Eur. J. Immunol. 1990. 20: 185-193 Rat tissue cultured mast cells zy 185 zy Bosco M. C. Chano+, Chisei Ran, Weining Huo Karol McNeillO, Hugh R. P. MillerA, Dean Befus", Jean Pierre Kineto and Arnold FkoeseO MRC Group in Allergy Research, Department of Immunologyo, University of Manitoba, Winnipeg, NIAMS, National Institutes of Healtho, Bethesda, Moredun Research Institutea, Edinburgh and Department of Microbiology and Infectious Diseasesv, University of Calgary, Calgary Characterization of rat tissue cultured mast cells* Twelve continuous rat tissue cultured mast cell (MC) lines were established by prolonged culture of rat peritoneal MC in the absence of added factors or feeder layers. Two of these lines, RCMCl and RCMC2, have been briefly described previously, seven others are now also described. Both RCMCl and RCMC2 lack a marker chromosomes present on RBL-CA10.7 cells. All lines were found to express the phenotype of mucosal MC as defined by alcian blue-positive and safranin 0-negative staining, the presence of rat MC protease I1 and a low histamine content. When analyzed for high-(FcsRI) and low-affinity (FcsRL) receptors for IgE, the various lines yielded a variety of receptor patterns. Northern blot analysis of the RNA of RCMCl, RCMC2 and RBL-CA10.7 revealed that all three cell lines contained the same mRNA species for the a , p and y subunits for FcsRI previously found in another rat basophilic leukemia cell line. Quantitation of the relative amounts of a , p and y mRNA did not correlate with the expression of the relative amounts of FceRI(a) in these cells.The relative amounts of mRNA for all these subunits of RCMC2 were equal or higher than those of RCMCl, suggesting that the low expression of FcsRI(a) on the former was a consequence of post-transcriptional events. Analysis of a RCMCl clone over a 6-month period revealed changes in the expression of both FceRI(a) and FCERL. 1 Introduction Experimental evidence indicates that in rats and mice two major types of mast cells (MC) can be distinguished. These are the connective tissue-type MC (CTMC) found in the skin, peritoneal cavity and the muscularis propria of the stomach, and the mucosal-type MC (MMC) abundant in the gut lamina propria [l-51. Phenotypically, CTMC stain alcian blue and safranin zyxwvutsrq 0 positive [6], have a high histamine content [7, 81, contain heparin [9] and, in the rat, contain rat MC proteinase I (Rh4CPI) [lo]. By contrast, MMC cannot be counterstained with safranin 0 [6]. Their hista- mine content is lower than that of CTMC 181, they contain chondroitin sulfate but not heparin [ll] and, in the rat, contain RMCPII but not RMCPI [lo]. Since it is relatively difficult to obtain large numbers of MMC from the lamina propria of the gut, studies of MMC-like cells of mice and rats have largely depended on [I 78091 * Incorporated, in part, into the Ph. D.Thesis of B. M. C. Chan and submitted in 1988 to the University of Manitoba. Supported by grants from the Medical Research Council of Canada, Ottawa, Ontario. + Present address: Tumor Virology Division, Dana-Farber Cancer Institute, Boston, MA, USA. Correspondence: Arnold Froese, Department of Immunology,The University of Manitoba, 730 William Avenue, Winnipeg, Manito- ba, Canada, R3EOW3 Abbreviations: MC: Mast cell CTMC: Connective tissue MC FcERI: High-affinity receptor for IgE FCERL: Low-affinity receptor for IgE, previously named H or FcERII. The latter designation was discontinued to avoid confusion with FcERII or CD23 found on B cells MMC: Mucosal MC RBL: Rat baso- philic leukemia RCMC: Rat tissue cultured MC RMCP: Rat MC protease R(M)pMC: Rat (mouse) peritoneal MC zyxwvut TfR: Transferrin receptor cultured MC and MC lines derived from the BM, and cultured in the presence of IL3 and/or IL4 [12, 131. Cell lines of the CTMC phenotype have become available only recently through co-culture of murine splenocytes with fibroblasts that produce a K-ras-containing murine sarcoma virus [14]. It appears that in the mouse the two MC phenotypes may be interconvertible.Thus, when factor-dependent, cultured MC derived from the BM of WBB6F*-+/+ mice are used to reconstitute MC-deficient WBB6F1-W/WV mice, they repopulate the various sites normally containing CTMC and also, based on staining, show phenotypic characteris- tics of such cells [ 151. Moreover, BM-derived MMC-like cells can be co-cultured with fibroblasts zyx in zyxw vifro t o yield cells expressing phenotypic characteristics of CTMC. Bidirec- tional changes in the phenotype of MC have also been observed [ 161. We have previously shown that prolonged culture of rat peritoneal MC (RpMC) in the absence of exogenous growth factors or feeder layers leads to the establishment of cell lines which exhibit the MMC phenotype [ 171.Two such cell lines have been partially characterized. We now de- scribe these cell lines further and report on the character- ization of additional RCMC cell lines. 2 Materials and methods 2.1 Rat basophilic leukemia cells (RBL), rat mast cells and their culture conditions Both RBL-CA10 and RBL-CA10.7 have been described previously and were maintained in tissue culture along with RBG2H3 according to established procedures but in an atmosphere of 3% COz [18]. RpMC were obtained form Wistar-ICI rats maintained in the Animal Care Facility, University of Manitoba, and purified as described previous- ly [19]. Preparations of zyxw 5 x 105 to 1 zy x lo6 cells at a purity of zy 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990 0014-2980/90/0101-0185$02.50/0