Isolation and Characterization of Heparin from Human Lung

Metcalfe, Dean D.; Lewis, Robert A.; Silbert, Jeremiah E.; Rosenberg, Robert D.; Wasserman, Stephen I.; Austen, K. Frank

doi:10.1172/jci109613

Cited by 148 publications

(59 citation statements)

References 26 publications

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“…Human mast cells have been estimated to contain 2.4 to 7.8 g heparin per 10 6 cells. 23 This observation, along with the knowledge that heparin is a negatively charged molecule helps explain why mast cell granules are preferentially stained with cationic dyes. Proteoglycans synthesized by human pulmonary mast cells contain chondroitin sulfate E and heparin chains at 1:1 to 1:2 ratio, while those in the human gastric mucosa have more chondroitin sulfate E, 24 contributing to variable staining characteristics among mast cell populations.…”

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confidence: 90%

Mast cells and mastocytosis

Metcalfe

2008

Blood

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484

515

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IntroductionMast cells have a rather unique position among cells of the immune response. Their progenitors are bone marrow derived, yet under normal conditions appear in the mature state only within vascularized tissues, where they are long-lived. Mast cells appear historically ancient, 1 yet their roles in mammalian biology, including disease pathogeneses and host defense mechanisms, often remain speculative and based on in vitro studies and animal models 2-4 with 2 primary exceptions-IgE-mediated immediate hypersensitivity reactions and mastocytosis. Complicating the understanding of the role of mast cells in human biology is that while other normal human immune cell functions often become more obvious in the absence of a specific cell type, such as with agranulocytosis, or in the absence of normal function of a specific pathway, as in autoimmune lymphoproliferative syndrome (ALPS) associated with defective lymphocyte Fas-mediated apoptosis, the single similar situation involving mast cells and human disease characterized to date is mastocytosis, resulting from disturbed control of mast cell proliferation.Mast cell research initially relied upon observation on mast cell appearance and numbers in tissue biopsies, sometimes correlated with tissue histamine levels. With time, methods were developed to obtain and study mast cells ex vivo. The most common protocols relied on obtaining mast cells from the peritoneal cavity of rodents, or enrichment of mast cells from tissue digests. These approaches initiated modern mast cell biology with the first work on histamine, slow-reacting substance of anaphylaxis (SRS-A), and other mast cell-derived mediators including proteases, and the early studies on the mechanisms of mast cell signal transduction. In more recent years, and with the identification of key mast cell growth factors, investigators have discovered how to culture mast cells in vitro from pluripotential precursors (Figure 1). This development has facilitated the further study of human mast cell gene expression, signal transduction, and production of mediators relevant to inflammation. HistoryThe most striking feature of mast cells is that their cytoplasm is filled with dense metachromatic granules that stain red or violet when treated with basic aniline dyes. Using this "metachromasia," Ehrlich in 1878 first clearly described mast cells or "mastzellen" (maestung-a root of the English word mastication; the active form "measten" is still in use). 5 He speculated that these granules were the product of overfeeding (die mast). Ehrlich also noted the tendency of mast cells to be associated with blood vessels, nerves, and glandular ducts. These observations contributed to Ehrlich's Nobel Prize in Medicine in 1908. In 1894, Unna reported that the cutaneous lesions termed urticaria pigmentosa (UP) were associated with increased mast cells below each lesion. 6 Thus, by the end of the 19th century, mast cells had been recognized both in the normal state and associated with pathology.Following a number of reports that ma...

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confidence: 90%

Mast cells and mastocytosis

Metcalfe

2008

Blood

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484

515

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“…Although most of the proteoglycans in mammals are located in extracellular matrices and are protease-susceptible, IgE-Fc-bearing cells contain proteaseresistant proteoglycans in their secretory granules (15,16). Heparin proteoglycans are present in the secretory granules of rat (15) and mouse (17) serosal mast cells, and human lung (18) and skin (19) mast cells. Chondroitin sulfate E proteoglycan and chondroitin sulfate di-B proteoglycan are present in the granules of interleukin 3-dependent mouse bone marrow-derived mast cells (17,20) and in rat basophilic leukemic cells (16), respectively.…”

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Proteoglycans in cell-mediated cytotoxicity. Identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis.

MacDermott¹,

Schmidt²,

Caulfield³

et al. 1985

The Journal of Experimental Medicine

143

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A clone of natural killer (NK) cells (JTB18) was found to be ultrastructurally similar to peripheral blood large granular lymphocytes (LGL). These cells incorporated [35S]sulfate into cell-associated proteoglycan molecules, which were then isolated by CsCl density gradient centrifugation. As assessed by gel filtration chromatography, the native 35S-labeled proteoglycan and its beta-eliminated 35S-labeled glycosaminoglycans were of Mr approximately 200,000 and 50,000, respectively. The 35S-labeled proteoglycans were resistant to proteolysis, since their Mr were apparently not altered by incubation with either pronase or S. aureus V8 protease. The purified NK cell 35S-labeled proteoglycans were degraded by approximately 90% to 35S-labeled disaccharides with either chondroitinase ABC or AC. High performance liquid chromatographic analysis of the digests revealed these disaccharides to be composed entirely of chondroitin sulfate A (glucuronic acid----N-acetylgalactosamine-4SO4). Whole 35S-labeled cells incubated with chondroitinase ABC failed to release 35S-labeled disaccharides into the supernatant, and x-ray energy-dispersive analysis revealed that sulfur-containing molecules were present in the intracellular granules, thereby localizing the NK cell-associated proteoglycan primarily in the granules of the cell, rather than on the plasma membrane. The 35S-labeled cloned NK cells incubated for 30 min to 4 h with K562 tumor cell targets at a 0.5:1 ratio exocytosed a mean of 49% of the granular 35S-labeled proteoglycans during the first 60 min of the culture. Proteoglycan release was maximal with an effector/target cell ratio of 0.5:1 for JTB18:K562. Significant proteoglycan release from JTB18 NK cells was also obtained with other sensitive target cells such as REX, Molt4, and CEM, but not with cells such as KG1 and Laz156, which have been shown previously to be resistant to killing by this NK cell. Thus, protease-resistant intracellular proteoglycans with chondroitin sulfate A side chains are specifically exocytosed from the granules of human NK effector cells upon contact with sensitive targets, suggesting that these proteoglycans may be involved in the mechanism of cytotoxicity.

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“…Human heparini glycosaminoglycan obtained from lung by Dowex and DEAE-cellulose chromatography exhibited all anticoagulant activity of 137 U/nmg (15).…”

Section: Introductionmentioning

confidence: 99%

“…The fractions containing heparin proteoglyean, as assessed by metachromasia (12) and assay of uronic acid content (13), were eluted with 3 M NaCl, dialyzed and lyophilized. This material was treated with chondroitinase ABC as described (14,15) to remove contaminating chondroitin sulfates, and rechromatographed on Dowex l-x2. The fractions eluted with 3 M NaCl that contained heparin proteoglyean were pooled, dialyzed against distilled water, and lyophilized.…”

Section: Introductionmentioning

confidence: 99%

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Structural Determinants of the Capacity of Heparin to Inhibit the Formation of the Human Amplification C3 Convertase

Kazatchkine¹,

Fearon²,

Metcalfe³

et al. 1981

J. Clin. Invest.

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152

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A B S T R A C T The ability of heparin glycosaminoglycan to prevent formation ofthe properdin-stabilized amplification C3 convertase is independent of antithrombin binding activity and requires substitution of the amino sugar and a degree of oxygen (0)-sulfation which could be on the uronic acid or the amino sugar. Preparations of heparin glycosaminoglycan isolated by different techniques from different species (rat, human, and porcine) exhibited an equivalent capacity to inhibit generation ofthe amplification C3 convertase. Hyaluronic acid, which is devoid of 0-sulfation, had no inhibitory activity; chondroitin 4-sulfate of rat and whale origins, chondroitin 6-sulfate of rat and shark origins, and dermatan sulfate from porcine skin are 0-sulfated on the galactosamine and had minimal activity. Porcine heparin glycosaminoglycan, isolated on the basis of affinity for antithrombin III, had no greater anticomplementary activity than porcine glycosaminoglycan, which failed to bind antithrombin III and had essentially no anticoagulant activity. Nitrogen (N)-desulfation of porcine heparin reduced anticomplementary activity to the level of the other sulfated mucopolysaccharides, and both N-resulfation and N-acetylation restored the original activities, thereby indicating a requirement for N-substitution, but not N-sulfation. N-resulfation of N-desulfated and 0-desulfated heparin did not restore any activity, thus indicating that 0-sulfation and N-substitution represent independent, critical structural requirements for the anticomplementary activity of heparin glycosaminoglycan.

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Isolation and Characterization of Heparin from Human Lung

Cited by 148 publications

References 26 publications

Mast cells and mastocytosis

Mast cells and mastocytosis

Proteoglycans in cell-mediated cytotoxicity. Identification, localization, and exocytosis of a chondroitin sulfate proteoglycan from human cloned natural killer cells during target cell lysis.

Structural Determinants of the Capacity of Heparin to Inhibit the Formation of the Human Amplification C3 Convertase

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