Development of mast cells in vitro. II. Biologic function of cultured mast cells.

Mast cells were obtained by long term culture of rat thymus cells on rat embryonic fibroblast monolayers. Pure mast cell preparations obtained culture were incubated with 125I-labeled rat E myeloma protein to study receptors for IgE on their surface. When the cells were obtained after 35 to 45 days culture, the average number of receptors per mast cell was 100,000 to 400,000. An equilibrium constant of the binding reaction between their receptor and rat IgE was in the order of 108 M-1. The histamine content of the cultured mast cells was 0.2 to 5 mug/106 cells. The measurement of histamine content in mast cells recovered after different periods of culture suggested that the histamine content increased with maturation. Even after 45 to 50 days culture, the histamine content of cultured mast cells was significantly lower than that in rat peritoneal mast cells. The cultured mast cells were passively sensitized in vitro with rat IgE antibody against Nippostrongylus brasiliensis. The sensitized cells released histamine upon incubation with the antigen. It was also found that cultured mast cells released histamine upon exposure to compound 48/80. These results indicated that cultured mast cells have physiologic functions similar to those of normal rat mast cells, but they have not reached full maturation.     

Development of rat mast cells in vitro. I. Differentiation of mast cells from thymus cells.

Mast cells were differentiated by long-term culture of rat thymus cells on rat embryonic fibroblasts monolayers. Mature mast cells obtained in the culture were morphologically similar to normal peritoneal and thoracic mast cells and possessed specific receptors for IgE on their surface. In culture, blast cells appeared on the monolayer several days after seeding of thymus cells. These cells developed into young mast cells in the monolayer and became free in the culture medium with maturation. Receptors for IgE were detected on the surface of mastoblasts which contained a small amount of metachromatic granules. Evidence was obtained which suggested that the number and/or affinity of the receptors for IgE increases with maturation of mast cells. It was found that some mast cells differentiated from monolayers of embryo cells without seeding thymus cells. The present experiments, however, clearly showed that mast cells can be differentiated from thymus cell culture without monolayer. It appears that both thymus and embryo tissues contain precursors of mast cells.     

Mucosal mast cells. I. Isolation and functional characteristics of rat intestinal mast cells

We have developed a procedure for the dispersion of mast cells from the intestinal lamina propria (LP) and epithelium of rats infected with the intestinal nematode, Nippostrongylus brasiliensis. The dispersed cells are morphologically and histochemically similar to intestinal mucosal mast cells (MMC) in situ and are distinguishable from peritoneal mast cells (PMC). MMC derived from the LP or epithelium of parasitized animals secrete histamine in response to the specific parasite antigens as well as anti-IgE. Unlike PMC, these cells are unresponsive to the basic secretagogues 48/80 and bee venom peptide 401. Similarly, bee venom peptide 401 conjugated with dansyl chloride binds to PMC and mast cells in the thymus and intestinal serosa, but not to mast cells in or derived from the intestinal LP and epithelium. Studies on PMC treated by the intestinal cell isolation procedure show that the functional characteristics of the MMC cannot be solely attributed to the isolation procedure. Thus, MMC have been isolated and shown to be morphologically, histochemically, and functionally different from PMC, as suggested by previous in vivo studies of the normal intestine.     

Mucosal mast cells. II. Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells

Functional mast cells have been isolated from the lamina propria of the small intestine of rats infected with the nematode Nippostrongylus brasiliensis. The cells released histamine on challenge with specific antigen, anti-rat IgE, concanavalin A, and calcium ionophores but were less responsive than peritoneal mast cells (MMC) from the same animals. Intestinal mucosa mast cells (PMC) were refractory to the action of the basic secretagogues peptide 401 from bee venom and compound 48/80. The anti-allergic compounds disodium cromoglycate (less than or equal to 10(-3) M), AH 9679 (less than or equal to 10(-4) M), and theophylline (less than or equal to 10(-2)) did not inhibit antigen-induced histamine secretion by MMC, although these compounds were effective against PMC. In contrast, doxantrazole (10(-5) to 10(-3) M) inhibited the secretion of histamine from both MMC and PMC in a comparable dose-dependent fashion. Thus, we have established that mast cells from different sites are functionally heterogeneous not only in their response to various stimuli for histamine secretion, but also in their responses to different pharmacologic modulators of secretion. It cannot be assumed that anti-allergic compounds effective against mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites. The extent of this functional heterogeneity must be established, and its investigation may provide new insights into the biochemical events involved in mast cell secretion.     

Long-term in vitro culture of murine mast cells. III. Discrimination of mast cells growth factor and granulocyte-CSF

Long-term in vitro growth of murine mast cells was dependent on the presence of a mast cell growth factor (MCGF) present in media conditioned by mitogen-activated splenic leukocytes or by various murine leukemic cell lines. MCGF shared a number of properties with granulocyte colony-stimulating factor (G-CSF). Both factors were present in media conditioned by the myelomonocytic leukemic WEHI-3 and the T cell lymphoma, LBRM-33 cell lines. They were relatively sensitive to trypsin treatment, and were resistant to boiling temperature. NZB mice that failed to respond to WEHI-3-derived G-CSF also failed to respond to MCGF. MCGF differed from G-CSF, however, in sensitivity to neuraminidase and lactoferrin, an inhibitor of macrophage CSF production, suppressed G-CSF production by WEHI-3 cells without affecting MCGF production. Furthermore, peritoneal cells produced G-CSF but not MCGF when stimulated with lipopolysaccharide. In vitro production of MCGF by normal spleen cells required the presence of T lymphocytes and is relatively macrophage-independent. The role of T cells in the maturation and growth of mast cells and the physiologic function of MCGF are discussed.     

Intestinal mast cells during experimental ancylostomiasis.

The alterations of mast cell counts induced in the gut of swiss albino mice by infection with Ancylostoma caninum larvae have been studied, both in single and multiple infections. It was found that mast cell rise was greater in females than males. In singly infected animals, mast cell increase was influenced by the infective dose of inoculated larvae. In case of repeatedly infected mice, mast cell counts were decreased because of their earlier degranulation.     

Uptake of 5-hydroxytryptamine by mast cells in vivo: a cytofluorometric study of mast cells and individual mast cell granules.

Uptake, distribution and turnover of 5-Hydroxytryptamine (5-HT) was studied by cytofluorometric analysis of whole mast cells and individual granules. Injection of 5-HT as well as 5-Hydroxytryptophan (5-HTP) intraperitoneally or subcutaneously resulted in a parallel uptake of 5-HT in cells and granules. Intraperitoneal injections of 5-HT in such small quantities that may be available under physiological conditions resulted in an increase in fluorescence intensity of the mast cells, indicating a very efficient uptake mechanism for 5-HT in vivo. Much larger doses of 5-HTP were required to obtain a corresponding uptake of 5-HT in the mast cells. The 5-HT was rather rapidly taken up in the granules and eliminated very slowly, at the same rate both from granules and mast cells. The low elimination rate confirms our previous findings that the turnover of 5-HT is much lower in mast cells than in other amine containing cell systems. The combination of an extremely efficient, rapid uptake of 5-HT with a slow elimination suggests a specific function for mast cells in the regulation of free amine concentrations in tissues.     

A selective stain for mast cells.

A selective stain for mast cells in tissue sections is presented. The procedure is based on the resistance to destaining with absolute ethanol-acetic acid of the complex acid mucopolysaccharide-Toluidine Blue reinforced with ferrioxamine B.     

Glycosaminoglycans in rat mucosal mast cells.

Rats were infected with the nematode Nippostrongylus brasiliensis, resulting in an approx. 5-fold increase in the number of mucosal mast cells and the histamine content of the intestinal (jejunum) wall. After injection of the infected animals with inorganic [35S]sulphate, a similar increase in the yield of labelled intestinal glycosaminoglycans was observed, compared with uninfected control rats. Autoradiography showed a highly selective labelling of the numerous mucosal mast cells and of the few connective-tissue mast cells in the subserosal region of the bowel. Analysis of the labelled polysaccharide from the infected animals showed that almost 60% of this material consisted of oversulphated galactosaminoglycan, whereas heparin-related polysaccharides accounted for only 13%. The galactosaminoglycan contained 4-monosulphated and 4,6-disulphated N-acetylgalactosamine residues in approx. 5:1 molar ratio, both being linked to D-glucuronic acid residues; the occurrence of L-iduronic acid units could not be excluded. No significant difference in structure was found between this polysaccharide and the corresponding component isolated from uninfected rats. It is concluded that the major polysaccharide produced by rat mucosal mast cells in vivo is an oversulphated galactosaminoglycan rather than heparin.     

Distribution of chymase-containing mast cells in human bronchi.

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.     

Ultrastructural studies of human basophils and mast cells.

Ultrastructural studies of human mast cells (HMCs) and basophils (HBs) are reviewed. Sources of HMCs include biopsies of tissue sites and in situ study of excised diseased organs; isolated, partially purified samples from excised organs; and growth-factor-stimulated mast cells that develop de novo in cultures of cord blood cells. Sources of HBs for study include partially purified peripheral blood basophils, basophils in tissue biopsies, and specific growth factor-stimulated basophils arising de novo from cord blood cells. The ultrastructural studies reviewed deal with identity, secretion, vesicles, recovery, and synthesis issues related to the biology of these similar cells.