Homology of the Rat Basophilic Leukemia Cell and the Rat Mucosal Mast Cell (Rat Connective Tissue Mast Ceil/Neutral Proteases) DAVID C

Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 3871-3875, June 1985 Medical Sciences

Homology of the rat basophilic leukemia cell and the rat mucosal mast cell (rat connective tissue mast ceil/neutral proteases) DAVID C. SELDIN*t, SCOrr ADELMANt, K. FRANK AUSTEN*t, RICHARD L. STEVENS*t, ANN HEINt, JOHN P. CAULFIELDt*, AND RICHARD G. WOODBURY§ Departments of *Medicine and *Pathology, Harvard Medical School and tDepartment of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA 02115; and Department of Biochemistry, University of Washington, Seattle, WA 98105 Contributed by K. Frank Austen, February 5, 1985

ABSTRACT Secretory granules of the rat basophilic leu- compound 48/80, (a condensation product of N-methyl-p- kemia (RBL-1) cell, a chemically- generated tumor cell line methoxyphenethylamine and formaldehyde), whereas the maintained in tissue culture, were shown to stain with alcian mucosal mast cell in situ (10) or isolated from intestine (11) blue but not with safranin counterstain and to have sparse, does not. Disodium cromoglycate and theophylline inhibit small, electron-dense granules. A Mr 25,000 protein was the the activation of the connective tissue mast cell but not of major [3H]diisopropyl fluorophosphate-binding protein in ex- the mucosal mast cell (12). Homogeneous populations of tracts of RBL-1 cells. Double-immunodiffusion analysis of ex- mast cells resembling the mucosal mast cell subclass have tracts revealed immunoreactivity for rat mast cell protease been obtained in vitro from rat bone marrow cultured in the (RMCP)-II, a Mr 25,000 neutral protase present in the secre- presence of conditioned medium from antigen-activated im- tory granules of rat mucosal mast cells and cultured rat bone mune mesenteric lymph nodes (13, 14). These mast cells marrow-derived mast cells, but no immunoreactivity for stain with alcian blue but not safranin, have low amounts of RMCP-I, the predominant neutral protease of rat connective histamine, contain RMCP-II, and appear to have a non-hepa- tissue mast cells. By radial immunodiffusion, there was 66.8 rin proteoglycan (15). ng of RMCP-ll per 106 cells. Whereas rat connective tissue Thymic-dependent proliferation of mast cells also occurs mast cells stain with alcian blue and safranin and contain hep- in the mucosa of helminth-infected mice (16), and mouse he- arm proteoglycan, rat mucosal and rat bone marrow-derived matopoietic stem cells cultured in vitro in the presence of mast cells stain with alcian blue only and contain a non-hepa- lymphocyte conditioned medium differentiate into mast cells rin proteoglycan and lesser amounts of histamine. Prolifera- resembling the mucosal mast cell subclass (17-22). The cul- tion of rat mucosal mast cells in vivo and rat bone marrow- tured cells depend on the T-cell lymphokine interleukin 3 for derived mast cells in vitro requires T-cell factors, whereas no growth (23) and contain an oversulfated chondroitin sulfate comparable requirement has been observed for connective tis- proteoglycan, termed chondroitin sulfate E proteoglycan sue mast cells. The transformed RBL-1 tumor cell, whose (24). The cultured interleukin 3-dependent mast cell and the growth is independent of factors other than those present in mouse connective tissue mast cell, which contains primarily standard tissue culture medium, has previously been shown to heparin proteoglycans (24), can be distinguished by differen- contain predominantly chondroitin sulfate di-B proteoglycans tial reactivities with a panel of monoclonal antibodies (25). and low amounts of histamine. The similar histology and se- The circulating histamine-containing, IgE receptor-bear- cretory granule biochemistry of the rat mucosal mast cell, rat ing cell characterized in humans and guinea pigs is the baso- culture-derived mast cell, and RBL-1 cell suggest that they phil (26, 27). Several early studies failed to identify basophil- comprise a single mast cell subclass distinct from the rat con- like cells in mouse blood (28-30), but recent investigations nective tissue mast cell. have reported basophils with a few metachromatic granules in mouse blood (31, 32) and bone marrow (33). Rat baso- Studies of mast cell populations in rats and mice have estab- phils, which represent <0.5% of the leukocytes in normal lished the existence of mast cell subclasses. Rat mast cells in rats, increase in number in the blood of normal (34, 35) but intestinal mucosa demonstrate fixation and staining proper- not athymic (36) rats infected with intestinal helminths; these ties different from those of mast cells in connective tissue are the same conditions under which mucosal mast cells pro- sites (1). Mucosal mast cells increase dramatically in number liferate in the intestine. in rats infected with intestinal helminths (2), provided that A leukemia composed of basophil-like cells was generated the animals have intact thymus function (3). Spectral analy- in rats by using the chemical carcinogen P-chlorethylamine sis of the secretory granule-associated toluidine blue me- (37) and was subsequently established as the RBL-1 cell line. tachromasia ofthese cells is not consistent with the presence The RBL-1 cell has been important for the physicochemical of heparin, the predominant proteoglycan of rat mast cells characterization ofthe IgE receptor (38, 39), for studying the from connective tissue sites such as skin or peritoneum (4). steps of the activation-secretion response (40, 41), and for Mast cells from connective tissues contain a Mr 29,000 pro- identification of the arachidonate-derived prostaglandins tease typed with monospecific antisera as rat mast cell prote- (42) and leukotrienes (43, 44). The intragranular proteogly- ase (RMCP)-I (5), whereas the homologous neutral protease cans of the RBL-1 cell maintained in tissue culture have been of rat mucosal mast cells, RMCP-11, is an antigenically and purified and recently characterized as bearing predominant- biochemically distinct molecule ofMr "25,000 (6-9). Both of ly oversulfated chondroitin sulfate di-B glycosaminoglycans these rat mast cell subclasses can be activated with IgE and (45). In this paper, we demonstrate that the RBL-1 cell stains specific antigen. The connective tissue cell also responds to with alcian blue but not safranin, has a sparsely granulated

The publication costs of this article were defrayed in part by page charge Abbreviations: iPr2P-F, diisopropyl fluorophosphate; HBSS, payment. This article must therefore be hereby marked "advertisement" Hanks' balanced salt solution; RBL-1, rat basophilic leukemia 1; in accordance with 18 U.S.C. §1734 solely to indicate this fact. RMCP, rat mast cell protease.

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ultrastructure, and contains RMCP-II as its predominant whereas mucosal mast cells stain with alcian blue only (1). neutral protease. These findings suggest that the RBL-1 cell Harvested RBL-1 cells were washed with Hanks' bal- line is an autonomous tumor resembling the mast cell that anced salt solution (HBSS) and sedimented at 400 x g; they increases in number at mucosal sites during the course of then were processed for transmission electron microscopy various helminth infections in vivo. by sequential fixation in mixed aldehydes and OSO4, staining in block with uranyl acetate, sectioning, and staining on grid MATERIAL AND METHODS with uranyl acetate and lead citrate (48). Identification of [3H]Diisopropyl Fluorophosphate (iPr2P- Light and Electron Microscopy. An adherent line of RBL-1 F)-Binding Proteins. Confluent monolayers of cells in flasks cells was seeded at 105 cells per flask in 175-cm2 tissue cul- (about 108 cells per flask) were washed with HBSS, harvest- ture flasks each containing 80 ml of Eagle's minimal essen- ed by scraping, divided into four aliquots, and centrifuged at tial medium (Earle's salts) supplemented with 10%o (vol/vol) 400 x g. The cells were resuspended in 0.5 ml ofHBSS alone fetal calf serum, 2 mM L-glutamine, 0.1 mM nonessential or in HBSS containing 0.15 M KCl, 0.5 M KCl, or 1.5 M KCI amino acids, 100 units ofpenicillin/ml and 100 ,g of strepto- [since hypertonic buffers are required to dissociate rat peri- mycin/ml (GIBCO) at pH 7.2 in a 370C incubator with a hu- toneal mast cell RMCP-I (chymase) from complexes with midified 6% CO2 atmosphere. The medium was changed proteoglycan (49)] and were sonicated with 201-sec pulses of twice weekly until the RBL-1 cells reached confluence (--108 a Branson Sonifier. The sonicates were incubated with cells per flask), at which time they were harvested by scrap- [3H]iPr2P-F (25 ,uCi/ml, >2 Ci/mmol, 1 Ci = 37 GBq; Amer- ing with a rubber policeman or passaged by trypsinization sham) for 2 hr at 37°C, centrifuged at 8000 x g (Microfuge, and reseeding in fresh flasks. The amount of histamine in Beckman) for 5 min at 4°C, and decanted. The supernatants RBL-1 cultures was determined by radioenzymatic assay were prepared for NaDodSO4/PAGE by boiling for 2 min (46). in 2% NaDodSO4/10% glycerol/0.5% 2-mercaptoethanol/ The staining properties of RBL-1 cells were evaluated by 0.05% bromophenol blue/62.5 mM Tris Cl, pH 6.8. 14C-la- fixation of cytocentrifuge (Shandon-Southern, Sewickly, beled molecular weight standards (Amersham) were pre- PA) preparations of RBL-1 cells in Mota's lead subacetate pared similarly. Radiolabeled samples and standards were for 1 min and staining with toluidine blue 0 (C.I. 52040, Sig- applied to wells in a 3% polyacrylamide stacking gel resting ma) at 5 mg/ml in 0.7 M HCO for 5 min (11) or by fixation and on a 10%-.15% polyacrylamide gradient slab gel 0.75 mm staining with alcian blue 8 GX (C.I. 74240, Sigma) at 5 mg/ml thick and were electrophoresed at 20 mA until the dye front in 0.3% acetic acid for 5 min and counterstaining with safra- reached =1 cm from the anodic end of the gel. The gel was nin 0 (C.I. 50240, Sigma) at 1 mg/ml in 1% acetic acid for 5 fixed in acetic acid/methanol/water (10:30:60, vol/vol) con- min (47). The granules of normal mast cells and basophils taining 10%6 trichloroacetic acid (wt/vol) for 1 hr, impregnat- stain metachromatically with toluidine blue. Connective tis- ed with EN3HANCE (New England Nuclear) for 1 hr, and sue mast cells stain with both alcian blue and safranin, then washed with distilled water to precipitate the scintillant.

FIG. 1. Electron micrograph of RBL-1 cells. The cells contain single nuclei (n), mitochondria (m), and some small granules (g). The cell at the bottom has many vacuoles (v). (x7000.) Inset: The granules (arrows) have electron-dense cores and lucent rims. (x35,000.) Downloaded by guest on September 30, 2021 Medical Sciences: Seldin et aL Proc. NatL Acad Sci USA 82 (1985) 3873 The gel was dried onto filter paper and loaded into a cassette RESULTS with XAR-5 film (Eastman Kodak); autoradiography was at -700C for 3 days. The relative amount of radioactivity in Morphology of the RBL-1 Cell. By light microscopy, cul- each resolved band ofthe autoradiogram was quantified with tured RBL-1 cells exhibited granules of variable size, which a Bio-Rad scanning densitometer. stained metachromatically with toluidine blue or with alcian Assays for Immunoreactive RMCP-I and -II. RBL-1 cells blue but not the safranin counterstain. By electron microsco- were harvested, sedimented, resuspended at a concentration py (Fig. 1), RBL-1 cells were mononuclear and contained of 3-5 x 107 cells per ml in 0.01 M Na2HPO4, pH 7.4/0.15 M electron-dense granules that usually comprised <5% of the NaCl (P1/NaCl) containing 0.15 M KCl, freeze-thawed, and cytoplasm. Most of the granules were small (<0.3 ,m) and centrifuged at 10,000 x g for 15 min. The supernatant was consisted of electron-dense cores surrounded by lucent rims decanted and the pellet was reextracted sequentially in 0.5 (Fig.lnset). Some cells also contained large vacuoles. and 1.5 M KCL. Samples were dialyzed against water and [WH~iPr2P-F-Binding Proteins. The major [3H~iPr2P-F-bind- concentrated 10-fold by lyophilization as needed. Double- ing protein, representing 61% of the total radiolabeled pro- immunodiffusion analyses were performed with monospecif- tein as assessed by densitometric scanning of the autoradio- ic rabbit anti-RMCP-I and anti-RMCP-II sera (7). A radial gram, was of Mr 25,000 (Fig. 2). Other prominent bands ap- immunodiffusion assay was used to quantify the amount of peared at Mr 32,000 and Mr 22,000, representing 19%o and RMCP-II in the extracts (8). Rabbit anti-RMCP-II serum (0.4 16% of the radioactivity, respectively. The Mr 25,000 protein ml) was mixed with 14 ml of 1% agarose in P1/NaCl contain- was largely extracted from RBL-1 cell sonicates in HBSS ing 0.02% sodium azide, and the mixture was poured onto alone (lane 1), but some bands of [33H]iPr2P-F-binding pro- glass plates. Ten-microliter wells were punched in the agar- teins were more marked when the cell sonicates were ex- ose support, the samples were applied, and diffusion was tracted with HBSS plus 0.15 M KCl (lane 2), 0.5 M KCl (lane allowed to occur for 24 hr at 37°C in a moist atmosphere; the 3), or 1.5 M KCl (lane 4). In a second experiment, the Mr plates were then washed in P,/NaCl for 12 hr, dried on filter 25,000 protein accounted for 82% of the radioactivity. paper, stained with Coomassie blue for 1 hr. destained, and RMCP Content of RBL-1 Cells. Although no precipitin air-dried. The amount ofRMCP-II in the RBL-1 cell extracts band was visible when double immunodiffusiou was per- was determined by comparing the diameter of the precipitin formed with RBL-1 cell extract and antiserum to RMCP-I rings to those of standards containing 2-100 ug of purified (data not shown), RBL-1 extract and purified rat mucosal RMCP-II/ml (8). Extracts of equal numbers ofrat peritoneal mast cell RMCP-II, diffused from adjoining wells, were pre- mast cells, mouse bone marrow-derived mast cells, and cipitated by the antiserum to RMCP-II in a single continuous mouse peritoneal mast cells were prepared in a similar fash- band indicative of antigenic identity (Fig. 3). By double ion and were assayed for immunoreactive RMCP-II. immunodiffusion, no immunoreactive RMCP-II was detect- ed in rat peritoneal mast cells, mouse bone marrow-derived 1 2 3 4 5 mast cells, or mouse peritoneal mast cells (data not shown). The RMCP-II content in extracts of 11 different cultures of RBL-1 cells, as assessed by radial immunodiffusion, was 66.8 + 26.2 ng per 106 cells (mean + SD). In a sequential extraction experiment, 92% of the RMCP-II was dissociated from the cell pellet in P1/NaCl with 0.15 M KCl, and the remaining 8% in P1/NaCl with 0.5 M KCL. The histamine content in the RBL-1 cell cultures was 101.3 ± 45.1 ng per 106 cells (mean ± SD, n = 11); its variation among cultures was not correlated with the variation in quantity of RMCP- II. _0- 92.5 RBL

%-69

BI BI *-46

"I''okft o. - ._ BI RMCP-II _ 2 L F _-~~~~~~~~~~30

14.3 -BPB RBL FIG. 2. Autoradiogram of RBL-1 cells extracted in HBSS alone (lane 1), in HBSS with 0.15 M KCI (lane 2), in HBSS with 0.5 M KCI FIG. 3. Double-immunodiffusion analysis for RMCP-II immuno- (lane 3), or in HBSS with 1.5 M KCI (lane 4); labeled with [3H]iPr2P- reactivity. Antiserum to RMCP-II was in the center well; a 10-fold F; and subjected to NaDodSO4/PAGE. Standards are in lane 5 (M, concentrated RBL-1 extract (RBL), purified RMCP-II, and blanks X 1o-3 at right). BPB, bromophenol blue tracking dye. (Bl) were in the surrounding wells. Downloaded by guest on September 30, 2021 3874 Medical Sciences: Seldin et aLPProc. Nad Acad ScL USA 82 (1985)

Table 1. Distinguishing characteristics of rat histamine-containing, IgE receptor-bearing cells* Mucosal Cultured Connective tissue mast Blood mast mast cell cell basophil cell RBL-1 cell Stainingt A, S A ND* A A T-cell factor dependence No Yes Yes Yes Transformed Heparin proteoglycan Yes No ND No No RMCP type I II ND II II Histamine content, pg per 106 cells 10-30 0.1-1 0.6-2 1-2 0.1 Activated by compound 48/80 Yes No ND ND No *See text for references. tA, alcian blue staining; S, safranin staining. *Not determined.

DISCUSSION mast cells and RBL cells (52) cannot. Connective tissue and mucosal mast cells also have different responses to other se- The RBL-1 cell has served as an important model system for cretagogues and to inhibitors of granule secretion (53); the the study of secretory cells that contain histamine and bear responses of the other cells have not yet been determined. IgE receptors. Staining properties, the presence of immuno- The common characteristics of the cells containing chon- reactive RMCP-II, and non-heparin proteoglycan content droitin sulfate proteoglycans and RMCP-II suggest that they are common features ofthe cultured RBL-1 cell, the mucosal comprise a single subclass of rat histamine-containing cells. mast cell in vivo, and the T-cell factor-dependent mast cell The murine thymus-dependent proliferating mucosal mast obtained in vitro, suggesting that these cells are members of cell appears to be represented in vitro by the T-cell factor- a single subclass of mast cells. dependent, bone marrow-derived mast cell, whose precur- By light microscopy, the RBL-1 cell exhibited fine gran- sors exist in many lymphoid tissues (54). The RBL-1 cell ules that stained with toluidine blue and alcian blue but not may be the transformed homologue of that cell type in the safranin, as is characteristic of the granules of the rat muco- rat and thus have escaped the requirement for exogenous sal (1) and bone marrow-derived (13) mast cells. The ultra- growth factors. The observation that both the rat blood baso- structure of the RBL-1 cell (Fig. 1) did not resemble normal phil and the mucosal mast cell undergo thymic-dependent rat, mouse, or human basophils or mast cells (50, 51). The proliferation during the course of helminth infection suggests relative paucity of granules in these cells is consistent with that these cells may also be closely related; there is not yet the histamine content, found to be 101.3 ng per 106 cells, enough evidence to conclude whether the rat blood basophil which is <1% the content in heavily granulated rat connec- is distinct from the mucosal mast cell or is its circulating cor- tive tissue mast cells (Table 1). Poor granulation and low me- relate. The heparin- and RMCP-I-containing mast cell of diator content may be due to the incomplete differentiation connective tissue appears to be less closely related to these of these tumor cells or to their rapid rate of division in tissue cell types, based on its morphology, biochemistry, and func- culture. By double immunodiffusion, extracts of RBL-1 cells tional characteristics. However, there is evidence that in the contained a protein indistinguishable from rat mucosal mast mouse it also is derived from bone marrow (55, 56), and the cell RMCP-II (6, 7) (Fig. 3) but lacked immunoreactive rat peritoneal mast cell has the capability to synthesize chon- RMCP-I, the predominant secretory granule protease of rat droitin sulfate E glycosaminoglycans in the presence of a connective tissue mast cells (5). A Mr 25,000 protein was the pharmacologic acceptor of polymerization (48). The hepar- major [3H]iPr2P-F-binding protein in RBL-1 cells (Fig. 2), in/RMCP-I/T-cell factor-independent mast cell subclass and consistent with the reported molecular weight of RMCP-II. the chondroitin sulfate/RMCP-II/T-cell factor-dependent The immunoreactive RMCP-II and the Mr 25,000 [3H]iPr2P- mast cell subclass may be derived from a common precursor F-binding protein were extracted from RBL-1 cells without through divergent differentiation or may have a progenitor- the addition of hypertonic buffers. These cultured RBL-1 progeny relationship. The classification of rat histamine- cells contained 66.8 ± 26.2 ng of immunoreactive RMCP-II containing mast cells into two subclasses, based on morphol- per 106 cells. ogy, granule and lipid-mediator biochemistry, and agonist/ The characteristics of five populations of rat histamine- antagonist responses, is a model that may be useful for un- containing IgE receptor-bearing cells are summarized in Ta- derstanding the role of such cells in any species and at any ble 1, including mast cells found primarily at connective tis- anatomical sue sites such as the peritoneal cavity, skin, and muscle site. (connective tissue mast cells); mast cells found primarily at This work was supported in part by Grants AI22531, AM20580, mucosal surfaces (mucosal mast cells); histamine-containing AM35984, GM15731, HL17382, and RR05669 from the National In- cells present in the blood (blood basophils); mast cells de- stitutes of Health and by Public Health Service National Research rived in vitro from bone marrow precursors cultured with T- Service Award Training Grant 2T32 GM07753-06 from the National cell factors (cultured mast cells); and histamine-containing Institute of General Medical Sciences. tumor cells (RBL-1 cells). Connective tissue mast cells stain with alcian blue and safranin and contain primarily heparin 1. Enerback, L. (1966) Acta Pathol. Microbiol. Scand. 66, 303- proteoglycans, RMCP-I, and large amounts of histamine. In 312. contrast, the other cell types stain with alcian blue alone and 2. Miller, H. R. P. & Jarrett, W. F. H. (1971) Immunology 20, contain predominantly non-heparin intragranular proteogly- 277-288. cans, RMCP-II, and less histamine. Mucosal mast cells, cul- 3. Mayrhofer, G. & Fisher, R. (1979) Immunology 37, 145-155. tured mast cells, and blood basophils are dependent upon T- 4. Tas, J. & Berndsen, R. G. (1977) J. Histochem. Cytochem. 25, cell factors for proliferation, there 1058-1062. whereas is no evidence 5. Woodbury, R. G., Everitt, M., Sanada, Y., Katunuma, N., that connective tissue mast cells have such a requirement. Lagunoff, D. & Neurath, H. (1978) Proc. Natl. Acad. Sci. All the cells studied in vitro can be activated with IgE and USA 75, 5311-5313. antigen or by calcium ionophores; connective tissue mast 6. Woodbury, R. G., Gruzenski, G. M. & Lagunoff, D. (1978) cells can also be activated by compound 48/80, but mucosal Proc. Nati. Acad. Sci. USA 75, 2785-2789. Downloaded by guest on September 30, 2021 Medical Sciences: Seldin et aL Proc. NatL Acad ScL USA 82 (1985) 3875

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