Biomedical subjects

R L Stevens

Publications and source records attributed to R L Stevens.

At least 109 records · Page 6Linked to original sources

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

Eosinophils cocultured with endothelial cells have increased survival and functional properties.

Human peripheral blood eosinophils, cells often associated with allergic and parasitic diseases, were maintained in vitro for at least 14 days when they were cocultured with bovine endothelial cells and for at least 7 days when cultured with either bovine or human endothelial cell-derived conditioned medium. The cocultured eosinophils became hypodense and generated about three times as much leukotriene C4 upon activation with calcium ionophore and killed about three times as many antibody-coated larvae of Schistosoma mansoni as freshly isolated normodense eosinophils. That these cells can be maintained in vitro by coculture with endothelial cells, and the surprising finding that the cocultured eosinophils have biochemical, cytotoxic, and density properties similar to those of eosinophils in patients with allergic and other disorders, will facilitate investigation of the regulation and role of these cells in health and disease.

Animals↗

PubMed1987

Co-sedimentation of chondroitin sulfate A glycosaminoglycans and proteoglycans with the cytolytic secretory granules of rat large granular lymphocyte (LGL) tumor cells, and identification of a mRNA in normal and transformed LGL that encodes proteoglycans.

Rat large granular lymphocyte (LGL) tumor cell lines were analyzed for the presence of proteoglycans and glycosaminoglycans in their cytolytic secretory granules. When isolated rat LGL tumor cells were incubated in vitro for 1 to 3 hr with [35S]sulfate, and the 35S-labeled macromolecules were purified by density-gradient centrifugation, they filtered on Sepharose CL-4B columns predominantly as approximately 500,000 m.w. macromolecules. After 19 hr of incubation with [35S]sulfate, however, an 85,000 m.w. species predominated. Pulse-chase experiments revealed that the larger macromolecules were proteoglycans that with time were processed to glycosaminoglycan-sized macromolecules. As assessed by their susceptibility to chemical and enzymatic degradation and by high pressure liquid chromatography of the chondroitinase ABC-generated unsaturated disaccharides, the cell-associated rat LGL tumor cell proteoglycans bore almost exclusively chondroitin sulfate A glycosaminoglycans. Northern blot analysis using a gene-specific probe revealed that both normal peripheral blood and transformed rat LGL expressed the same approximately 1.3-kb mRNA that encodes the peptide core of the proteoglycans in the secretory granules of rat and mouse mast cells. In vivo radiolabeling of rat LGL tumor cells and isolation of their intact granules after nitrogen cavitation and density sedimentation established that glycosaminoglycans compartmentalized with cytolytic activity. Thus these negatively charged macromolecules may play a role in the regulation of the packaging and delivery of the cytolysins and basically charged serine proteases that have been identified in the cytolytic secretory granules of LGL.

Aggrecans↗

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

Regulation of human eosinophil viability, density, and function by granulocyte/macrophage colony-stimulating factor in the presence of 3T3 fibroblasts.

Normodense human peripheral blood eosinophils were isolated under sterile conditions from the 22/23 and 23/24% interfaces and the cell pellet of metrizamide gradients. After culture for 7 d in RPMI media in the presence of 50 pM biosynthetic (recombinant) human granulocyte/macrophage colony-stimulating factor (rH GM-CSF), 43 +/- 7% (mean +/- SEM, n = 8) of the cells were viable; in the absence of rH GM-CSF, no eosinophils survived. The rH GM-CSF-mediated viability was concentration dependent; increased survival began at a concentration of 1 pM, a 50% maximal response was attained at approximately 3 pM, and a maximal effect was reached at concentrations of greater than or equal to 10 pM rH GM-CSF. In the presence of rH GM-CSF and mouse 3T3 fibroblasts, 67 +/- 6% (mean +/- SEM, n = 8) of the eosinophils survived for 7 d. In a comparative analysis, there was no difference in eosinophil viability after 7 and 14 d (n = 3) in the presence of 50 pM GM-CSF and fibroblasts. Culture with fibroblasts alone did not support eosinophil survival. The addition of fibroblast-conditioned media to rH GM-CSF did not further improve eosinophil viability, indicating a primary role for GM-CSF in supporting these eosinophil cell suspensions ex vivo and a supplementary role for 3T3 fibroblasts. Eosinophils cultured for 7 d localized on density gradient sedimentation at the medium/18, 18/20, and 20/21 interfaces of metrizamide gradients, indicating a change to the hypodense phenotype from their original normodense condition. In addition, the cultured eosinophils generated approximately 2.5-fold more LTC4 than freshly isolated cells when stimulated with the calcium ionophore A23187 and manifested sevenfold greater antibody-dependent killing of S. mansoni larvae than the freshly isolated, normodense cells from the same donor. Thus we demonstrate the rH GM-CSF dependent conversion in vitro of normodense human eosinophils to hypodense cells possessing the augmented biochemical and biological properties characteristic of the hypodense eosinophils associated with a variety of hypereosinophilic syndromes. In addition, these studies provide a culture model of at least 14 d suitable for the further characterization of hypodense eosinophils.

Animals↗

PubMed1987

Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia.

Basophilic leukocytes from two patients with myelogenous leukemia were enriched to a purity of 10 to 45% by density gradient centrifugation. Ultrastructurally, these basophilic leukocytes contained segmented nuclei and granules with reticular patterns resembling those of normal basophils, and other granules with scroll and grating patterns resembling those of normal connective tissue mast cells. The 35S-labeled macromolecules isolated from these cells were approximately 140,000 m.w. Pronase-resistant proteoglycans bearing approximately 15,000 m.w. glycosaminoglycans. On incubation with chondroitinase ABC, nitrous acid, and heparinase, the 35S-labeled proteoglycans were degraded 50 to 84%, 16 to 43%, and 8 to 37%, respectively, indicating the presence of both chondroitin sulfate and heparin. As assessed by high performance liquid chromatography, the 35S-labeled chondroitin sulfate disaccharides liberated by chondroitinase ABC treatment were approximately 95% monosulfated chondroitin sulfate A and approximately 5% disulfated chondroitin sulfate E. The presence of heparin was confirmed by two-dimensional cellulose acetate electrophoresis of the 35S-labeled glycosaminoglycans. Cell preparations, enriched to 75% basophilic leukocytes by sorting for IgE+ cells, also synthesized 35S-labeled proteoglycans containing chondroitin sulfate and heparin. In one experiment, treatment of the cells with 1 microM calcium ionophore A23187 resulted in a 12% net release of both chondroitin sulfate and heparin containing 35S-labeled proteoglycans, a 57% net release of histamine, and the de novo generation of 8, 8, and 0.16 ng of immunoreactive equivalents of prostaglandin D2, leukotriene C4, and leukotriene B4, respectively, per 10(6) cells. Because only mast cells have been found to contain Pronase-resistant heparin proteoglycans, to generate PGD2 on cell activation, and to contain granules with scroll and grating patterns, these findings indicate that in some patients with myelogenous leukemia there are basophilic cells that possess properties of tissue mast cells.

Arachidonic Acid↗

PubMed1987

Phenotypic changes of bone marrow-derived mast cells after intraperitoneal transfer into W/Wv mice that are genetically deficient in mast cells.

The ability of mouse IL-3-dependent, bone marrow culture-derived mast cells (BMMC) to generate serosal mast cells (SMC) in vivo after adoptive transfer to mast cell-deficient mice has been defined by chemical and immunochemical criteria. BMMC differentiated and grown from WBB6F1-+/+ mouse progenitor cells in medium containing PWM/splenocyte-conditioned medium synthesized a approximately 350,000 Mr protease-resistant proteoglycan bearing approximately 55,000 Mr glycosaminoglycans, as defined by gel filtration of each. Approximately 85% of the glycosaminoglycans bound to the cell-associated BMMC proteoglycans were chondroitin sulfates based upon their susceptibility to chondroitinase ABC digestion; HPLC of the chondroitinase ABC-generated unsaturated disaccharides revealed these glycosaminoglycans to be chondroitin sulfate E. As determined by heparinase and nitrous acid degradations, approximately 10% of the glycosaminoglycans bound to BMMC proteoglycans were heparin. In contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient WBB6F1-W/Wv mice 15 wk after intraperitoneal injection of BMMC synthesized approximately 650,000 Mr protease-resistant proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 105,000 Mr. Thus, after adoptive transfer, the SMC of the previously mast cell-deficient mice were like those recovered from the normal WBB6F1-+/+ mice that were shown to synthesize approximately 600,000 Mr proteoglycans that contained approximately 80% heparin glycosaminoglycans of approximately 115,000 Mr. As assessed by indirect immunofluorescence staining and flow cytometry using the B1.1 rat mAb (an antibody that recognizes an epitope located on the neutral glycosphingolipid globopentaosylceramide), approximately 5% of BMMC bound the antibody detectably, whereas approximately 72% of the SMC that were harvested from mast cell-deficient mice 15 wk after adoptive transfer of BMMC were B1.1-positive; approximately 82% of SMC from WBB6F1-+/+ mice bound the antibody. These biochemical and immunochemical data are consistent with the results of previous adoptive transfer studies that characterized mast cells primarily on the basis of morphologic and histochemical criteria. Thus, IL-3-dependent BMMC developed in vitro, cells that resemble mucosal mast cells, can give rise in vivo to SMC that express phenotypic characteristics of connective tissue mast cells.

Animals↗

PubMed1987

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta.

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.

Amino Acids↗

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↗

PubMed1986

Complexes of heparin proteoglycans, chondroitin sulfate E proteoglycans, and [3H]diisopropyl fluorophosphate-binding proteins are exocytosed from activated mouse bone marrow-derived mast cells.

The predominant [3H]diisopropyl fluorophosphate (DFP)-binding proteins that are released from the secretory granules of activated mouse bone marrow-derived mast cells (BMMC) are demonstrated to have an isoelectric point of approximately 9.1 and to be complexed to proteoglycans. Upon Sepharose CL-2B chromatography of the supernatants of calcium ionophore-activated BMMC, 67-78% of the total exocytosed [3H]DFP-binding proteins co-eluted in the excluded volume of the column as a greater than 1 X 10(7) Mr complex bound to 4-7% of the total exocytosed proteoglycans. The remainder of the exocytosed proteoglycans, which filtered in the included volume of the gel filtration column with a Kav of 0.66, contained chondroitin sulfate E glycosaminoglycans. After dissociation of the large Mr complexes of [3H]DFP-binding proteins-proteoglycans with 5 M NaCl and removal of the proteins via phenyl-Sepharose chromatography, the proteoglycans filtered from the Sepharose CL-2B column as a single peak with a Kav of 0.66. The susceptibility of 24-59% and 36-76% of the glycosaminoglycans in the large Mr complex to degradation by nitrous acid and chondroitinase ABC, respectively, indicated the presence of proteoglycans that contained heparin and chondroitin sulfate glycosaminoglycans. Disaccharide analysis revealed that the chondroitin sulfate in the high Mr complex was chondroitin sulfate E. Following chondroitinase ABC treatment of the large Mr complex, the residual heparin proteoglycans filtered on Sepharose CL-4B under dissociative conditions with the same Kav as the original, untreated proteoglycans. Thus, the protein-proteoglycan complexes that are exocytosed from activated mouse BMMC contain approximately equal amounts of proteoglycans of comparable size that bear either predominantly heparin or predominantly chondroitin sulfate E glycosaminoglycans. The demonstration of these secreted complexes indicates that the intragranular protease-resistant heparin and chondroitin sulfate E proteoglycans in the T cell factor-dependent BMMC bind serine proteases throughout the activation-secretion response.

Animals↗

PubMed1986

Secretory granules of heparin-containing rat serosal mast cells also possess highly sulfated chondroitin sulfate proteoglycans.

Rat serosal mast cells, which synthesize only heparin proteoglycans as detected by intrinsic labeling with [35S]sulfate, were analyzed for the presence of intracellular chondroitin sulfate proteoglycans by chemical and immunochemical means. Rat serosal mast cells of greater than 99% purity were treated with Zwittergent 3-12 and 4 M guanidine HCl, and the extracted nonradiolabeled proteoglycans were purified by density gradient centrifugation. As assessed by quantification of the unsaturated disaccharides released from the proteoglycans by chondroitinase ABC treatment, 10(6) rat serosal mast cells contained 2.4-4.5 micrograms of chondroitin sulfate proteoglycans. Analysis of the chondroitinase ABC digests by high performance liquid chromatography revealed the unsaturated disaccharides delta Di-4S, delta Di-diSB, and delta Di-diSE which were derived from GlcA----GalNAc-4-SO4, iduronic acid-2-SO4----GalNAc-4-SO4, and GlcA----GalNAc-4,6-diSO4, respectively. The molar ratio of the monosulfated to disulfated disaccharides was approximately 2:1 with delta Di-diSE greater than delta Di-diSB. When analyzed with a mouse anti-chondroitin sulfate monoclonal antibody and fluorescein-labeled F(ab')2 goat anti-mouse IgG, approximately 91% of permeabilized and chondroitinase ABC-treated cells in the mast cell preparations exhibited intracellular fluorescence, and the pattern of staining indicated that the chondroitin sulfate molecules were located in the secretory granules. The specificity of the monoclonal antibody for the unsaturated double bond created by chondroitinase ABC treatment of the proteoglycan in situ was established by the absence of fluorescence when the chondroitinase ABC step was omitted or when heparinase digestion was substituted for chondroitinase ABC. Furthermore, the ability of the anti-chondroitin sulfate monoclonal antibody to mediate fluorescence in situ was markedly reduced by absorption with solid-phase chondroitin sulfate proteoglycan that had been chondroitinase ABC-treated, but not by absorption with undigested proteoglycan or with solid-phase heparin. The highly sulfated chondroitin sulfate proteoglycans of rat serosal mast cells are the same type synthesized by the rat mucosal mast cell subclass.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Intestinal mucosal mast cells from rats infected with Nippostrongylus brasiliensis contain protease-resistant chondroitin sulfate di-B proteoglycans.

Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.

Animals↗

PubMed1986

Inhibition of post-translational modification and surface expression of a melanoma-associated chondroitin sulfate proteoglycan by diethylcarbamazine or ammonium chloride.

Cultured human melanoma M21 cells were treated with diethylcarbamazine (DEC), an inhibitor of proteoglycan biosynthesis in rat chondrosarcoma cells, to examine the assembly and transport of a chondroitin sulfate proteoglycan to the plasma membrane. Pretreatment of melanoma cells at 37 degrees C for 15 min with increasing doses of DEC followed by a 60-min pulse with [35S]sulfate in the presence of DEC resulted in a dose-related inhibition of incorporation of [35S]sulfate into macromolecules. In cells incubated for 75 min with both 1 mM beta-D-xyloside and 15 mM DEC, synthesis and secretion of beta-D-xyloside-bound 35S-glycosaminoglycans were inhibited by more than 80% as compared to cells treated with beta-D-xyloside alone; this inhibition was reversible. As assessed by [3H]serine incorporation into protein, overall protein synthesis was not substantially inhibited by DEC treatment. Detergent lysates from [35S]methionine-labeled melanoma cells were incubated with a monoclonal antibody (9.2.27) that specifically recognizes the peptide core of the melanoma proteoglycan. As assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the immunoprecipitate, a 240,000 Mr endoglycosidase H (Endo-H)-sensitive intermediate was the only form of the proteoglycan present inside the cells when the cultures were treated for 60-120 min with 10-15 mM DEC. When the melanoma cells were incubated for 10 min with 15 mM DEC and 100 mu Ci/ml of [35S]methionine, washed, and then chased for 15 min to 4 h in radioactive-free medium, the 240,000 Mr Endo-H-sensitive intermediate was slowly converted to a 250,000 Endo-H-resistant intermediate but not to a mature proteoglycan molecule that possessed chondroitin sulfate glycosaminoglycans. SDS-PAGE analysis of cell surface immunoprecipitates revealed that only a small amount of the 250,000 Mr intermediate was transported to the plasma membrane within 5 h of incubation in the presence of DEC. Proteoglycan synthesis was also inhibited when the melanoma cells were incubated for 60-120 min with ammonium chloride, but unlike DEC-treated cells the majority of the synthesized peptide core was converted to a 245,000 Mr Endo-H-resistant intermediate that was detected on the cell surface. Light and electron microscopic analysis of DEC-treated melanoma cells revealed large vacuoles and a distended Golgi and endoplasmic reticulum. Ammonium chloride-treated cells contained fewer vacuoles than DEC-treated cells but more vacuoles than normal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonium Chloride↗

PubMed1986

Biochemical and phenotypic characterization of human basophilic cells derived from dispersed fetal liver with murine T cell factors.

Metachromatically granulated cells were generated from human fetal liver stem cells cultured in heterologous mouse conditioned medium rich in interleukin 3. After 2 to 3 wk of culture with biweekly changes of medium and selection of nonadherent cells, all cells present in five cultures had cytoplasmic granules, and 60 to 95% of the cells stained metachromatically with toluidine blue or with alcian blue but not with the safranin counterstain. Ultrastructurally, many granules contained fibrillar material or electron-dense cores with fibrils and vesicular fragments. In addition, the granules of many cells were filled with electron-dense material, which in some cases had a fine structure consisting of concentric whorls or a reticular pattern. Analysis of high-affinity IgE receptors on the cultured cells by flow cytometry demonstrated a unimodal fluorescence pattern, suggesting that most cells were in the basophil or mast cell lineage. The cultured cells lacked the lymphoid cell surface determinants B1, B4, T3, and T11, the myeloid determinants Mo2 and MY9, the natural killer cell determinant 901, and Ia histocompatibility antigens, but expressed the myeloid determinant MY7. The cells contained 52 ng/10(6) cells of histamine and incorporated [35S]sulfate at an average rate of 31,300 cpm/10(6) cells/4 hr into 175,000 m.w. chondroitin sulfate A proteoglycans. Upon activation with 1 microM calcium ionophore A23187, the cultured cells released 53% of their cell-associated histamine and metabolized arachidonic acid to 15.0 ng/10(6) cells of immunoreactive leukotriene C4 equivalents, 0.5 ng/10(6) cells of leukotriene B4, and 3.1 ng/10(6) cells of prostaglandin D2 (means, n = 3). Thus, stem cells present in human fetal liver give rise, as do stem cells in mouse fetal liver, to metachromatically granulated cells when cultured in the presence of mouse interleukin 3. In both species, the cultured cells bear IgE receptors, lack characteristic lymphoid and most myeloid cell surface determinants, and contain histamine and chondroitin sulfate proteoglycans. The human fetal liver-derived cells are similar in morphology and T cell factor dependence to basophil-like cells derived from umbilical cord blood, but are novel in their capacity to generate leukotrienes and prostaglandin D2.

Animals↗

PubMed1986

Secretory granule proteoglycans of mast cells and natural killer cells.

Proteoglycan research on cells that participate in immune responses has progressed from the early novel finding that heparin proteoglycans are present in the secretory granules of the connective tissue mast cell to the more recent findings that mucosal mast cells and natural killer (NK) cells possess chondroitin sulphate proteoglycans in their granules. Characterization studies of these intracellular proteoglycans have revealed that they all possess peptide cores which are very resistant to proteolytic degradation. Their glycosaminoglycans, however, differ in such parameters as the type of hexosamine, location of sulphation degree of sulphation, or extent of epimerization of the uronic acid. Amino acid compositional analyses of heparin proteoglycans from rat connective tissue mast cells and chondroitin sulphate E proteoglycans from mouse mucosal mast cells indicate that their peptide cores are homologous to, but possibly distinct from one another. It is not yet known if these differences reflect a species variation, are due to different post-translational proteolytic processing, or are the result of expression of distinct genes coding for different peptide cores. The proteoglycans of mast cells and natural killer cells are packaged in the granules with cationic proteins. In mast cells these proteins have been shown to be serine proteases, and when bound to the acidic proteoglycans their enzymic activity is inhibited. Since the type of glycosaminoglycan linked to the proteoglycan has been found to be a characteristic of that cell, the structure of the cell-associated proteoglycan has become one of the markers used to distinguish cells phenotypically. By following the expression of different proteoglycans during differentiation, the relationship of the two subclasses of mast cells has been determined.

Animals↗

PubMed1986

Coculture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells.

The heparin-containing mast cells that reside in the connective tissue of the mouse, but not the chondroitin sulfate-containing mast cells in the gastrointestinal mucosa, stain with safranin when exposed to alcian blue/safranin. Mouse bone marrow-derived mast cells (BMMC), the probable in vitro counterparts of in vivo mucosal mast cells, were cultured for 14 days with mouse skin-derived 3T3 fibroblasts in RPMI 1640 medium containing 10% fetal calf serum and 50% WEHI-3 conditioned medium. Although the BMMC adhered to the fibroblast monolayer, they continued to divide, probably due to the presence of interleukin 3 in the conditioned medium. The mast cells remained viable throughout the period of coculture, since they failed to release lactate dehydrogenase and because they increased their histamine content approximately 15-fold. After 12-14 days of coculture, greater than 50% of the BMMC changed histochemically to become safranin+; 30-40% of the 35S-labeled glycosaminoglycans on the proteoglycans synthesized by these cocultured mast cells were heparin, whereas heparin was not detected in the initial BMMC. In the absence of WEHI-3 conditioned medium, BMMC adhered to the fibroblast monolayer, and after 8 days of coculture, the number of mast cells did not change and their histamine content remained the same. However, these mast cells also became safranin+ and synthesized 40% heparin glycosaminoglycans. Thus, coculture of BMMC with fibroblasts induces a phenotypic change so that the resulting mast cells stain safranin+ and synthesize heparin proteoglycans, whereas the presence of WEHI-3 conditioned medium stimulates proliferation and an increase in histamine content.

Animals↗

PubMed1986

A single gene in mast cells encodes the core peptides of heparin and chondroitin sulfate proteoglycans.

The diversity of the genes encoding mammalian proteoglycan peptide cores was explored using a cDNA clone that encodes the partial sequence of a cell surface/pericellular matrix-localized chondroitin sulfate proteoglycan. Thus we were able to detect the expression of the gene(s) encoding the intracellular chondroitin sulfate proteoglycan produced by a variety of rat and mouse mucosal-like mast cells and the intracellular heparin proteoglycan synthesized by rat serosal mast cells. The cDNA from the proteoglycan cDNA clone pPG-1 was fractionated into two discrete fragments, one of which contained the nucleotides encoding the serine-glycine repeat sequence (pPG-B) and the other of which contained sequences on the 3' side of the repeat (pPG-M). As assessed by Southern blot analysis, pPG-B identified a large gene family, whereas pPG-M identified a single DNA fragment in the rat genome. When the pPG-1 insert and the two subcloned probes pPG-B and pPG-M were used to analyze RNA extracted from the rat and mouse mucosal-like mast cells and the rat serosal mast cells, the same major RNA species was detected at 1.3 kilobases with both probes. These data suggest that the gene responsible for the peptide core of the extracellular chondroitin sulfate proteoglycan synthesized by the rat yolk sac cell line is also the gene that encodes the core peptides of the secretory granule-localized chondroitin sulfate and heparin proteoglycans.

Aggrecans↗

PubMed1986

The identification of a plasma membrane 3,3',5-triiodo-L-thyronine binding protein on the cultured Swarm rat chondrosarcoma chondrocyte and the lack of its up-regulation by insulin in vitro.

Primary cultures of Swarm rat chondrosarcoma chondrocytes were examined for the presence of T3 plasma membrane binding proteins and their possible regulation by insulin. Incubation of this tumor cell with [125I]T3 at 4 C yielded saturable and reversible binding of the radioligand. As assessed by LIGAND computer analysis, the binding data in one experiment revealed two classes of [125I]T3 binding sites with association constants of 2.2 X 10(9) M-1 and 4.8 X 10(6) M-1, and binding capacities of 4.9 X 10(3) and 1.9 X 10(6) sites per cell, respectively. Whole cells and a preparation enriched for plasma membrane were affinity labeled with N-bromoacetyl-[125I]T3 (N-BrAc-[125I] T3), and the molecular weights of the radiolabeled proteins were analyzed both by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by Sephadex G-200 gel filtration chromatography. One major chondrosarcoma protein of 55,000 mol wt and two minor proteins of 47,000 and 33,000 mol wt bound N-BrAc-[125I]T3, suggesting that the mol wt of the T3 binding protein was 55,000. As assessed by isoelectric focusing, the 55,000 mol wt protein had an isoelectric point of 5.1. The radiolabeled 55,000 mol wt chondrosarcoma protein was immunoprecipitated with anti-T3 and anti-GH3 plasma membrane T3 antisera. The high degree of homology between this chondrocyte N-BrAc-[125I]T3 binding protein and the protein on rat GH3 cells was demonstrated by a comparison of the peptide maps of Staphylococcus aureus V8 protease and elastase digested radiolabeled binding protein. Although culture of the chondrocytes in medium containing insulin resulted in an approximate 400% increase in plasma membrane [125I]insulin binding, no significant increase in [125I]T3 binding was observed. Thus, expression of the T3 plasma membrane binding protein was similar to that observed previously for the insulin-like growth factor-II receptor on these chondrosarcoma chondrocytes in not being influenced by the concentration of insulin in the culture medium.

Affinity Labels↗