Establishment of Two Dog Mastocytoma Cell Lines in Continuous Culture Rebekah DeVinney and Warren M. Gold Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco, California

We have previously characterized dog mastocytoma cells propagated in nude mice. We have established two of these lines (C, and C2 ) in continuous culture. Freshly dis aggregated mastocytoma cells were cultured in Dulbecco's modified Eagle's medium (DME)-HI6 mixed with 50% Ham's F12 and supplemented with histidine and 5% allergic dog serum (ADS). Cells were fed every 3 d and passaged weekly. Growth was assessed by cell count. Cell growth was best supported by culture in 5 % ADS. C, cells grow in suspension in ADS and have been passaged 55 times with a doubling time of 37.4 ± 18.7 h (mean ± 1 SD; n = 15). C, cells adhere to tissue culture plastic in ADS and have been passaged 26 times with a doubling time of 49.3 ± 12.5 h (n = 13). Morphologic and functional characteristics are unchanged from those described in cells propagated in nude mice. Histamine content for C, is 0.46 ± 0.18 pg/cell (n = 12) and 0.07 ± 0.04 pglcell (n = 6) for Cs. Both lines contain the neutral protease tryptase and C z contains chymase. Calcium ionophore A23187 or ragweed antigen caused concentration-dependent histamine release from both cell lines. C, and C, generate prostaglandin D, in response to A23187. We conclude that dog mastocytoma cells can be established in continuous culture, thus providing a system for studying mast cell biology, including growth and development.

Mast cells are thought to play an important role in a variety of diseases, including asthma, interstitial fibrosis, and neoplasia, as well as inflammatory, metabolic, and reparative reactions (1-4). They produce a variety of granule-associated and newly formed mediators in response to both immunologic and nonimmunologic stimuli. Until recently, no preparation of pure cultured human or dog mast cells was available, and previous studies have relied on rodent mast cells (5-8), or on acutely isolated human lung mast cell lines (9, 10). Studies of rodent mast cell lines have provided basic information about mast cell biology, but there are important anatomic, biochemical, immunologic, and pharmacologic differences between rodent and human or dog mast cells. These differences indicate that dog mast cells are more closely related to human mast cells than to rodent mast cells. The isolated human lung mast cell preparation has generated a great deal of important information about mast cell biology, but the cell yield is very small and the cells are ob-

(Received in original form December 28, 1989 and in revised form May 15, 1990) Address correspondence to: Warren M. Gold, M.D., Cardiovascular Research Institute, Box 0130, University of California, San Francisco, CA 94143-0130. Abbreviations: allergic dog serum, ADS; bovine serum albumin, BSA; calcium- and magnesium-free, CMF; calcium- and magnesium-free phosphate-buffered saline, CMF-PBS; Dulbecco's modified Eagle's medium, DME; Hanks' balanced salt solution, HBSS; lactate dehydrogenase, LDH; modified Eagle's medium, MEM; sodium chloride, NaCl; prostaglandin O 2 , PGD2 ; supplemented calf serum, SCS. Am. J. Respir. Cell Mol. BioI. Vol. 3. pp, 413-420, 1990

tained from different donors who may vary in genotype and phenotype. We have previously established four lines of dog mastocytoma cells propagated in athymic nude mice. These cells resemble normal dog and human mast cells both morphologicallyand functionally (11, 12). They release histamine in response to calcium ionophore A23187 and compound 48/80, and generate lipoxygenase- and cyclooxygenase-derived metabolites of arachidonic acid in response to calcium ionophore A23187 and exogenously added arachidonic acid (13, 14). The presence of functional IgE receptors is evidenced by release of histamine in response to ragweed antigen, following passive sensitization with IgE-rich allergic dog serum (ADS). We have characterized protease content and releasability (15-18) and proteoglycan content (19) in these cells. The availability of a permanent source of well-characterized cells has allowed us to study a wide range of effects of mast cell mediators on airway cells and systems. We have investigated the effects of mast cell-derived mediators on ion flux in tracheal epithelial cells (20), airway smooth muscle responsiveness (21), modulation of nonadrenergic neural control of airway tone (22), and regulation of secretion by serous cells (23). The major experimental limitation of these acutely isolated dog mastocytoma cells is that they remain functionally viable for less than 1 wk after disaggregation, do not proliferate in culture, and therefore cannot be used to study the regulation of mast cell growth, differentiation, and expression of phenotype. We describe here the successful establishment of two lines of dog mastocytoma cells in permanent culture in vitro. These lines have been in continuous culture for more than

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1 yr and provide us with a permanent source of wellcharacterized cells to study the regulation of mast cell biology, including regulation of mast cell growth and differentiation.

Materials and Methods The experimental protocols were developed in accordance with the Revised PHS Policy on Humane Care and Use of Laboratory Animals, the published "Guiding Principles in the Care and Use of Laboratory Animals" approved by the Council of the American Physiological Society, and specific protocols approved by the Committee on Animal Research of the University of California, San Francisco. Reagents Dulbecco'smodified Eagle's medium (DME)-H16 mixed with 50% Ham's F12, modified Eagle's medium-Hanks' balanced salt solution (MEM-HBSS), Hepes buffer, Joklik-modified MEM, penicillin, streptomycin, amphotericin B, glutamine, and supplemented calf serum (SCS) were all purchased from the Cell Culture Facility, University of California, San Francisco. Unless noted otherwise, all other reagents used were from Sigma Chemical Co. (S1. Louis, MO). Preparation of Cell Cultures Isolated dog mastocytoma cells from C 1 and C2 lines passaged serially 30 to 50 times in athymic nude mice were prepared by methods reported previously (11). Briefly, mastocytoma tumors were excised from anesthetized mice under sterile conditions, minced finely with iris scissors, and incubated in 50 ml Joklik-modified MEM containing 25 mM Hepes buffer, 2 g/100 ml bovine serum albumin (BSA), 80 U/ml collagenase type vn, and 1 Jtg/ml DNAse type I (Calbiochem, San Diego, CA) in a Gyrotory" Water Bath Shaker (Model 076; New Brunswick Scientific Co., Inc., Edison, NJ) at 37° C with a gas mixture of 95 % O2 and 5 % CO2 introduced directly into the flask. After 90 min, the disaggregated tumor was filtered through cheesecloth, and the filtrate washed 3 times in calcium- and magnesiumfree (CMF) Tyrode's buffer to remove all traces of collagenase. Mast cells were identified by metachromatic staining with toluidine blue dye, and viability was assessed by exclusion of erythrosin B dye. Cell Culture Conditions Freshly isolated mastocytoma cells were plated at 2 X 1{)5 cells/ml in DME-HI6 mixed with 50% Ham's F12 supplemented with 2 mM glutamine, 250 mg/liter histidine, 25 mM Hepes buffer, 1 Jtg/ml amphotericin B, 50 Jtg/ml gentamicin, and either SCS or IgE-rich ADS obtained from the University of Califomi a, Davis allergic dog colony (24). Cultures were observed daily, fresh media added every 3 d, and the cells passaged weekly. Cultures were incubated at 37° C in a water-saturated atmosphere containing 5 % CO2 in room air. Cell growth and viability were measured by hemocytometer counts using erythrosin B dye. For studies examining the effect of serum concentration on growth, cells were plated at 1 X 10s/ml in DME-HI6 mixed with 50% Ham's F12 containing 0 to 10% ADS. After 7 d in culture,

cells were harvested and cell number and viability determined. Histochemical Staining Cell pellets containing 5 X 1{)6 mastocytoma cells were fixed in Mota's basic lead acetate for 18 h. Pellets were then processed by dehydration in 50 to 100% acetone for 20 min at each concentration and embedded in glycol methacrylate, and 2 .5-Jtmsections were cut using a JB-4 retractable microtome. Sections were stained with 1.0% toluidine blue for 5 min. Biochemical and Functional Characterization For studies of histamine content and release, cells were washed 3 times in CMF Tyrode's and then suspended in complete Tyrode's buffer containing 25 mM Hepes and 0.1% BSA. Replicate aliquots of cells were incubated with calcium ionophore A23187 (0.01 to 3.0 JtM), compound 48/80 (0.05 to 50 Jtg/ml), or ragweed antigen (0.43 to 4,300 ng/ml; Hollister Stier, Spokane, WA). Included in each study were tubes containing cells but no releasing agent for calculation of spontaneous histamine release. Incubations were conducted at 37° C in polypropylene tubes with a final reaction vol of 1 ml. After 30 min, the reaction was stopped in ice and the cells were centrifuged at 450 X g for 10 min. Supernatants were separated from cell pellets and perchloric acid was added (0.2 M, final concentration) to lyse the cells. Samples were stored at -20° C until assayed. Histamine was measured by a spectrofluorometric method modified for automated analysis by Siraganian (25). Histamine release was calculated as the amount of histamine present in the supernatant expressed as a percentage of the total histamine present in both the supernatant and pellet fractions. Tryptic and chymotryptic activity were measured as described previously (16-18). Briefly, cell pellets containing 1 X 107 mastocytoma cells were extracted in 10 mM bis2hydroxyethylimino-trishydroxymethyl methane (bis-Tris) (pH 6.1) containing 2 M sodium chloride (NaCl) and sonicated for 15 s. The resulting supernatants were assayed for tryptic activity with 0.11 mM N-benzoyl-L-val-gly-arg-p-nitronilide in 60 mM Tris-HCI (pH 7.8), 2 mg/ml heparin, and, for chymotryptic activity, 0.11 M N-succinyl-L-phe-L-pro-Lphe-p-nitroanilide (Bachem Inc., Torrance, CA) in 30 mM Tris-HCI (pH 8.0) and 1 M NaCl. Both assays were performed at 37° C by following the change in optical density at 394 om. Specific activities of 1,740 OD/min·ml-1·mg- 1 (tryptase) and 110 OD/min·ml-1·mg- 1 (chymase) were used to compute milligrams of protease activity per sample. Results are expressed as nanograms of protease per lQ6 cells. For measurement of prostaglandin D2 (PGD 2) generation, replicate aliquots of 2 X 106 cells were incubated in tubes containing calcium ionophore (0.01 to 3.0 JtM) in 2 ml HBSS (pH 7.4) containing 3 g/liter bovine gamma globulin, for 15 min at 37° C. At the end of the incubation, the reaction was stopped on ice, and cells centrifuged at 450 X g for 10 min. PGD 2 was measured by radioimmunoassay of unextracted supernatant within 30 min of completion of the experiment, using an antiserum raised in rabbits in our laboratory (14). Data are expressed as nanograms of PGD 2 per

DeVinney and Gold: Continuous Culture of Dog Mastocytoma Cells

415

1()6 cells. Lactate dehydrogenase (LDH) was measured by a spectrofluorometric method using Sigma LD-L reagent. Statistical Analysis Data are expressed as mean ± 1 SD. Effects on growth and mediator release were evaluated using one-way analysis of variance (ANOVA), and differences between groups were evaluated using the Newmann-Keuls multiple range test (26). A probability of P < 0.05 was considered significant.

CELLS/ML

Results Both C\ and C2 cells have been in culture in our laboratory for over 1 yr. Optimal cell growth in both C 1 and C2 occurred in DME-H16 mixed with 50% Ham's F12 supplemented with IgE-rich ADS. The effect of ADS on cell growth is concentration-dependent' (Figure 1). Cell growth in both C\ and C2 was supported best in DME-HI6 mixed with 50% Ham's F12 containing at least 2% ADS with no significant difference between 2, 5, and 10% ADS. Culture in SCS supported cell proliferation, but not as well as ADS (Figure 2). Cell density after 7 d in culture was significantly more (54% more for C 1 and 91% more for C2 ; P < 0.05) in 5 % ADS-supplemented media than in DME-HI6 mixed with 50% Ham's F12 supplemented with 5% SCS for both C\ and C2 • Log-phase doubling time was also enhanced by culture in ADS, increasing by 72 % for C 1 and 20% for C2 • C\ cells grow in suspension and are passaged weekly by dilution to 1 to 2 X lOS cells/ml with fresh culture media. At this plating density, cells reach a saturation density of 1.5 to 1.7 x 1()6 cells/ml after 10 to 12 d in culture. Under these conditions, C\ cells have been in continuous culture for 55 passages, show sigmoidal log-phase growth, and have a log-phase doubling time of 37.4 ± 18.7 h (Figure 3). C z cells show a characteristic growth pattern (Figure 4a, inset). At low densities (l to 5 X lOS/ml) , greater than

SFM

5% SCS

5% ADS

Figure 2. Effect of serum type on cell growth in C. (closed bars) and Cz (open bars) cells. Data are presented as cells/ml 7 dafter plating in DME-HI6 mixed with 50% Ham's F12 alone (serum-free media [SFM]) or supplemented with either 5 % ADS or 5 % SCS and are the mean ± 1 SD for six experiments (C\) and three experiments (C2) . ANOVA showed that the increase in cell number in ADS-supplemented media was significantly higher than that in SCS-supplemented or serum-free media for both cell lines (P < 0.05).

90 % of the cells adhere to tissue culture plastic, either as single cells or small groups of cells. As cell density increases to 1 x 1()6 cells/ml, the cells become associated closely with one another in morulae of various sizes, with approximately 30 % of the cells growing in suspension. When these suspension cells are transferred to a fresh culture flask, about 90 % of the cells adhere. C, cells are passaged weekly by pipetting off the nonadherent cells and removing the adherent cells by treatment with 0.05% trypsin for 5 min. Adherent and nonadherent cells are pooled, spun at 200 X g for 5 min to pellet cells, and plated in fresh culture flasks at 1 to 2 x lOS cells/ml. Cells show sigmoidal log-phase growth (Figure 3) and reach a saturation density of 1.0 to 1.2 X 1()6 cells/ml after 10 to

6

CELLS/ML

10

CElLS/ML

4

10 -J-----r-----r----.-------,......-----. 2 4 6 10 8 o

SERUM CONCENTRATION (% vlv)

o

5

10

15

DAY IN CULTURE

Figure 1. Effect of serum concentration on cell growth in C 1 (closed squares) and C2 (open squares) dog mastocytoma cells. Data are presented as cells/ml 7 d after-plating in DME-HI6 mixed with 50% Ham's F12 supplemented with 0% to 10% ADS and are the mean ± 1 SD for n = 6 experiments (C\) and n = 3 experiments (C2) .

Figure 3. Growth curves for cultured C\P38 (closed squares) and C2P18 (open squares) cells cultured in DME-HI6 mixed with 50% Ham's F12 supplemented with 5% ADS. Data are expressed as mean ± 1 SD of three replicate cell counts (P = passage number).

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tain 0.27 ± 0.15 j.tg/l06 cells tryptic activity (n = 24) but no chymotryptic activity (n = 23). C2 cells contain 1.61 ± 0.44 j.tg/1Q6 cells tryptic activity (n = 23) and 8.11 ± 2.95 j.tg/l06 cells chymotryptic activity (n = 23). Functional Characterization Spontaneous histamine release was measured by incubating cells in Tyrode's buffer alone under conditions otherwise identical to the rest of the experiments. Spontaneous histamine release for all experiments was 4.2 ± 2.3 % (n = 30) and did not differ between cell lines (P = 0.43). Calcium ionophore A23187 (0.01 to 3.0 j.tM) caused a concentration-dependent release of histamine from both cell lines (Figure 6). Maximum histamine release from C 1 was 69.8 ± 16.9% (n = 11), and ranged from 46.7 to 91.3%. Maximum release from C2 was 33.4 ± 16.4% (n = 7) and ranged from 14.2 to 56.1%. We measured LDH release from cells as an indicator of cytoxicity. LDH release from C 1 cells after stimulation with 0.3, 1.0, and 3.0 j.tM A23187 was not significantly different from unstimulated cells (unstimulated: 3.2 ± 2.2%; 3j.tM A23187-stimulated: 3.0 ± 2.8%). In C2 cells, 1 j.tM A23187 was not cytotoxic (10 ± 0.4% LDH release), but 3 j.tM was. Compound 48/80 (0.1 to 50 j.tg/ml) caused histamine release from C 1 cells that was not cytotoxic or concentration-dependent, but was prevented by pretreatment with 2-deoxyglucose. Compound 48/80 did not cause significant release from C2 cells. Both C\ and C2 cells have functional IgE receptors as evidenced by the ability to release histamine after challenge with ragweed antigen after the cells have been in culture in IgE-rich ADS for at least 48 h. Ragweed antigen caused histamine release from both cell lines (Figure 7). Maximum histamine release from C 1 cells was 17.5 ± 11% (n = 9), and from C2 , 13.7 ± 2.2% (n = 6). This response was Figure 4. Photomicrograph of cultured mastocytoma cells stained with toluidine blue as described in the text. (a) C 2P19; (b) C 1P43. Total original magnification = x l ,575; bar = 10 #Lm. Inset shows characteristic growth pattern of C2P19 cells on tissue culture plastic, 6 d after plating. P = passage number. Total original magnification = x225; bar = 50 #Lm.

12 d. C2 cells have been in continuous culture for 26 passages and have a log-phase doubling time of 49.3 ± 12.5 h. Morphology C 1 and C2 cells contain cytoplasmic granules that stain metachromatically with toluidine blue (Figures 4a and 4b). Both C 1 and C2 are approximately the same size, with cell diameters ranging from 10 to 20 j.tm. Cells from the C 1 line are more heavily granulated than C2 cells. Biochemical Characterization Cells of both the C 1 and C2 lines contain histamine as measured by spectroftuorometric methods. C\ cells contain 0.46 ± 0.18 pg histamine/cell (n = 12), and C2 cells contain OJ17 ± 0.04 pg/cell (n = 6). Both C 1 and C2 cells contain the neutral protease tryptase and C2 also contains chymase (Figure 5). C 1 cells con-

12 10 Protease Content (Activity in 119/10 6 cells)

8 6 4 2 0 C1

C2

Figure 5. Content of granule-associated proteases in cultured C 1 and C 2 mastocytoma cells. Values represent tryptic (hatched bars) and chymotryptic (open bar) activity extractable at high ionic strength. Tryptic substrate is N-benzoyl-L-val-gly-arg-p-nitroanilide and chymotryptic substrate is N-succinyl-L-phe-L-pro-L-phe-p-nitroanilide. Data are expressed as mean ± 1 So.

DeVinney and Gold: Continuous Culture of Dog Mastocytoma Cells

100

417

30

80 HISTAMINE RELEASE (%)

PGD 2

60

20

GENERATION (ng/l0 6 cells)

40

10

20

,

0Q:=~:;:j-==~~~-~--......,i .01

0.1

1.0

o

10

A23187 CONCENTRATION (jJ.M)

significantly greater than ragweed-induced histamine release from cells cultured in 5 % SCS: maximum histamine release was 3.8 ± 1.3% (n = 4) from C, cells and 1.3 ± 1.7% (n = 18) from C2 cells. Cells from both the C, and C 2 lines generate PGD 2 in a concentration-dependent manner after exposure to calcium ionophore (Figure 8). Maximum PGD 2 generation from C, cells was 4.1 ± 1.9 ng/l O' cells (n = 5) and 25.1 ± 3.4 ng/IO' cells (n = 3) from C 2 cells. The establishment of a permanent cell line depends not only on the ability to propagate cells; but also on the ability to successfully freeze and thaw cells. To test this possibility,

30

20

10

.01

.1

0.1

1,0

10

A23187 CONCENTRATION (uM)

Figure 6. Calcium ionophore A23187-induced histamine release from C, (closed squares) and C2 (open squares) cells in cultures. Data are expressed as mean ± 1 SD, n = 13 experiments (C,) and n = 5 experiments (C2) . ANOVA showed that the effect of calcium ionophore A23187 was statistically significant (P < 0.05 relative to 0 ionophore), and the threshold concentration of calcium ionophore A23187 was 0.1 JlM for C, and 0.3 JlM for C2 •

HISTAMINE RELEASE (0/0)

.01

10

100

RAGWEED ANTIGEN (Ilg/ml)

Figure 7. Antigen-induced histamine release from C, (closed squares) and C2 (open squares) cells. Data are expressed as mean ± 1 SD, n = 7 experiments (C,) and n = 6 experiments (C2) . ANOVA showed that the effect of antigen was statistically significant (P < 0.05relative to 0 antigen), and the threshold concentration of antigen was 4.3 x 10-3 Jlg/ml for both cell lines.

Figure 8. Calcium ionophore A23187-induced PGD2 generation by C, (closed squares) and C2 (open squares) cells. Data are expressed as mean ± 1 SD, n = 4 experiments. ANOVA showed that the effect of A23187 was statistically significant (P < 0.05 relative to 0 A23187), and the threshold concentration of calcium ionophore A23187 was 0.3 JlM for both cell lines.

C, and C 2 cells in log-phase growth were frozen in 1 ml 90% FCS-I0% DMSO at 4 X lQ6 cells/vial, kept at -70 0 C for 3 wk, and thawed. After five passages, growth characteristics for C. and C 2 were unchanged after thawing.

Discussion In this report, we describe the successful establishment of two lines of dog mastocytoma cells in continuous culture in vitro. Cells proliferate in culture and have a population doubling time of 40 to 60 h. Both lines resemble normal dog mast cells morphologically and functionally. They contain cytoplasmic granules that stain metachromatically with toluidine blue. Cultured dog mastocytoma cells contain histamine and generate PGD 2 , both of which are released in response to calcium ionophore A23187. These cells have functional IgE receptors and release histamine in response to antigen challenge. Like human mast cells, cultured dog mastocytoma cells contain granule-associated tryptic and chymotryptic proteases. Media supplemented with IgE-rich ADS supports cell growth and viability better than media supplemented with 5 % SCS. Although both sera supported growth, culture in 2 to 10% IgE-rich serum caused the greatest increase in cell number, and maintained cell viability at greater than 90 % over 7 d in culture. IgE is known to play a role in mast cell growth and differentiation. Kawanishi (27) found that IgE acts as a co-factor for growth and differentiation of mouse IL-3-dependent mast cells. Although IgE alone did not support cell proliferation, addition of IgE to culture media containing IL-3 enhanced mast cell proliferation. IgE can also regulate the expression of IgE receptors in rat basophilic leukemia (RBL) cells (28). Cross-linking of IgE receptors has been shown to stimulate IL-3 and IL-4 production in both IL-3-dependent and -independent mast cell lines (29, 30). Sensitization of murine IL-3-independent cells with IgE, followed by culture in the presence of specific antigen or anti-IgE, caused cell proliferation, measured by PH]thy-

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TABLE 1

Comparison of three different mast cell preparations Mast Cell Preparation

Histamine Content (pglcell)

C I dog mastocytoma cell C 2 dog mastocytoma cell Human lung mast cell Rat peritoneal mast cell Mouse bone marrow-derived mast cell

0.46 0.07 3.6* 15.2 0.14

* Reference

PGD z Generation (ng per 10 6 cells)

Histamine Release (%) A23187-induced

± 0.18 ± 0.04

69.8 33.4

± 5.8* ± 0.14**

64.6

± 16.9 ± 16.4

± 0.9§ 28 ± 7.2§§

Antigen-induced

± ± 25 ± 6 ± 11 ±

17.5 13.7

11 2.2 9t 4*

2.2§§

A23187-induced

4.1 25.1

± 1.9 ± 3.4

± 33.41 5.8 ± 3.3§§

50.6

Antigen-induced

50 20 1.6

± 37t ± 6* ± 0.2§§

9.

t Reference 10.

*Reference 31.

Reference 33. , Reference 32. ** Reference 29. §§ Reference 30. Note: Plain numbers indicate mean §

± SD; bold italicized numbers indicate mean ± SEM.

midine incorporation in the absence of an exogenous source of IL-3. Proliferation was shown to be due to autocrine production ofIL-3 and IL-4: neutralizing antibodies inhibited proliferation. It is possible that cross-linking of IgE receptors by IgE aggregates found in ADS may be stimulating autocrine production of IL-3 and IL-4 in our dog mastocytoma cell lines. Culture of the cells in IgE-containing media prevented the elimination of IgE receptors from the cell surface, and increased the expression of new receptors. IgE alone does not act as a growth factor but appears to act synergistically with IL-3 and other serum factors as a mast cell differentiation factor. Whether IgE is the only factor in ADS responsible for the enhanced proliferation and maintenance of cell viability has yet to be determined. The two cell lines we describe differ in granularity and histamine content. Mediator content and release in these cells are within the ranges reported for human (9, 10) and rodent (31-35) mast cells (Table 1). Histamine content in C 1 falls between that reported for mouse bone marrow-derived mast cells and human lung mast cells, and in C 2 is greater than that reported for mouse mastocytoma P815 cells (36). Calcium ionophore A23187-induced histamine release, PGD 2 generation, and IgE-mediated histamine release from both lines are in the range of that reported for human, mouse, and rat mast cells. Mediator content has remained stable in these cells over the time in culture and has not changed significantly from levels reported previously in nude mouse-propagated cells. Histamine content for C 1 from acutely disaggregated cells and cultured cells, passages 2, 21, 30, and 46, was 0.45 ± 0.07, 0.48 ± 0.02, 0.33 ± 0.06, and 0.46 ± 0.04 pg/cell (mean ± 1 SD), respectively. Histamine content for C 2 from acutely disaggregated cells and cultured cells, passages 1, 11, and 14, was 0.8 ± 0.04,0.06 ± 0.07, and 0.11 ± 0.09, respectively. These cell lines were isolated from the same dog at two different times during the animal's illness: C 1 was obtained by excisional biopsy of a single mastocytoma lesion at the presentation of the tumor, whereas C2 was obtained during the final illness when the mastocytoma had recurred at the site of the original lesion, metastasized to liver, spleen, and lymph nodes, and was associated with mast cell leukemia.

Both specimens were adapted successfully in athymic nude mice before establishment in continuous culture. In both samples obtained from the original tumor and from the nude mouse-adapted lines, we found C 1 cells to be more heavily granulated, contain more histamine than C2 cells (11), and to differ in protease content (17) (G. H. Caughey, personal communication). These differences are not due to establishment in continuous culture but to differences inherent in the original tumor material. Until recently, most studies of mast cells have relied on rodent mast cells. These studies have provided basic information about mast cell biology, but there are important major differences between rodent and human or dog mast cells. Rodent mast cells differ from human and dog mast cells in their release mechanisms, modulation by autonomic receptors, content of histamine and other mediators, and regulation of growth and differentiation (37-39). Rodent mast cells also differ from human and dog mast cells in protease type and content. Rat mast cells contain two proteases (RMCP 1 and RMCP 2) that show chymotryptic substrate specificity and another protease with tryptic substrate specificity (4042). Human and dog mast cells contain both tryptic and chymotryptic proteases. Schecter and associates (43) found that antibodies against human tryptase react with normal dog skin mast cells but not with rat skin mast cells. Antibodies isolated against dog mastocytoma chymase react with chymase isolated from normal dog and human mast cells but not with rat mast cell chymase. These immunologic, biochemical, and pharmacologic observations indicate that dog mast cells are more closely related to human mast cells than to rodent mast cells. One of the most exciting areas of mast cell research involves the regulation of mast cell phenotype. Investigators have described two classes of rodent mast cells based on morphologic appearance, histochemical staining, mediator content, and response to degranulating stimuli and pharmacologic agents that modulate degranulation (44). Both phenotypes appear to develop from a single precursor cell in the bone marrow (45). Studies in vivo (46) and in vitro (31, 32) have shown that the mast cell phenotype is not fixed, but can change depending on its microenvironment. Dog (47-51) and human mast cells (52-60) do not conform to the strict

DeVinney and Gold: Continuous Culture of Dog Mastocytoma Cells

two-type classification of rodent mast cells, but do appear to have phenotypes that can respond to changes in the microenvironment (33). Because there is no permanent source of cultured human mast cells, we believe our cultured dog mastocytoma cells provide an excellent source of cells (closely resembling human mast cells) to study this phenomenon. In summary, we have shown that dog mastocytoma cells can be established in continuous culture in vitro. These are the only lines of nonrodent mammalian mast cells in continuous culture. Cells have remained functionally stable after as many as 55 passages and resemble normal dog and human mast cells morphologically and functionally. Cultured dog mastocytoma cells will provide us with a permanent source of well-characterized cells to study the regulation of mast cell growth and differentiation. Acknowledgments: The work discussed in this report was supported, in part, by

Program Project Grant HL-24136 from the National Institutes of Health. The writers thank Aundrea Wilson for excellent technical assistance, Linda Prentice for preparing samples for histochemistry, Lynne Calonico for her assistance with photomicroscopy, and Marguerite Santy for her assistance in preparation of the manuscript.

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