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.

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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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in detail (2, 25, 26). Briefly, PT-18 is an IL-3-dependent cellline derived from C3H.SW mouse spleen cells (26). IL-3-dependent BMCMC were derived from the femoral bonemarrow cells ofBALB/c mice, genetically mast cell-deficient(WBB6)F1 W/W' mice, or the congenic normal (WBB6)Fl+/+ mice-i.e., [WB/ReJ(W/+) x C57BL/6J(WV/+)]F1WIW' +/+ mice (The Jackson Laboratory)-were main-tained in IL-3-containing conditioned medium (CM), consist-ing of Dulbecco's modified Eagle's medium with 10%6 heat-inactivated fetal bovine serum, 50 AtM 2-mercaptoethanol,and 2 mM glutamine (complete medium) supplemented with20% (vol/vol) supernatants of concanavalin A-activatedspleen cells as described (2, 25). PMC were purified from theperitoneal cavities of retired breeder BALB/c mice (TheJackson Laboratory) as described (25). Mast cells werecounted after staining with neutral red (5).SCF. In vitro experiments were performed with rrSCF1M

purified from Escherichia coli as described in detail (19). Invivo experiments were performed with E. coli-derivedrrSCFL64, which was modified by the covalent attachment ofpolyethylene glycol (20).

Effect of SCF on Dermal Mast Cells in Vivo. Groups of fivefemale 8- to 12-wk-old (WCB6)F1 +/+ mice-i.e., [WB/ReJ(Sl+) x C57BL/6J(Sld/+)]Fl +/+ mice (The JacksonLaboratory)-recieved for 3 wk a daily subcutaneous (s.c.)injection ofrrSCF1M [30 or 10 ,ug/kg in 0.20-0.25 ml of sterile0.9% NaCl containing 0.1% bovine serum albumin (fractionV, fatty acid free; ICN)] or vehicle alone as described (20).Blood for determination of hematocrit (Hct) was obtained byretroorbital puncture under light ether anesthesia on the daybefore initiation of treatment and on the day of sacrifice.After sacrifice by cervical dislocation, one portion of thecutaneous injection site was processed for preparation ofGiemsa-stained Epon-embedded sections (1 ,um) for quanti-fication of mast cells in an area of dermis including thepanniculus carnosus (-"0.6 mm2) as described (27), whereasanother portion was fixed in Carnoy's fixative and processedfor staining with the fluorescent, heparin-binding dye ber-berine sulfate (5, 28) or alcian blue/safranin (5).

Stimulation ofMast Cell Proliferation and/or Maturation inVitro. PT-18 cells or BMCMC were washed free of CM andresuspended at 4.0 x l0- cells per ml in complete medium,which lacks IL-3 or other cytokines, for 18 h. The cells werethen washed and placed in 96-well plates at 5.0 x 103 cells perwell in 0.2 ml of complete medium. rrSCF1M was then added,to the final concentrations indicated, for 24 h, after which[3H]thymidine (6.7 Ci/mmol; 1 Ci = 37 GBq; NEN) wasadded to a final activity of 2.0 ,Ci/ml. Four hours later,cultures were collected onto glass filter strips and assayed byliquid scintillation counting. Purified PMC were immediatelyplaced in 96-well plates at 5.0 x 103 cells per well in 0.2 mlof complete medium containing various concentrations ofrrSCFl64, or in 0.2 ml of CM containing IL-3, for 5 days.Incorporation of [3H]thymidine over a 4-h period was thendetermined as described above. In each experiment, somewells contained cells in only complete medium (negativecontrol) or IL-3-containing CM (as described above). Toidentify proliferating cells in these preparations, cells werepermitted to incorporate 5-bromodeoxyuridine (3 ,ug/ml;Boehringer Mannheim) over a 30-min incubation period at37°C, and then cytocentrifuge preparations were processedfor colocalization of nuclear staining with a monoclonalantibody to 5-bromodeoxyuridine (Becton Dickinson), usedaccording to the supplier's instructions, and mast cell cyto-plasmic granules were stained with alcian blue (29).For assessment of effects of SCF on mast cell phenotype,

BMCMC or purified PMC were maintained in completemedium supplemented with rrSCFlM (50 ng/ml, replaced twoor three times weekly) for 2-6 wk. Some BMCMC were alsomaintained in IL-3-containing CM. For assessment of histo-

chemical characteristics, mast cells were analyzed in cyto-centrifuge preparations fixed in Carnoy's fixative for stainingwith berberine sulfate and in air-dried cytocentrifuge prepa-rations stained with alcian blue and safranin (5). Mast cellhistamine content was measured by the fluorometric methodof Shore (30). To assess the content of 35S-labeled proteo-glycans, aliquots of 2.0 x 106 mast cells in CM or rrSCFM4-containing medium were labeled overnight with 300 gCi ofNa235SO4 (482 Ci/mmol; NEN) per 1.0 x 106 cells per ml. Thecells were then washed three times in complete medium andfrozen at -80'C. For analysis of 35S-labeled proteoglycans,specimens were thawed, sonicated for 20 sec, centrifuged at10,000 x g for 10 min to remove cell debris, and chromato-graphed on a PD-10 gel filtration column under dissociativeconditions to remove free [35S]sulfate. 35S-labeled proteogly-cans appearing in the excluded volume were extracted,isolated, characterized, and identified by their susceptibilityto degradation by treatment with NaOH and nitrous acid,purified heparinase, or chondroitin ABC lyase as described indetail (31, 32).

RESULTSrrSCF64 Induces the Development of Dermal Mast Cells in

Vivo. rrSCF164 greatly expanded the numbers of dermal mastcells in normal mice: the values in mice treated with rrSCF164at 30 or 10 uggkg-1day-1 were 8.4 or 3.1 times that in thevehicle-injected control mice (Table 1). The effect of s.c.administration of rrSCF164 on mast cell populations wasrestricted to the vicinity of the injection site; no increase inmast cell numbers was seen in sections of other cutaneoussites or the stomach of these mice. And in +/+ mice, incontrast to S1/Sd mice (20), injection ofrrSCF11 had little orno effect on Hct (Table 1). Administration of rrSCF11 togenetically mast cell-deficient (WBB6)F1 W/W' mice, atdoses up to 100 ,uggkg-1 day-1 s.c. for 3 wk, failed to repairthe dermal mast cell deficiency of these mutants (data notshown).When representative sections from SCF injection sites

were examined by histochemistry, we found that most of themast cells exhibited cytoplasmic reactivity with berberinesulfate and that many, but not all, of the mast cells stainedwith safranin. The presence of increased numbers of mastcells positive for berberine sulfate or safranin in the skin ofmice injected with SCF does not necessarily indicate thatSCF promotes mast cell maturation, however; this findingmay have reflected simply the SCF-induced proliferation ofthe population ofberberine sulfate-positive, safranin-positiveCTMC that ordinarily resides in the dermis of normal mice.To assess separately the effects of rrSCF1M on the prolifer-ation or maturation of mast cells, we therefore analyzed mastcell populations exposed to rrSCF11 in vitro.

rrSCF'64 Induces Proliferation of Immature or Mature MastCells in Vitro. We first demonstrated that rrSCF164, like c-kit

Table 1. rrSCF'" increases numbers of dermal mast cells innormal mice

Hct, %

rrSCF'1, Before After Mast cells perAg-kg-'-day-l treatment treatment mm2 of dermis

30 43.4 + 2.1 46.1 ± 1.1 290 ± 71*10 42.0 ± 0.6 43.7 ± 1.5 109 ± 17*0 43.7 ± 1.8 40.8 + 3.0 35 ± 6*

(WCB6)F1 +/+ mice received a daily s.c. injection of rrSCF164, orvehicle alone, for 3 wk and were killed for determination of Hct anddermal mast cell number at the injection site 24 h after the lastinjection.*P < 0.001 vs. each of the other values by Student's t test (two-tailed).

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FIG. 1. Proliferation of various mouseresponse to different concentrations of rrIL-3-dependent PT-18 mast cell line. (B)populations of IL-3-dependent BMCMC IFour-week-old populations of BMCMCW/WI mice (o) or the congenic normal (+oPMC from retired breeder BALB/c mice.SEM (n = 3-5 per point) of [3H]thymi(incorporated over a period of 4 h. The 4-hmidine observed after incubating cells in ILof in rrSCF'" was 11,384 + 420 cpm for PTfor BALB/c BMCMC, 6656 441 cpm ICMC, 10,065 899 cpm for (WBB6)F1 W,30 cpm (not significant vs. the value forgrowth factors: 193 29 cpm) for PMC.

ligand purified from BALB/3T3 fibinduce proliferation of mature as wedependent mast cells (Fig. 1). rrSCF16of the IL-3-dependent cloned mast ceIL-3-dependent BMCMC from either Ior (WBB6)F1 +/+ mice (Fig. 1C) andwhich comprise an IL-3-independentCTMC mast cells (reviewed in ref. Iderived from (WBB6)F1 WIW" miceproliferative response to rrSCFlM, v

B significant (P = 0.014 vs. cells maintained without rrSCF164by two-tailed Student's t test) only at a rrSCF164 concentra-tion of 500 ng/ml (Fig. 1D). This result is consistent withprevious work indicating that the W and W" alleles encodec-kit receptors that express no (W) or markedly diminished(WV) tyrosine kinase activity (33) and indicates that theeffects of rrSCF164 on mast cell proliferation require theinteraction of the cytokine with a functional c-kit receptor.The cells in Fig. 1 that responded to the proliferative effectsof rrSCFl64 were morphologically identifiable mast cells

, , , , , rather than less mature precursors. Ninety percent [(WBB6)-0 0.5 5 500 2500 F1 +/+ BMCMC, PMC] to 100lo (PT-18) of the cells tested

were mast cells according to staining with neutral red (5). And>98% of the cells proliferating in response to rrSCF64 were

D identified as mast cells by colocalization of immunohisto-chemical staining for bromodeoxyuridine incorporated intonuclear DNA and mast cell cytoplasmic granule staining withalcian blue (29).Both dermal CTMC and PMC are regarded as T-cell-

independent mast cell populations that do not proliferate inresponse to IL-3 (reviewed in ref. 1). As shown in the legendof Fig. 1, we confirmed that purified PMC did not proliferatein response to IL-3-containing medium, whereas all of theIL-3-dependent populations, including BMCMC derived

. , . , , from (WBB6)F1 W/WI mice, gave the expected proliferative0.5 5 50 500 2500 response. IL-3-dependent BMCMC and PMC are repre-

sentative of immature and mature stages of mast cell devel-opment (reviewed in ref. 1). Thus, when used as the only

mast cell populations in exogenous cytokine, rrSCF16" can stimulate proliferation ofrSCF164. (A) The cloned, the mast cell lineage at a later period of its development than) Four-week-old primary that influenced by the proliferative effects of IL-3 alone.from BALB/c mice. (C) rrSCF'" Induces IL-3-Dependent Immature Mast Cells to

+)mice (n). (D)Purified Mature and Synthesize Heparin in Vitro. In accord withData shown are means previous work (2, 5, 6, 31), we found that BMCMC main-dine, expressed as cpm, tained in IL-3-containing medium (Table 2) had a very lowincorporation of [3H]thy- histamine content and were consistently negative for cyto--3-containingCM instead plasmic staining with either berberine sulfate (Fig. 2a) or'-18 cells, 3234 ± 214 cpm safranin (Fig. 2d). By contrast, freshly isolated PMC (Tablefor (WBB6)F1 +/+ BM- 2) exhibited strong cytoplasmic staining for berberine sulfatelWv BMCMC, and 222 ± (Fig. 2b) or safranin (Fig. 2e). We found that when BMCMCr cells incubated without were maintained in rrSCF'1 (50 ng/ml) for 4 wk (Table 2),

many of the cells acquired reactivity for berberine sulfateiroblast CM (24), can (Fig. 2c) (Fig. 2f). Moreover, BMCMCroblast Cmma(24, can3- tained in rrSCF11 had a histamine content =30 times that of:ll as immature, IL-3- BMCMC maintained in IL-3-containing medium (Table 2).induced proliferation To confirm that the acquisition of reactivity with berberine

11 line PT-18 (Fig. 1A), sulfate by BMCMC maintained in rrSCF1" reflected anBALB/c mice (Fig. 1B) increased ability of these populations to synthesize heparin,I purified mouse PMC, we biochemically analyzed the 35S-labeled proteoglycanspopulation of mature synthesized by BMCMC maintained in the presence or

1) (Fig. 1D). BMCMC absence of rrSCFl64. In accord with previous work (2, 4, 6,exhibited a very weak 31, 34), we found thatBMCMC maintained in IL-3-containingvhich was statistically medium incorporated [35S]sulfate predominantly into chon-

Table 2. Histochemical characteristics and histamine content of various populations of mouse mast cellsAlcian blue (AB)/safranin (S), % Berberine sulfate, % Histamine,

Mast cells AB+/S- AB+ > S' S+ > AB+ - + + ++ pg per cellBMCMC (CM 4 wk) 100 0 0 100 0 0 0 0.08 ± 0.02BMCMC (CM 8 wk) 100 0 0 100 0 0 0 0.08 ± 0.04BMCMC (CM 4 wk/SCF 4 wk) 33 ± 3 47 ± 3 20 ± 2 44 ± 16 16 ± 2 40 ± 15 0 2.23 ± 0.39PMC(SCF2wk) 0 5±1 95±1 6± 1 23±7 69± 2 2±1 2.53±0.23PMC (freshly isolated) 0 0 100 0 0 0 100 20.9 ± 2.7BMCMC were maintained in IL-3-containing CM as in Fig. 1 for 4 or 8 wk or were maintained in CM for 4 wk and then transferred to medium

lacking IL-3 but containing rrSCF1" (50 ng/ml) for an additional 4 wk. PMC were used immediately after purification or after 2 wk in mediumcontaining rrSCF16 (50 ng/ml). Medium containing rrSCF161 was replaced two or three times per wk. Mast cells in cytocentrifuge preparationswere stained for alcian blue/safranin or berberine sulfate; at least 100 cells were examined in each preparation. AB+/S-, all granules AB+, nogranules S+; AB+ > S+, more AB' than S+ granules; S+ > AB', more SI than AB+ granules. Berberine sulfate: -, no berberine sulfate positivegranules; ±, occasional weakly positive granules; +, many positive granules; + +, many intensely positive granules. Histamine content wasdetermined by a fluorometric method. Data shown are means ± SEM (n = 3-8 per point).

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FIG. 2. Histochemical characteristics of mast cells. (a-c) Mastcells stained with the fluorescent heparin-binding dye berberinesulfate. (a) BALB/c BMCMC after 8 wk ofculture in IL-3-containingmedium exhibit weak nuclear fluorescence but little or no cytoplas-mic fluorescence. (b) A freshly isolated PMC exhibits intense cyto-plasmic fluorescence. (c) After 4 wk in IL-3-containing medium andthen 4 wk in medium supplemented only with rrSCF1' (50 ng/ml; asin Fig. 1). BALB/c BMCMC populations contain many cells withbright cytoplasmic fluorescence. (d-f) Same mast cell populations asin a-c, respectively, but after staining with alcian blue (AB)/safranin(S). (d) BALB/c BMCMC in IL-3-containing medium are alcian bluepositive, safranin negative (AB+/S-). (e) Most of the cytoplasmicgranules of a PMC are intensely safranin positive (S' > AB'). (f)After 4 wk in rrSCF14-containing medium, BMCMC populationsinclude some cells that appear AB+/S- (solid arrowheads), othersthat have more AB' than S+ granules (AB' > S+; solid arrows), andothers that have more S+ than AB' granules (AB' < S+; openarrows). (a-c, x900; d-f, x1350.)

droitin sulfates. Indeed, these 35S-labeled macromoleculeswere susceptible to complete degradation by treatment withchondroitin ABC Iyase and thus contained no detectable[35S]heparin. By contrast, BMCMC maintained 4-6 wk inrrSCF164 (50 ng/ml) incorporated a substantial amount of[35S]sulfate into heparin, as judged by degradation of the35S-labeled macromolecules by treatment with either hepa-rinase or nitrous acid. Thus, 41-70% (mean + SD = 51% +13%; n = 4) of the 35S-labeled proteoglycans synthesized byBMCMC maintained 4-6 wk in rrSCF11 were susceptible todegradation by treatment with heparinase and 45-55% (mean+ SD = 50%o ± 4%; n = 4) of the 35S-labeled proteoglycanswere susceptible to degradation by nitrous acid. The 35S-labeled macromolecules were identified as proteoglycans bytheir susceptibility to degradation byNaOH into molecules ofsmaller molecular weight (31, 32).rrSCF11 also induced phenotypic changes in PMC. Nocka

et al. (24) reported that a c-kit ligand (21) purified from CMofBALB/3T3 fibroblasts stimulated the proliferation ofPMCin vitro and that the cytoplasmic granules of these cellsstained with berberine sulfate. We also found that many PMCmaintained in vitro with rrSCF1" retained reactivity withberberine sulfate (Table 2). However, our results indicatethat some PMC maintained in rrSCF1M had no detectablecytoplasmic staining with berberine sulfate or exhibited di-minished reactivity with safranin (Table 2). In addition, PMCmaintained in rrSCF11 had a histamine content very similarto that of BMCMC maintained in rrSCFlM but only ==12%that of freshly isolated PMC (Table 2).

DISCUSSIONOur findings show that rrSCFL4 not only can promote theproliferation of either immature or mature mast cells, but alsocan induce changes in the phenotype of these populations.BMCMC maintained in rrSCF1M both proliferated and be-came more mature. This finding identifies SCF as the firstsingle cytokine that can induce immature, IL-3-dependentmast cells to acquire phenotypic characteristics of CTMC,including the ability to synthesize substantial amounts ofheparin. This observation also represents a direct demon-stration that SCF alone can influence both the proliferationand the maturation of a cellular lineage expressing c-kit.Mature, IL-3-dependent PMC also proliferated in response torrSCF164. But in this case, the cells maintained in rrSCF164had a substantially lower histamine content and, as a popu-lation, exhibited less mature histochemical characteristicsthan the mast cells initially placed in culture. However, PMCmaintained in rrSCF1M had a much higher histamine contentand more mature histochemical staining characteristics thanPMC propagated in medium containing IL-3 and IL-4 (31).We do not know why the characteristics of BMCMC or

PMC maintained in rrSCF'" differed from those of freshlypurified PMC. Perhaps the soluble form of SCF used in ourexperiments (19) influences mast cell maturation differentlythan the membrane-associated form of SCF (22), which maybe encountered by mast cells in vivo. However, both thehistochemical staining characteristics and the histamine orheparin content of BMCMC maintained in soluble rrSCF1"were very similar to those reported for BMCMC coculturedwith 3T3 fibroblasts (6), a system that favors contact betweenmast cells and membrane-associated c-kit ligand (9, 10, 17,22). For example, when BMCMC that became adherent to3T3 fibroblasts during coculture for 14 days were analyzed,39%o ± 7% of the cells' 35S-labeled proteoglycans weresusceptible to degradation by nitrous acid treatment (6). Inour study, the corresponding value for BMCMC maintainedfor 4-6 wk in rrSCF164 was 50% ± 4%. Thus, according tomultiple criteria including heparin synthesis, exposure tosoluble rrSCF164 can reproduce the mast cell maturation-inducing effect of coculture of BMCMC with 3T3 cells.

Nevertheless, mast cells cultured with rrSCF164 neitheracquired nor retained a phenotype fully identical to that ofresident PMC. This finding may indicate that additional mastcell maturation factors remain to be identified. Alternatively,this observation may reflect the active proliferation of mastcell populations maintained in SCF. PMC proliferating invitro in response to IL-3 and IL4 contained much lesshistamine and exhibited less mature histochemical charac-teristics than freshly isolated PMC (31, 35, 36). And we foundthat the expanded population of dermal mast cells at sites ofs.c. injection of rrSCF164 in (WCB6)F1 +/+ mice exhibitedmore variable intensity of cytoplasmic staining with berber-ine sulfate or safranin than did the nonproliferating dermalmast cell populations in control mice. Taken together, theseobservations support other findings indicating that popula-tions of proliferating CTMC contain mast cells exhibiting

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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.

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