Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium

Journal of hnmum~hJgi('al Methods. 149(19¢92) 173-181

173

~) 1992 ElsevierScience PublishersB.V. All rights reserved(1022-175t)/~12/5115.1111

JIM 1)h263

Induction and identification of mast cells from long-term culture of mouse spleen cells without conditioned medium Z h i - Q i n g Hu, T a k a h i k o Y o s h i d a and T a d a k a t s u S h i m a m u r a [h7utrtnwnt of Mic'rohiohJ~,Tam/hnmunoh~gy. Showa Uttit e'r~it,."School of Ah,¢h¢ira'. Tokyo 14Z Japttn

(Received 2(1 M;ly 1991, revisedreceived7 ()chlhcr It)t)l. itcccplctl 13 December PAt,H)

A simple method is described for the preparation of large numbcrs of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4-5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 2(1 days. Using this method, 2 × I(1" mast cells could be induced from I × 107 nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thyl,2, L3T4, Ly-2, Ig, B220, Asialo GM~, and WGA receptors were all negative but functional igE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 + 0.024 ~ g stored histamine in 1 × 10" of the cells. Histamine was released from the cells in an antigen-induced and lgE-mcdiated proccss. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA. Key words: Spleen cell. mnuse: Mast cell induction: Mast cell identification:Milogenrcspcmseof mast cells

Introduction

Mast cells are now considered to play a pivotal role not only in allergic reactions but also in many protec:ivc and pathological immune responses and biological processes (Stevens ct al., 1989; Galli, t990; Gordon et al., 19901. For re-

('orrespotldt'tzci' to: "I'. Shimamura, Department of Microbiology and Immunology. Showa University School eft Medicine, 1-5-1.;Ilatanodai. Shinagawa-ku,Tokyo 142, Japan. Ahbreriatkm.~: FCS, fetal calf serum: FACS, fluorescenceactivated cell ,',otter" FSC, forward angle-lightscatter count: SSC, side angle-light scatter count; ConA, concanavalin A; LPS, lipopulysaccharidc:('AS. ConA-stimulatedspleen cellconditioned medium: WGA. wheat germ agglutinin: cpm, coun's per minute; IL. interleukin: DNP-OVA. 2.4-dinitrophenylatcdovalbumin.

search into the mechanisms underlying those functions, cultured mast cells have been shown to be a very convenient and important resource. Hasthorpc reported that mast cells could be induced from the spleen cells of mice previously injected with the cell-free supernatant from Friend virus producing erythroleukemia cells (Hasthorpc. 19801. The growth of these mast cells was promoted by factor(s) present in the supernatant of pokeweed mitogen-stimulated spleen cells. But the mast cells contained C-type virus particles. This was confirmed by electron microscopy. Thercafter, several laboratories simultaneously discovered that apparently pure populations of mast cells could be generated by culturing normal mouse hematopoietic cells in conditioned media derived from mitogen-activated T cells, cloncd Lyl "2- inducer T cells or WEHI-3B

tumor cclls (Nabcl ctal,, 1981; Nagao et al., 1981; Razin c t a l , 1981; Schradcr ct al., 1981; Tertian ctal., 1981). The cytokine in those T cell conditioned media that induced the preferential growth and differentiation of precursor cells to mast cells was shown to be IL-3 (lhle et al., 1983). It was also verified that cultured mast cells dcpended absolutely on the conditioned medium-derived growth factor(s) for growth and viability (Tertian et al., 1981). However, a simple method has recently been established in our laboratory for the induction of large numbcrs of mast cells from mouse spleen cells using medium without any conditioned supcrnatant. This mast cell induction required neither T cells nor conditioned supernatant dcrived from T cells. In the present report, we describe thc methodology used for mast cell induction and idcntification. The mechanism and significance of this finding are also discussed.

oclonal anti-Thyl,2, clone F7D5, Serotec, Kidlington, Oxford, UK) and anti-L3T4 (rat monoclonal anti-L3T4, clone GKI.5, a kind gift from Drs. S. Tamauchi and S. Habu) for 30 min at room temperature. After washing, the cells were treated in the same volume of I/1(1 diluted guinea pig complement (Kyokuto Pharmaceutical Industries, Tokyo, Japan) for 30 rain at 37°C. The remaining cells were washed twice and cultured in the same way for mast cell intmction.

Preparation of CAS Spleen cells of C3H mice were cultured at 5 × 106/ml in RPMI 1640 medium containing 10% FCS and other additives. ConA (5 ~ g / m l ) was added to the culture and the cells were cultured for 48 h at 37°C in a humidified incut~ator containing 5% CO,. The cell-free supernatalzt was collected and kept at -20°C.

Antibodies and conjugates for phenotype analysis Materials and method.~

Mice Male C 3 H / H c N . BALB/c, and C57BL/6N mice. 7-10 weeks old, were purchased from Charles River Japan, Atsugi, Japan.

hzduction of mast cells from normal mouse spleen cells Spleen cell suspensions were prepared as described previously (Shimamura et al., 1982). The spleen cells were adjusted to 6-10 × l0 s, cells/ml in RPMi 1640 medium (Gibco, Grand Island, NY, USA) containing 10% FCS (inactivated, lot 407, Bockneck, Outario, Canada), 25 mM Hepes, 200 mM L-giutamine (Gibco), 100 U / m l penicillin, 100 ~t,.g/ml streptomycin (Gibco), and 5 × 10 -s M 2-VIE. The cultures (1 ml/well) were incubated in 2~, well culture plates (Linbro, Flow Lab., Mclean. USA) at 37°C in a humidified atmosphere with 5% CO~. Half of the total volume of the culture medium was changed every 4-5 days.

Elimination of T cells with antibodies plus complemetll To remove T cells, I × 107/ml spleen cells in RPMI 164(I medium were incubated simultaneously with optimally diluted anti-Thyl,2 (rat mon-

Rat monoclonal anti-Thyl,2 conjugated with fluorescein (anti-Thyl,2 FL, clone 30-HI2), rat monoclonal anti-L3T4 conjugated with phycoerythrin (anti-L3T4 PE, clone GK1.5), rat monoclonal anti-Lyt2 conjugated with biotin (anti-Ly2 Biotin, clone 53-6.7), and avidin conjugated with phycoerythrin (avidin PE) were purchased from Becton Dickinson, Sunnyvale, USA. Rat monoclonal anti-B220 conjugated with biotin was a gift from Dr. Habu. Rabbit anti-mouse immunoglobulin conjugated with fluorescein (anti-lg FL) was purchased flora Cedarlane Lab., Ontario, Canada. Rabbit anti-asialo G,~t~ conjugated with biotin was prepared in our laboratory. WGA conjugated with fluorescein isothiocyanate (WGA-FITC) was purchased from Vector Lab., Burlingame, CA, USA. Purified mouse monoclonal lgE against DNP-OVA (M-IgE) was purchased from Japan lmmuno-monitoring Center, Tokyo, Japan.

Phe.oO'pe analysis by FACS Histopaque-1077 (Sigma) was used to separate large cells from dead and small lymphoc3'tes of the cultured spleen cells. The large mast cells were adjusted to 2 × 107 cells/ml. 50 pA of the cell suspension (1 × 10 f' cells) were mixed with optimally diluted conjugates (10 p.I volumes) in tubes. After 20 min at 4°C, the cells were washed

three times. For Ly2, asialo Gmt, and B22(1, the ceils were treated at 4°C with avidin PE for another 20 min followed by three washes. For IgE receptors, I x 10~' mast cells in II111/.tl RPMI 1640 medium containing 5% FCS and (1.01 M E D T A were incubated with 2.5/.tg M-igE at 37°C for i h. After washing with cold PBS, the cells were incubated with anti-lg FL at 4°C for 20 rain followed by three washes. The control was set tip in the same way but without M-IgE. After the resuspended cells were fixed in !% buffered paraformaldehyde for I - 2 days, they were washed, filtered, and again suspended in 3(11) ~1 PBS. The phenotypes of the cells which were gated according to scatter (FSC and SSC) were then analyzed by FACStar (Becton Dickinson). (~ell collnl.~ Cell viability was determined by trypan blue exclusion. Mast cells were counted by a staining method using alcian blue specificity for counting basophils and mast cells (Gilbert et al., 19751. Stabzb~g o f mast cells The smears were stained conventionally with Wright-Giemsa solution and with alcian bluesafranin as described elsewhere (Pretlow et al., 1970). Peritoneal mast cells were stained with alcian blue-safranin at the same time for comparison. Degranulation in&wcd by compound 4 8 / 8 0 and A 23187 Compound 4 8 / 8 0 (Sigma) and the calcium ionophore A 23187 (Sigma) were used for degranulation of mast cells as described elsewhere (Jazaki et al., 19901. The morphologic changes induced were examined by light microscopy after degranulation. Antigen-induced and IgE-mediated histamine release A method described elsewhere (Dvorak e t a l . , 19871 was used. Briefly, mast cells separated by Histopaque-1077 were washed twice with Hanks' solution containing 2 mM Hopes and passively sensitized with monoclonal mouse lgE anti-DNP. 1 x 10 ~' cells in 100 p.I Hanks" solution supplemented with 10% FCS were incubated with 2.5

p.g IgE at 37°C for 60 min. The cclls were thcn washed thrcc times in Hanks" solution and I × 106 cells/ml wcrc incubated for 10 min at 37°C with antigen (DNP-OVA, containing about 21 tool DNP per mol OVA) prepared in our laboratory according to a published method (Little et al., 19671. The controls included untreated cells, cells sensitized with IgE but not stimulated with DNPOVA, and cells incubated with DNP-OVA only. The supernatants and cells in triplicate samples were then recovered separately after centrifugation. Histamine assay A fluorometric assay (Shore, 1971) was used as described. For complete recovery of histamine the cells were diluted with 0.1 N HCI and boiled for 3 rain. The samples were then centrifuged at 8(1t1(I X g for 20 min and the supernatants collected for histamine assay. Purification o f mast cells using a cell sorter The cultured mast cells wcrc further purified using a cell sorter. The scatter gates were set to exclude residual small lymphocytes on the basis of FSC and SSC (size). After sorting, the cells were collccted under sterile conditions. Mitogen response o f mast cells The mast cells purified by the cell sorter were cultured in 96 well flat bottom plates at different densities in a total volume of 200 /~1. ConA (Sigma) or LPS (Difco Lab., Detroit, MI, USA) were added to thc cell suspensions to achieve a final concentration of 2.5 g.g/ml or 5 tzg/ml, respectively. Cultures without mitogens were set up as a control. The cells were incubated for 48 h and pulsed for thc final 4 h with 0.5 p, Ci 3H-TdR (ARC, St. Louis, USA). The cells were then harvested and 3H-TdR incorporation was determined by scintillation counting.

Results

Time course o f mast cell induction To determinc the peak appearance and kinetics of mast cell induction, the viable cells and mast cells in the cultures were examined every 4

days in addition to observations by invt:rse microscopy. As shown in Fig. I, there were few large cells in the culture on the 5th day (Fig. IA). After 15 days, the large cells had increased and many red blood cells and small lymphocytes had died (Fig. I/3). The large cells proliferated for 3 weeks and were separated from the dead cells and small lymphocytes by Histopaquc separation. Almost all of the cells were large cells (Fig. 1C) and they were confirmed to be mast cells by a!cian blue staining. As the culture time continued, the viable cell number in the culture decreased, but the mast cell number increased (Fig. 2). The number of mast cells peaked between the 16th anti 20th day of culture. When the fresh spleen cells were examined almost no mast cells could be found. On the 4th day of culture only 1.2,/1000 of the viable cells were mast cells but the ma;t cells increased to represent 4.5%, 26.7%, 65.8%, ¢,7.5%, and 71% of the total viable cells on the ~',th, 12th, 16th, 20th, and 24th days, respectively. On the basis of the mast cell numbers and the ratios of mast cells to viable cells, 3 weeks was considered to be the optimal time to harvest the mast cells. Mast cells were induced from the spleen cells of three strains of mouse. However, fewer mast cells could be induced from C57BL/6N mice than from C 3 H / H c N and B A L B / c mice as indicated in Table 1. The experiment was repeated 56

•

,~

¢"

Fig. I. Photomicrographs of cultured spleen cells. A: 5th day; B: l Sth day; C: ~fter 3 w'eeks, the dead cells and small lympho~'tes v.ete removed by liislopaquc separation

4oI

~\\

i '"1

.---.

-.

Fig. 2, Kinetics of mast cell induction. (o) total viable cells; (#) mast cells; (A) total viable cells following culture with CAS (25% v/v); ( • ) mast cells after culture with CAS (25% v/v).

177 TABLE I MAST CELL INDUCTION FROM SPLEI~N CEt.LS OF DIFFERENT STRAINS OF MICE Mouse

Mast cells/culture ( x 10%

C3II C57BL BALB/c

Day 8 15.2_+ I.I 13.8+_0.6 30.3 .+_6.6

Day 12 62.7_+ 11.3 31.6_+ 3.8 I 13.11_+ 9.3

Day 16 138.5+ 18.8 59.7± 4.8 158.11+_28.6

Day 211 157.9+37.1 6X.5± 3 184.1)+_22.8

l)ay 24 21198-t 9.5 95.9-tl4.8 158.5 ~c18.I

t i m e s on C 3 t t / H e N mice, a n d successful induction of m a s t cells was o b t a i n e d every time.

g r a n u l e s of the cultured mast cells stained only blue.

The role of T cells in mast cell induction

Phenotype ana&sis of mast cells by FACS

T o observe t h e effects of T cells on m a s t cell induction, T cells were r e m o v e d from spleen cells by a n t i - T h y l , 2 a n d a n t i - L 3 T 4 plus c o m p l e m e n t . T h e rate of cell recovery after this t r e a t m e n t was 3 2 - 4 5 % a n d t h e r e m a i n i n g T cells were less t h a n 1% w h e n c h e c k e d by F A C S analysis. A f t e r T cell-depletion, t h e r e m a i n i n g cells were c u l t u r e d a n d large n u m b e r s of m a s t cells were induced. T h e s e results c o n f i r m e d that the m a s t cell induction r e q u i r e d n e i t h e r T cells nor c o n d i t i o n e d s u p e ~ n a t a n t derived from T cells. However, the m a s t cell n u m b e r d e c r e a s e d after 24 days (Fig. 2). W h e n t h e culture m e d i u m was s u p p l e m e n t e d to 25% of t h e v o l u m e with C A S on the 40th day of culture, t h e mast cell n u m b e r s increased again in r e s p o n s e to the C A S (Fig. 2). T h e m a s t cells were cloned by limiting dilution a n d using m e d i u m c o n t a i n i n g 2 5 - 5 0 % ( v / v ) of CAS. T h r e e of the clones were c u l t u r e d in vitro for 3 m o n t h s . Witho u t CAS, the mast cells could not be c u l t u r e d for m o r e t h a n 40 days (Fig. 2). T h i s result s u g g e s t s that s o m e factor(s) in C A S is n e e d e d for continu e d proliferation of t h e mast cells.

As s u m m a r i z e d in T a b l e il, the cultured mast cells did not express m a r k e r s of T, B, or N K cells. However, 79% of the cells were positive for IgE receptors (Fig. 4). T h e purity of the gated mast cells by F A C S t a r was 93%, verified by c o u n t i n g with the alcian blue m e t h o d . T h e r e f o r e , a n o t h e r 14% of the mast cells were negative for lgE

Morphological characterization of the mast cells U n d e r light m i c r o s c o p y , W r i g h t - G i e m s a s t a i n e d s m e a r s of t h e c u l t u r e d mast cells app e a r e d as large, r o u n d or oval cells c o n t a i n i n g m o n o n u c l e i s u r r o u n d e d by cytoplasm with num e r o u s basophilic g r a n u i e s (Fig. 3 A t . T h e c u l t u r e d m a s t cells a n d peritoneal m a s t cells were e x a m i n e d with alcian b l u e - s a f r a n i n at t h e s a m e time a n d w h e r e a s the g r a n u l e s in peritoneal mast cells s t a i n e d both blue a n d red the

"FABLE II C I l A R A C T E R I S T I C S OF

MAST CELLS

Characteristic

Mast cells

Phenotypc lgE receptor Thy- 1,2 L3T4 Ly-2

+ (79.11%) - ( 97.6'7r ) - (97.qc~ )

lg

- (99.5%) - (98.3r/r )

B2211 Asialo-GM~ W G A Receptor

- ( 99.6¢~.) - ( 99.9"7r ) - ( 100% )

Adhe.',ion t,.) plastic plate T cell dependence for induction Cytoplasmic granules ttistamine Antigen-induced and lgE-mcdiated histamine release Degranulatior,

+ +

Compound 48/811 A 23187 Mitogen response ConA LPS Staining with alclan blue

+

Staining with safranm

+

+

+ +

ID

|

•

Log FIuoto$¢Qnce INlen$11y Fig. 4. lgE receptor analysis by FACS. ( . . . . . . ) mast cells + anti-lg FL as control; ( ) mast cells+ IgE+anti-lg FL. 79% of the cells were positive ft~r lgE receptors.

Degranulation of mast cell.~ Both c o m p o u n d 4 8 / 8 0 and A 23187 were able to induce degranulation of the mast cells in a d o s e - d e p e n d e n t fashion. T h e mast cells were more responsive to A 23187. and less responsive to c o m p o u n d 48/8(I. Fig. 3B shows the degranulation induced by 1 / z g / m l of A 23187.

tb

~.

d,.

Antigen-hzduced and lgE-mediated histamhte release O n e characteristic of mast cells is the presence of histamine in the granules. It was shown that 1 x 10" of the mast cells contained 1.632 + 0.024 /zg of histamine and both a n t i - D N P igE plus D N P - O V A were able to induce histamine release (Table 1111.

Mitogen response of mast cells" The cultured mast cells were purified by Histopaque separation and by FACS. Fig. 5 indiTABLE III ANTIGEN-INDUCED AND TAMINE RELEASE I-tg. 3. Morphc~logyol mast cell.', (Wnght-~lemsa). A: mast cells from long-term culture: B: mast cells degranulated by I /,tg/ml of A 23187: C: mast cells after ConA stimulation. receptors. This may have been because some immature mast cells did not yet exoress IgE receptors as previously reported (Razin et al., 19841.

IgE-MEDIATED HIS-

Treatment of mast cells(10 ~)

Histamine(/ag) ¢,; Histamine Cells Supernatant release

Untreated IgE÷DNP-OVA lgE DNP-OVA

1.632 +_0.024 1.433+_(I.I115 0.175+_0.015 1.583+11.175 I).073+0.[106 1.566_+11.1157().I)7tA_+0.(102

111.7 4.4 4.8

8/

17q

6.

5

10

25

Mast Cells/WeN (x 10'1 Fig. 5. Resl:~)nse of cultured mast ceils to milogens. (~,) control without mitogens;(o) ConA (2.5 .u.g/ml); ( • ) LPS (5 ~ag/ml). cates that the purified mast cells (purity 93%) responded to ConA but not to LPS. The incorpor_tion of 3H-TdR was greatly enhanced by ConA stimulation. After ConA was added to the cultures, the mast cells became larger and exhibited fewer granules in their cytoplasm (Fig. 3C). However, the cell surface phenotype did not change after ConA stimulation.

Discussion

Based on morphology, eytochemistry, histochemistry and function, the cultured large cells described in this study were idcntified as mast cells (summarized in Table 11). Mast cells have been divided into three populations, connective tissue or serosal mast cells (peritoneal mast cells), mucosal mast cells, and growth factor-dependent cultured mast cells (Galli, 199{I). Mucosal ma:it cells and growth factor-dependent cultured mast cells share characteristics such as T cell dependence, staining by alcian blue but not by safranin, and a small quantity of stored histamine ( 0 . 1 - 2 / , t g / l ( l ~' cells). But the growth factor-dependent cultured mast cells are derived from non-mucosal sites. The peritoneal mast cells differ from the other populations in some properties such as T cell independence, staining not only by aleian blue but

also by safranin, and the possession of a large quantity of stored histamine ( 10-30 ~gfl I()~' cells) (Stevens et al., 1989). The mast cells in our system should bek)ng to either the growth factor-dependent cultured mast cell or mucosal mast cell populatk,n based on (i) the histochemical pronerty of being alcian bluepositive, safranin-negative; (ii) the property of being cloned by limiting dilution in response to CAS which contains factor(s) for mast cell growth and (iii) the quantity of stored histamine ( 1.632 + 0.024/.tg/10 ¢' cells). It has bccn confirmed that mast cells are derived from precursors that originate in bone marrow and then reside in bone marrow, spleen, lymph node, blood, and other peripheral tissucs (Kitamura et al., 1981; Nakano et al., 1987; Ashman et al., 1991). The precursors in these sites are present in very small numbers (less than 3/105 nucleated normal spleen cells) (Crapper et al., 1981) and cannot be identified as mast cells by morphology. We also confirme0 that almost no mast cells could be identified in fresh splenic lymphocytcs, Nevertheless, large numbers of mast cells could bc induced by culturing the precursors with conditioned medium derived from T cells (Nabel et al., 1981; Nagao et al., 1981; Razin et al., 1981; Schrader et al., 1981; Tertian et al., 1981). The cytokine responsible for the proliferation and differentiation of the precursors has been shown to be IL-3 (lhle et al., 1983). Two other factors derived from T cells, IL-4 and IL-9, have also recently been shown to be mast cell growth factors (Mosmann et al., 1986; Hiiltner et al., 1990). Howcver, in our system for mast cell induction, conditioned medium was not used. Even T cells in the spleen cell preparation were unnecessary for induction. This suggests some unknown mechanism(s) of mast cell proliferation and differentiation. One possibility is that IL-3 mainly affects the mast cells at some stage of differentiation (probably a late stage). We have noticed that after 24 days, the cultured mast cells need CAS for further proliferation (Fig. 2). in the early stages, some factor(s) o r / a n d some stimulation from non-T cells, such as cell-cell contact, may be rcsponsible for the proliferation and differentiation. Another possibility is that the precursors or the mast cells produce some factor(s)

t h e m s e l v e s at an early stage o f d ifferen tiatio n . The various s t udi e s of mast cells as a source of m u l t i f u n c t i t m a l cytokines have b e e n rev iew ed ( G o r d o n ct al., 1990) and it is clear that mast cells can re l e a s e m a ny c yt oki ne s in clu d in g 1L-3, IL-4, IL-6 etc. However, the biological significance of mast cell cytokine p r o d u c t i o n is at present unclear. The possibility that FCS may act as an i m p o r t a n t s t i m u l a n t for p r e c u r s o r s of mast cells is not e xc l ude d by the p r e s e n t data. It has be e n r e p o r t e d that C o n A is not e s s e n t i a l for the gro w t h of growth f a c t o r - d e p e n d e n t cult u r e d mast cells ( T e r t i a n ct al., 1981), but the c u l t u r e d mast cells in our system r e s p o n d e d to C onA. T h e i n c o r p o r a t i o n ot" t H - T d R in the mast cells was e n h a n c e d by C onA . A f t e r C o n A stimulation, the m a s t cells b e c a m e l a r g e r an d h ad fewer g r a n u l e s in t h e i r cytoplasm. S u p e r n a t a n t d e r i v e d from m a s t cells s t i m u l a t e d by C o n A was able to s u p p r e s s the m i t o g e n r e s p o n s e s of thymocytes and B cells ( u n p u b l i s h e d o b s e r v a t i o n s ) sugge st i ng that the m a s t cells w e r e a c t i v a t e d by Co~A. T h e m e t h o d of mast cell i n d u c t i o n p r e s e n t e d he re should prove to be a c o n v e n i e n t way to obt a i n large n,_,.m,b e t s of mast cells in vitro. Furt h e r m o r e , the c u l t u r e system may be a useful m o d e l for r e s e a r c h into m a s t cell p r o l i f e r a t i o n a nd di ffe re nt i a t i on.

Acknowledgements We t h a n k Dr. A. S i m ps on for rev iewin g this manuscript.

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