Biomedical subjects

F Levi-Schaffer

Publications and source records attributed to F Levi-Schaffer.

At least 91 records · Page 5Linked to original sources

PubMed1991

Morphological evidence for chronic mast cell activation after prolonged exposure with supernatants from chronic graft-versus-host splenocytes.

Chronic graft-vs.-host disease (cGVHD) includes a syndrome of inflammatory and fibrotic changes in some respects resembling scleroderma. In the present study we have quantitated the number of peritoneal mast cells (MC) in mice with cGVHD induced across minor histocompatibility barriers. MC were evaluated by staining with toluidine blue. The number of MC decreased significantly (by 25%) at the onset of the cGVHD fibrosis (day 12). Around day 35, MC were virtually undetectable, and started to reappear on day 130. Upon clinical recovery (day 200) a dramatic increase in MC numbers was found (about 8-fold). In addition, we evaluated by electron microscopy the morphology of peritoneal MC, obtained from normal mice and rats, that had been co-cultured on 3T3 fibroblast monolayer in the presence of splenocyte supernatants from mice with cGVHD or from control mice. After 6-8 days of continuous incubation with the cGVHD splenocyte supernatant, MC appeared to be activated, since they displayed an array of heterogenous granules. Few of the granules were dense; many were swollen and pale. Rare granule extrusion was evident. This would indicate that MC underwent a slow activation process due to a factor(s) present in the cGVHD supernatant, different from the classical acute anaphylactic activation.

Animals↗

PubMed1991

Long-term cultures of mast cells: a new model for studying the allergic response.

Mast cells are the key cells of allergic reactions and probably play also a role in chronic inflammatory reactions resulting in fibrosis. Although their biochemical and functional properties have been extensively investigated, several studies have been hampered by the absence of a tissue culture system to keep these cells alive and functionally active for long periods of time. Recently we have developed an in vitro system in which rat peritoneal mast cells are cocultured with 3T3 mouse skin derived fibroblasts (MC/3T3). Under these tissue culture conditions, mast cells do not proliferate and maintain their viability and functional activity for more than a month. This system allowed us to carry out long-term studies on the functional properties of mast cells. We have found that mast cells activated both by IgE-dependent and IgE-independent stimuli survive, and slowly replenish their histamine content. After a non-immunological challenge mast cells retain their full potential to release histamine upon a repeated similar challenge. In contrast, immunologically challenged mast cells become partially unresponsive to a similar activation event for up to 3 weeks. By exploiting this long-term culture system we described a novel type of mast cell activation induced by cytokines. The onset of this activation is slow and its course appears to be chronic-continuous, very different from the classical anaphylactic type activation that is completed within few minutes. Since MC/3T3 release higher amounts of histamine, this system is a sensitive tool to investigate the antiallergic properties of various drugs. By employing MC/3T3 cultures we were able to show that the gold salt auranofin inhibits histamine release from mast cells stimulated by different secretagogues. In addition, salbutamol inhibited histamine release from repeatedly challenged mast cells; and nedocromil sodium was effective in preventing mast cell activation when incubated for a week with MC/3T3.

3T3 Cells↗

PubMed1991

Xanthines inhibit 3T3 fibroblast proliferation.

It is believed that xanthines can inhibit cell proliferation by elevating intracellular cAMP. Therefore, these compounds can be useful in hyperproliferative disorders. Our aim was to assess the efficacy of theophylline, caffeine and dyphylline on 3T3 fibroblast proliferation. 3T3 subconfluent cultures were incubated for 3 days with the xanthine derivatives (1 mM-1 microM). At 1 and 0.1 mM the three derivatives exhibited marked inhibition of fibroblast proliferation. Theophylline and caffeine were more potent than dyphylline. At a concentration of 1 mM, the three xanthine derivatives also inhibited proliferation of psoriatic dermal fibroblast, albeit to a lower extent. Caffeine was the most active compound followed by theophylline and dyphylline. We conclude that xanthine derivatives are good candidates for use as fibroblast antiproliferative drugs. We also conclude that 3T3 fibroblasts appear to be a valid system for the pharmacological screening of fibroblast antiproliferative drugs.

3T3 Cells↗

PubMed1991

Increased numbers of mast cells in pemphigus vulgaris skin lesions. A histochemical study.

We have used a histochemical technique to study mast cells (MC) in skin biopsies of 8 patients suffering from pemphigus vulgaris (PV) and from 4 control volunteers. The MC were stained for 30 min with 0.5% toluidine blue, pH 0.5, counted and then restained for 5 days under the same conditions. This staining method allows the identification of two groups of MC, one that stains promptly (30 min) and one that stains after longer incubation times (5 days). After 30 min of staining, a slight increase was found in the number of MC in PV sections, in comparison with normal controls. However, when the 30 min stained sections were reincubated under the same conditions for 5 days, a significant increase in the number of MC in PV was found in comparison with 5-day-stained normal skin sections (p less than 0.005) and in comparison with 30-min-stained PV sections (p less than 0.005). The MC were distributed throughout the dermis and were concentrated in the upper dermis near hair follicles and vessels. The possible importance of the increased numbers of MC in PV is discussed.

Adult↗

PubMed1991

Effects of interleukins on connective tissue type mast cells co-cultured with fibroblasts.

We investigated the effects of interleukin-2 (IL-2), interleukin-3 (IL-3) and interleukin-4 (IL-4) on mouse and rat peritoneal mast cells (MC) co-cultured with 3T3 fibroblasts (MC/3T3). The continuous presence of these cytokines for 7-9 days in the culture media was neither toxic nor caused proliferation of MC, as determined by the stability of MC numbers in culture. Long-term incubation of mouse MC/3T3 with IL-2 (100 U/ml), IL-3 (50 U/ml), IL-4 (50 U/ml) or a mixture of IL-3 and IL-4 (25 U/ml) induced an increase in basal histamine release of 79.3 +/- 19.0%, 41.0 +/- 17.3%, 25.2 +/- 10.4% and 30.2 +/- 3.2%, respectively, over control cells incubated with medium alone. When rat MC/3T3 were incubated for 7 days with the various interleukins an enhancement in histamine release similar to that observed with mouse MC/3T3 was found. Preincubation (1 hr) of rat MC/3T3 with interleukins prior to immunological activation with anti-IgE antibodies enhanced histamine release. The highest effect was observed with IL-3 + IL-4 (60.4 +/- 10.8% increase) followed by IL-2 (51.5 +/- 4.5%), IL-4 (28.6 +/- 10.3%) and IL-3 (13.2 +/- 4.2%). This study demonstrates that when mouse and rat peritoneal MC are cultured with fibroblasts in the presence of interleukins they do not proliferate, suggesting that they preserve their connective tissue type MC phenotype. Moreover, interleukins display a pro-inflammatory effect on these cells by enhancing both basal and anti-IgE-mediated histamine release.

Animals↗

PubMed1990

Unresponsiveness of rat peritoneal mast cells to immunologic reactivation.

Rat peritoneal mast cells cocultured with 3T3 fibroblasts (MC/3T3) were activated with Ag or with anti-IgE antibodies in the presence of Ca2+, and their responsiveness to a second similar challenge was evaluated. MC/3T3 were presensitized with IgE anti-DNP antibodies and activated with DNP-human serum albumin. When these MC/3T3 were reactivated with the same Ag 2 and 6 h later, they released only a minimal percentage of histamine. A gradual recovery of responsiveness was detected during the first 7 days after activation, and a full recovery was attained by days 14 to 21. A similar pattern of unresponsiveness was observed when MC/3T3 were challenged and rechallenged with cercarial Ag after presensitization with anticercarial serum. Activation of MC/3T3 with one Ag (DNP-human serum albumin or cercarial) and rechallenge 3 days later with the other Ag did not overcome the state of partial unresponsiveness. Challenging MC/3T3 with anti-IgE led to a subsequent unresponsiveness to rechallenge with the same ligand, regardless of whether or not the cells were presensitized with IgE antibodies. Cross-linkage with anti-IgE resulted in a more intense and prolonged state of unresponsiveness in comparison with that observed with Ag. When MC/3T3 activated with anti-IgE were rechallenged with various IgE-independent agents they released a percentage of histamine comparable to that of control cultures challenged with these secretagogues for the first time. MC/3T3 partially resynthesized their histamine content during the two-week period after activation. Our results suggest that MC undergo a temporary state of "physiologic" unresponsiveness after immunologic activation in the presence of calcium ions.

Animals↗

PubMed1990

Histamine release from mouse and rat mast cells cultured with supernatants from chronic murine graft-vs-host splenocytes.

There is growing interest in studying pathways of mast cell activation. In a mouse model of chronic graft-vs-host disease (cGVHD) extensive mast cell activation and degranulation occurs in vivo coincident with the development of dermal fibrosis. An interesting feature of this model is that the mast cell reaction is slow to develop, occurring over a period of weeks and waning by 300 days. The aim of our work was to investigate the effects of supernatants from splenocytes of such cGVHD mice (cGVHD sups) on mouse and rat peritoneal mast cells cocultured with 3T3 skin fibroblasts. We found that cGVHD sups are able to release histamine from both mouse and rat cultured mast cells in a slow fashion. Histamine release became evident only after 5-8 days of coculture of the mast cells with the cGVHD supernatants and thereafter decreased to basal levels. Mast cell activation due to cGVHD supernatants was a noncytotoxic event as demonstrated by mast cell counts in the cocultures and by the ability of mast cells to exclude trypan blue. Mast cells that had been activated by incubation with the cGVHD sups were as responsive to stimulation with either anti-IgE antibodies or compound 48/80 as were mast cells incubated with control sups. Supernatants from mice early in GVHD (Days 11-28) were most active in promoting histamine release. Supernatants from spleens of mice which had GVHD for 290 days and where the mast cells had returned to full granulation in vivo were inactive. This is the first in vitro study demonstrating slow mast cell histamine release instituted by other cells, namely the splenocytes of cGVHD mice.

Animals↗

PubMed1990

Effects of prolonged incubation of rat peritoneal mast cells with compound 48/80.

Rat peritoneal mast cells (MC) co-cultured on a monolayer of 3T3 fibroblasts (MC/3T3) were continuously exposed to compound 48/80 for 14 days. As early as 2 days following continuous exposure to compound 48/80, the MC/3T3 appeared as a heterogeneous population, with various MC appearing partially or fully degranulated or intact; this morphological pattern continued throughout the duration of the experiment. MC/3T3 remained functionally active as demonstrated by their ability to secrete histamine 15 min after each replacement with fresh medium containing compound 48/80, although this capacity diminished towards the end of the 14-day experiment. Concomitant with the histamine release, a significant increase in cellular histamine pools was observed. When MC/3T3 continuously exposed to compound 48/80 for 7 or 14 days were acutely challenged with anti-IgE antibodies, they were able to secrete histamine and prostaglandin D2 in amounts similar to those produced by control MC. In contrast, when these cells were challenged on day 7 or 14 with a higher dose of compound 48/80 or with substance P, the release of histamine was partially inhibited. Our results indicate that continuous in vitro exposure to compound 48/80, and the resulting MC degranulation product histamine, does not adversely affect the ability of MC/3T3 to synthesize histamine and to respond to activation stimuli of a related secretagogue for 7 days and a non-related one for at least 14 days.

Animals↗

PubMed1990

Mast cells enhance migration and proliferation of fibroblasts into an in vitro wound.

The effects of mast cells (MC) in an in vitro wound model were studied. The model consisted of rat peritoneal MC cultured on confluent monolayers of 3T3 fibroblasts (MC/3T3). A linear wound was performed by cutting along the midline and scraping one half of the monolayer. After 42 h fibroblasts were counted in the scraped area of the wound. In the MC/3T3 cocultures 27.6 +/- 2.1 fibroblasts were found compared to 16.6 +/- 0.9 in the 3T3 cultures. The most significant increase in the number of fibroblasts was obtained upon activation of the MC with anti-IgE antibodies immediately after wound production (39.9 +/- 2.1). Stimulation with compound 48/80 had a weaker effect (32.7 +/- 1.5). Incubation of 3T3 wounded monolayers with supernatants of anti-IgE- or compound 48/80-activated MC, or with sonicated MC, induced an increase in fibroblast number similar to that found in unactivated MC/3T3. [3H]Thymidine incorporation followed by autoradiography was performed to assess fibroblast mitosis. The highest number of labeled fibroblasts beyond the wound line was found in immunologically activated MC/3T3 (29.7 +/- 4.4), followed by compound 48/80-activated MC/3T3 (18.4 +/- 1.5), MC/3T3 (15.1 +/- 3.6), and 3T3 (10.6 +/- 2.6). After addition of aphidicolin, which inhibited fibroblast mitosis, MC were still effective in enhancing fibroblast migration. In all the cocultures MC were observed to have migrated alongside fibroblasts. Thus merely the presence of MC adhering to wounded fibroblast monolayers significantly enhanced migration and proliferation of the fibroblasts. A further increase was achieved by immunological activation of the MC. We therefore suggest that MC have a facilitating role in this in vitro wound model.

Animals↗

PubMed1990

Auranofin inhibits histamine release from rat peritoneal mast cells.

The effect of auranofin on histamine release from immunologic and non-immunologic activated rat peritoneal mast cells cocultured with 3T3 fibroblasts (MC/3T3) was investigated. When MC/3T3 were preincubated with 2 x 10(-5) M auranofin and thereafter challenged with anti-IgE antibodies, a maximal inhibition of histamine release (86.2%) was obtained. Non-immunological histamine release induced by compound 48/80, substance P and bradykinin was inhibited to a lesser degree, i.e. 36.0%, 37.6% and 24.0% respectively. Simultaneous incubation of auranofin and the stimulating agents resulted in a higher inhibition of histamine release: anti-IgE antibodies--92.0%; compound 48/80--73.5%; substance P--46.1%. We conclude that auranofin effectively reduces histamine release from immunologic and non-immunologic activated mast cells. This may be relevant to the control of allergic reactions.

Animals↗

PubMed1990

Osteoblast-like cell line maintains in vitro rat peritoneal mast cell viability and functional activity.

We studied the ability of the rat osteosarcoma derived cell-line with osteoblastic properties ROS-17/2.8 (ROS) to maintain in vitro rat peritoneal mast cells (MC) in a functional state. Highly purified (greater than 95%) MC were seeded on confluent ROS cells. The MC adhered tightly to the monolayers within a few hours and remained viable for at least 2 weeks, but did not proliferate. The MC retained their typical appearance, exhibiting highly granulated resting morphology when stained with alcian blue followed by safranin or with acidic toluidine blue. Furthermore, after 2 weeks, the MC were fully responsive to activation with compound 48/80 (3 micrograms/ml), releasing 75% of their histamine content, as compared to 3% in the absence of the secretagogue. Utilizing metabolically inactive ROS cells and prevention of contact between ROS and MC, we found that both release of factor(s) and cell-cell contact were required by ROS to exhibit their MC supporting activity. Various other cells and cell lines were unable to support MC viability. On the other hand, as demonstrated before, 3T3 fibroblasts were capable of promoting MC viability. Thus, MC viability and functional activity are specifically maintained by fibroblastic and osteoblastic cells. The abundance of MC in bone, and their participation in bone remodelling raise the possibility of physiological and pathological significance to interactions between MC and osteoblasts.

Animals↗

PubMed1989

In vitro regeneration of activated rat peritoneal mast cells cocultured with 3T3 fibroblasts.

Rat peritoneal mast cells (MC) cocultured with 3T3 fibroblasts (MC/3T3) were activated by compound 48/80 to release most of their histamine content (approximately 90%). These activated MC could be kept viable in vitro for as long as 2 weeks, during which their regeneration had been followed. As early as 3-4 hr following activation, the MC started reacquiring their resting morphology. In 1-2 weeks they were more densely granulated grossly resembling nonactivated, control MC. Reactivation of MC/3T3 with compound 48/80 on Day 7 or 14 of culture resulted in a percentage histamine release which was similar to that of Day 0, and to control MC/3T3, indicating that these activated MC were functionally responsive. Activated MC/3T3 could partially resynthesize histamine during the 14 days of culture following activation. On Day 7 their histamine content was approximately 23% and on Day 14 approximately 40% of the initial amount they had on Day 0, prior to activation. Thus, MC/3T3 activated by compound 48/80 in vitro can regenerate by resuming their initial morphology, becoming responsive to a subsequent reactivation and resynthesizing histamine.

Animals↗

PubMed1989

Differential release of histamine and prostaglandin D2 in rat peritoneal mast cells activated with peptides.

Rat peritoneal mast cells co-cultured with mouse 3T3 fibroblasts (MC/3T3) are fully responsive to immunologic stimuli. To assess their nonimmunologic activation MC/3T3 were challenged with various peptides. Optimal concentrations of substance P (10(-4) M) and bradykinin (5 x 10(-5) M) induced histamine release of 58.2 +/- 9.3 and 66.8 +/- 6.6%, respectively, while neurotensin (10(-4) M) released only 16.6 +/- 3.7% histamine. Freshly isolated mast cells (F-MC) challenged with the same concentrations of peptides released lower percentages of histamine (substance P 45.6 +/- 5.1%, bradykinin 32.5 +/- 5.3%, neurotensin 11.3 +/- 6.0%). In both MC/3T3 and F-MC, only minute amounts of prostaglandin D2 (PGD2) were produced. In contrast, activation with anti-IgE antibodies and compound 48/80 caused both histamine release and PGD2 generation. Compound 48/80-stimulated MC/3T3 and F-MC released 80.2 +/- 3.4 and 51.8 +/- 6.2% histamine, respectively, and produced 15.4 +/- 2.8 ng/10(6) mast cells and 3.9 +/- 1.4 ng/10(6) mast cells PGD2, respectively. These findings indicate that peptides and bradykinin induce selective release of histamine with no PGD2 production in both F-MC and MC/3T3. Moreover, MC/3T3 preserve their functional characteristics of connective tissue mast cells since they are fully responsive to these peptides as F-MC.

Animals↗

PubMed1988

Coculture of mouse IL-3-dependent mast cells with 3T3 fibroblasts stimulates synthesis of globopentaosylceramide (Forssman glycolipid) by fibroblasts and surface expression on both populations.

Mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mouse 3T3 fibroblasts, cultured separately or together, were examined for their cell surface expression and biosynthesis of globopentaosylceramide, a marker of the mouse serosal mast cell. As assessed by flow cytometric analysis, BMMC cultured for up to 7 wk in 50% WEHI 3-conditioned medium containing IL-3 did not bind the B1.1 anti-globopentaosylceramide mAb (six experiments). A total of 10 +/- 4% (mean +/- SD, three experiments) of 3T3 fibroblasts that had reached confluence in medium without IL-3 bound B1.1 antibody and, after an additional approximately 28 days of culture in that medium or in 50% WEHI 3-conditioned medium, 12 +/- 3% (mean +/- SD, five experiments) and 16 +/- 7% (mean +/- SD, three experiments) of the cells, respectively, bound the antibody. After coculture of BMMC and confluent 3T3 fibroblasts for 28 days in 50% WEHI 3-conditioned medium, followed by dispersal and purification of the cells, 92 +/- 18% of the mast cells and 92 +/- 16% (mean +/- SD, seven experiments) of the fibroblasts were B1.1+. Whereas the increase in the expression of the epitope bound by B1.1 antibody on fibroblasts was noted by day 14 of coculture, expression of the epitope on mast cells did not occur until day 21 (three experiments). Biosynthesis of globopentaosylceramide was assessed by intrinsic radiolabeling of each cell population and identification of the extracted neutral glycosphingolipids by TLC and autoradiography. Synthesis of globopentaosylceramide was not detected in extracts of 9 x 10(6) BMMC, 1 x 10(6) confluent 3T3 fibroblasts cultured alone for 28 days, or 9 x 10(6) mast cells purified from 28-day cocultures but was readily detected in extracts of 3 x 10(5) fibroblasts purified from the same cocultures. These findings indicate that BMMC stimulate an increase in the synthesis and expression of globopentaosylceramide on 3T3 fibroblasts and suggest that the subsequent appearance of this neutral glycosphingolipid on the surface of the mast cells is due to its secretion by fibroblasts and adsorption to the mast cell surface. Thus, the interactions between mast cells and fibroblasts during coculture alter the biochemical and Ag phenotypes of both populations.

Animals↗

PubMed1988

3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency.

As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated inter-leukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. We now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approximately equal to 50-fold and their carboxypeptidase A content approximately equal to 100-fold, and augment approximately equal to 45-fold their biosynthesis of proteoglycans bearing 35S-labeled heparin relative to 35S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

Animals↗

PubMed1987

Mouse bone marrow-derived mast cells cocultured with fibroblasts. Morphology and stimulation-induced release of histamine, leukotriene B4, leukotriene C4, and prostaglandin D2.

Mouse bone marrow-derived mast cells (BMMC), cultured for 2, 7, or 14 days in WEHI-3 conditioned medium in the absence or presence of mouse 3T3 fibroblasts, were examined morphologically and for their functional responses to IgE-Fc-mediated and calcium ionophore-mediated activation. The 7- and 14-day fibroblast-adherent and non-fibroblast-adherent populations of cocultured BMMC had more granules per cell and the granule contents were more electron dense than non-cocultured BMMC or BMMC cocultured for only 2 days. The adherent cocultured BMMC were usually located within multiple layers of fibroblasts, but did not form junctions with the fibroblasts. When activated immunologically, the adherent cocultured mast cells generally discharged their granules singly, but compound exocytosis was occasionally seen. Both the non-adherent cocultured BMMC and the BMMC that were cultured in the absence of fibroblasts were similar to one another in that they exocytosed 9 to 11% of their histamine when sensitized with anti-dinitrophenyl IgE and challenged with dinitrophenyl-bovine serum albumin and 27 to 29% of their histamine when challenged with calcium ionophore. In contrast, adherent cocultured BMMC exocytosed 27 and 61% of their histamine upon immunologic and calcium ionophore activation, respectively, representing a significant two- to three-fold increase relative to that obtained from the other populations of BMMC. When activated immunologically, BMMC cultured in WEHI-3 conditioned medium alone generated a mean of 12 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 1.6 ng of leukotriene B4 (LTB4), and 1.0 ng of prostaglandin D2 (PGD2)/10(6) cells. The immunologic response of the nonadherent 7-day cocultured BMMC was similar. Fibroblast-adherent cocultured BMMC, on the other hand, generated 56 ng of immunoreactive C-6-sulfidopeptide leukotrienes, 6.4 ng of LTB4, and 5.6 ng of PGD2/10(6) mast cells, representing a significant increase for each product. When calcium ionophore was used as the agonist, the adherent cocultured mast cells also generated significantly more arachidonic acid metabolites than nonadherent cocultured BMMC or BMMC cultured in the absence of fibroblasts. Retention times on high performance liquid chromatography confirmed that the generated immunoreactive products were LTB4, PGD2, and LTC4. Thus, coculture of BMMC with fibroblasts induces an alteration in the composition of the secretory granules of the mast cells, as well as an augmentation of the activation-secretion response of the BMMC.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes.

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.

Animals↗

PubMed1987

Ontogeny of in vitro-differentiated mouse mast cells.

Rat and mouse heparin-containing connective tissue mast cells (CTMC) are stained by both alcian blue and safranin, whereas the chondroitin sulfate-containing mucosal mast cells (MMC) are stained by alcian blue but not by safranin. Mouse bone marrow-derived mast cells (BMMC) (the presumptive in vitro counterpart of the in vivo-differentiated MMC) were derived by culture of progenitors in the presence of 50% WEHI-3-conditioned medium and 10% fetal calf serum and were then cultured for up to 14 days with confluent skin-derived mouse 3T3 fibroblasts in the same culture medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably because of the presence of interleukin 3 in the conditioned medium. After 14 days of coculture, their cellular histamine content increased approximately 15-fold, and greater than 50% of the BMMC changed histochemically from safranin negative to safranin positive. At this time 30-50% of the glycosaminoglycans of the proteoglycans synthesized by these cocultured mast cells were heparin, whereas the initial BMMC synthesized proteoglycans containing chondroitin sulfate E. When activated immunologically, the cocultured mast cells generated approximately fivefold more prostaglandin D2 than did the activated starting BMMC. Thus, interleukin 3-dependent mouse BMMC can be induced to undergo phenotypic changes in staining characteristics, histamine content, glycosaminoglycan structure, and metabolism of arachidonic acid to resemble heparin-containing CTMC. These findings suggest that the tissue microenvironment determines the phenotypic characteristics of mast cells. This demonstration of the functional diversity of different populations of mast cells adds an important dimension to the understanding of the role of mast cells in biological processes.

Animals↗