Mast cell clones: a model for the analysis of cellular maturation.

Abstract

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc~R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance Ig secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of I mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35S04 into chondroitin sulfates rather than heparin. 1-hese findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc, R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.We have previously reported methods to clone mast ceils with normal karyotypes from mouse hematopoietic tissue in vitro (30). Our cloned mast cells contain less histamine than normal mouse peritoneal mast cells and resemble immature mast cells by morphology. Others have described similar findings with uncloned cells (22,32,38,42,43,51,53). Although some mast cells synthesize heparin when fully differentiated (23, 54), certain mast cell tumors are devoid of heparin and incorporate 3BSO4 into chondroitin sulfate exclusively (18). Histochemical evidence suggests that normal immature connective tissue mast cells (6) and the mast cells in the intestinal lamina propria of rodents (25, 50) also may synthesize glycosaminoglycans other than heparin, but this notion has not been confirmed directly.In this report, we show that cloned mast cells closely resemble, by ultrastructure, immature mast cells found in vivo.Cloned mast cells incorporate 35804 preferentially into chondroitin sulfates, confirming histochemical evidence that immature mast cells contain little heparin. In addition, cloned mast cell proliferation, cytoplasmic granule synthesis, and mediator storage can be modulated in vitro, permitting direct analysis of mast cell maturation. MATERIALS AND METHODS AntiseraLyt-1.2 and Lyt-2.2 antisera, prepared as described (45), were kindly donated by F. W. Shen~; monoclonal antibody against Thy-l.2 (mc-a-Thy-l.2) was donated by Ed Clark2; and mc-a-Lyt-1 and mc-a-Lyt-2 were gifts from J. Ledbetter and L Herzenberg a. Mast cells were examined for la antigens using ATH-a-ATL alloantisera (16) or mc-a-Ia (10-3.6, reference 35) generously provided by John Freed and J. M. Kupinski 4. Mast Cells...

Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell-deficient W/Wv mice. Evidence that cultured mast cells can give rise to both connective tissue type and mucosal mast cells.

“…We (1,2,15) and several other investigators (10,14,17) have noted that cultured murine mast cells express certain characteristics similar to those of MMC. MMC contain low levels of histamine (21,22), and microspectrophotometric evidence suggests that their granules contain a poorly sulfated glycosaminoglycan similar to chondroitin sulfate (23).…”

“…These findings strongly suggest that cultured mast cells or their progeny acquire the ability to synthesize and store heparin when they are transplanted to appropriate anatomical locations in vivo. This represents an important observation, since under usual conditions of suspension culture (2,10,17,19), or even when exposed to the inducing agent sodium butyrate (2), mouse mast cells synthesize chondroitin sulfates rather than heparin. It should be acknowledged, however, that histochemistry represents an indirect approach to the identification of cellular constituents.…”

“…First, cultured mast cell populations, even when not cloned, for the most part exhibit remarkably uniform characteristics (reviewed in 15). Expression of type II major histocompatibility complex (MHC) products may vary, but probably in response to differences in levels of soluble factors such as y-interferon (2,5,15,56). Second, in a recent collaborative study with Dr. T. Nakahata and his colleagues at Shinshu University, Matsumoto, Japan, we injected cloned mast cells derived from individual methylcellulose colonies into both the skin and the gastric mucosa of WBB6F~-W/W v mice (manuscript in preparation).…”

“…Although some authors have suggested that cultured mast cells may be committed to express the MMC phenotype, we (1,2,15) and Yung and Moore (8) entertained an alternative hypothesis: that many of the properties of cultured mast cells might reflect their immaturity. However, attempts to induce further maturation of cultured mast cells in vitro met with only limited success.…”

“…However, attempts to induce further maturation of cultured mast cells in vitro met with only limited success. The inducing agent sodium butyrate caused a marked inhibition of cultured mast cell proliferation, resulted in increased storage of histamine and chondroitin sulfate, and favored partial maturation of cytoplasmic granules (2,30). But the cells did not appear fully mature by ultrastructure nor did they synthesize detectable amounts of heparin.…”