T., Yamamura,
T.-i.,Asai, H., Kitamura, Y., Metcalfe, D. D. & Galli, S. J.
(1988)Blood 72, 877-885.
32. Thompson, H. L., Schulman, E. S. & Metcalfe, D. D.
(1988) J.Immunol. 140, 2708-2713.
33. Nocka, K., Tan, J., Chiu, E., Chu, T. Y., Ray, P.,
Traktman,P. & Besmer, P. (1990) EMBO J. 9, 1805-1813.
34. Sredni, B., Friedman, M. M., Bland, C. E. & Metcalfe, D.
D.(1983) J. Immunol. 131, 915-922.
35. Nakahata, T., Kobayashi, T., Ishiguro, A., Tsuji, K.,
Na-ganuma, K., Ando, O., Yagi, Y., Tadokoro, K. & Akabane,
T.(1986) Nature (London) 324, 65-67.
36. Hamaguchi, Y., Kanakura, Y., Fujita, J., Takeda, S.-I.,
Na-kano, T., Tarui, S., Honjo, T. & Kitamura, Y. (1987) J.
Exp.Med. 165, 268-273.
37. Galli, S. J., Tsai, M. T., Langley, K. E., Zsebo, K. M.
&Geissler, E. N. (1991) FASEB J. 5, A1092 (abstr.).
38. Plaut, M., Pierce, J. H., Watson, C. J., Hanley-Hyde,
J.,Nordan, R. P. & Paul, W. E. (1989) Nature (London)
339,64-67.
39. Wodnar-Filipowicz, A., Heusser, C. H. & Moroni, C.
(1989)Nature (London) 339, 150-152.
40. Burd, P. R., Rogers, H. W., Gordon, J. R., Martin, C.
A.,Jayaraman, S., Wilson, S. D., Dvorak, A. M., Galli, S. J.
&Dorf, M. E. (1989) J. Exp. Med. 170, 245-257.
41. Galli, S. J., Wershil, B. K., Gordon, J. R. & Martin, T.
R.(1989) in IgE, Mast Cells and the Allergic Response,
CibaFoundation Symposium No. 147, eds. Chadwick, D., Evered,D.
& Whelan, J. (Wiley, Chichester, U.K.), pp. 53-73.
42. Gordon, J. R., Burd, P. R. & Galli, S. J. (1990)
Immunol.Today 11, 458-464.
6386 Medical Sciences: Tsai et al.
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