Identification of Human Eosinophil Lysophospholipase As the Constituent of Charcot-Leyden Crystals (Phospholipase B) PETER F
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Proc. Natl. Acad. Sci. USA Vol. 77, No. 12, pp. 7440-7443, December 1980 Medical Sciences Identification of human eosinophil lysophospholipase as the constituent of Charcot-Leyden crystals (phospholipase B) PETER F. WELLER*t, EDWARD J. GOETZL*t, AND K. FRANK AUSTEN* Department of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, Massachusetts 02115; and tHoward Hughes Medical Institute Laboratories for the Study of Immunological Diseases ofHarvard Medical School, Boston, Massachusetts 02115 Contributed by K. Frank Austen, September 15,1980 ABSTRACT Since the initial descriptions of Charcot-Ley- protein in the large granule of the eosinophil (12). The intra- den crystals more than 100 years ago, the presence of these cellular source of the eosinophil CLC protein remains uncertain, slender, di yramidal c stals in human tissues and biologic although nuclear (9), granular (10), and cytoplasmic (12) origins fluids has become a hallmark of eosinophilic leukocye infil- tration, especially in association with allergic and helminthic have been proposed. Further, no biochemical activity or bio- diseases. The formation of these crystals in vitro after disruption logic function has been established for the protein composing of human eosinophils, but not of other cell types, in hypotonic the crystals (11). The findings that chromatographically puri- saline or detergent established the eosinophil as the unique fied, homogeneous eosinophil lysophospholipase (lysolecithin cellular source of the crystalline protein. Charcot- yden acylhydrolase, EC 3.1.1.5) and CLC protein express a common crystals have now been found to express lysophospholipase ac- tivity (lysolecithin acylhydrolase, EC 3.1.1.5), and the solubilized enzymatic activity, exhibit the same physicochemical charac- Charcot-Leyden crystal protein presents a single stained protein teristics of size and charge, and form crystals of identical band that is coincident with the lysophospholipase activity morphology establish human eosinophil lysophospholipase as eluted from replicate gels on alkaline polyacrylamide gel elec- the constituent of CLC. trophoresis. On sodium dodecyl sul ate/polyacrylamide gel electrophoresis, the solubilized Charcot-Leyden crystal protein migrates with a molecular weight of 17,400, which is compa- MATERIALS AND METHODS rable to that of eosinophil lysophospholipase purified chroma- Materials. Reagents and equipment were obtained from the tographically to homogeneity; further, on combination, the two proteins comigrate as a single staining band. Finally, the chro- sources indicated: L-1-[1-'4C]Palmitoyl lysophosphatidylcholine matographically purified eosinophil lysophospholipase in hy- (specific activity about 50 mCi/mmol; 1 Ci = 3.7 X 1010 bec- potonic buffer forms dipyramidal crystals morphologically querels) and Aquasol scintillation fluid from New England identical to Charcot-Leyden crystals. The findings that chro- Nuclear; Sephadex G-100, heparin-Sepharose, gel filtration and matographically purified, homogeneous eosinophil lysophos- electrophoresis molecular weight calibration proteins, 6% pholipase and Charcot-Leyden crystal protein express the same enzymatic activity, are of the same size and charge, and form dextran 70 in normal saline (Macrodex), and diatriazoate-Ficoll crystals of identical morphology indicate that human eosinophil (Ficoll-Paque) from Pharmacia; synthetic palmitoyl lyso- lysophospholipase is the constituent of Charcot-Leyden crys- phosphatidylcholine, egg-yolk phosphatidylcholine, iodo- tals. acetamide, and glycine from Sigma; organomercurial agarose (Affi-Gel 501), NaDodSO4, acrylamide, N,N'-methylene- Prominent blood or tissue accumulations of eosinophilic leu- bisacrylamide, N,N,N',N'-tetramethylethylenediamine, urea, kocytes occur in many allergic reactions and helminthic in- dithiothreitol, ammonium persulfate, Coomassie brilliant blue fections as well as in the course of some neoplastic, inflamma- stain, and Bio-Rad protein assay from Bio-Rad; Na2EDTA, tory, and immunodeficiency diseases (1). Charcot-Leyden chloroform, methanol, heptane, isopropanol, Spectrapor dialysis crystals (CLC), which are characteristically long, slender, di- membranes, and Gelman SA ITLC thin-layer chromatography pyramidal crystals, were initially described in the mid-nine- sheets from Fisher; calcium- and magnesium-free Hanks' teenth century (2, 3) and are hallmarks of eosinophil involve- balanced salt solution from Microbiological Associates (Walk- ment in some tissue reactions. CLC may be prominent in spu- ersville, MD); 10,000 Mr retention collodion bags from tum of patients with asthma (3), pulmonary ascariasis (4), and Schleicher & Schuell; Branson model 350 sonifer from Branson tropical eosinophilia (5), in the feces of patients with amebic, Sonic Power; and UM-05 Diaflo membranes from Amicon. Trichuris, or ulcerative colitis (6), and in tissues containing Blood from normal donors provided neutrophils, mononu- eosinophilic granulomas of bone (7) and granulomas associated clear cells, platelets, and erythrocytes (13), and donors with with tissue-invading helminths (8). blood eosinophilias due to trichinosis, filariasis, drug allergies, Disruption of eosinophils either by detergents (9) or by ho- and a hypereosinophilic syndrome provided granulocytes en- mogenization and suspension in hypotonic saline (5) allows for riched in eosinophils (14). Eosinophils from a donor with hyp- the formation of CLC in vitro. Similar disruption of other ereosinophilia (leukocyte count of 28,000-36,000/mm3 of blood classes of leukocytes or of eosinophils derived from nonprimate with 87-94% eosinophils) were used without further purifica- mammalian species fails to result in CLC formation (10). Thus, tion as a source of protein for formation of CLC and for isolation eosinophils, specifically those from primate species, have been of enzyme. established as the unique cellular source of this crystalline Assay of Lysophospholipase. The cell preparations were material (11). Human CLC are composed of a single protein, disrupted by sonication and dialyzed against 0.1 M NaCl which differs from the major basic protein, the predominant overnight before assay of cellular lysophospholipase. A 100 to 200-,1 portion of cell extract or chromatographic fraction was The publication costs of this article were defrayed in part by page of 1 con- charge payment. This article must therefore be hereby marked "ad- added to a reaction mixture with a final volume ml, vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: CLC, Charcot-Leyden crystals. 7440 Downloaded by guest on September 24, 2021 Medical Sciences: Weller et al. Proc. Nati. Acad. Sci. USA 77 (1980) 7441 taining 250 nmol of synthetic palmitoyl lysophosphatidylcho- pH 8.9,7.5% acrylamide gels (17) with the modifying addition line and 0.96 nmol of [14C]palmitoyl lysophosphatidylcholine, of 2 mM dithiothreitol to the electrode buffer. Gels were elec- both of which had been sonicated for 2 min at4VC at setting trophoresed at 2.5 mA per gel for 3 hr before use. 5 with a microtip (Branson model 350 sonifier) in 0.1 M Tris- HCI, pH 7.5/2 mM EDTA. Five hundred micrograms of RESULTS phosphatidylcholine, sonicated in buffer for 9min as above, was For the same numbers of disrupted human peripheral blood included in the assay of chromatographic fractions. The reac- leukocytes, lysophospholipase activity in the eosinophil-en- tion mixture was incubated at 370C for 1 hr and was then ex- riched granulocytes was 8-fold greater than that in the neu- tracted with acidified isopropanol/heptane (15) to partition the trophil-enriched granulocytes and 3-fold greater than that of released fatty acid into the upper organic layer, from which a the mononuclear leukocytes (Fig. 1). Relatively trivial activity sample was taken and its radioactivity was measured in Aquasol was present in platelets and erythrocytes. (14). The identification and quantitation of the released fatty In a preliminary experiment, 200 Ml of pelleted CLC con- acid was confirmed by extracting replicate incubation mixtures tained 184 units of lysophospholipase activity when assayed with chloroform/methanol/water (10:10:9, vol/vol) and re- directly. To determine if this enzyme activity was a major solving the extracted lipids by thin-layer chromatography on component of the crystal, lyophilized CLC were solubilized in silicic acid with a neutral lipid solvent system in comparison 0.1 M Tris-HCI, pH 7.5/2 mM EDTA, and 40-jg quantities of with purified standards (14). One unit of enzyme activity protein, as assessed by the Bio-Rad assay using human IgG as represents 1 nmol of fatty acid liberated per hour at 37"C, at a reference standard, were subjected to alkaline disc gel elec- pH 7.5. trophoresis in two replicate gels. In the gel stained with Coo- Purification of Eosinophil Lysophospholipase. Approxi- massie brilliant blue, a single stained protein band was present. mately 1 X 109 leukocytes (94% eosinophils, 6% neutrophils) The parallel gel was sliced into 2-mm sections and each section were disrupted by sonication in 0.05 M Tris-HCI, pH 8.5/2 mM was macerated in 500,ul of 0.1 M Tris-HCl, pH 7.5/2 mM EDTA. After the cells were centrifuged at 750 X g for 10 min, EDTA/2 mM dithiothreitol and dialyzed overnight against the the supernatant fluid was subjected to gel filtration on Sephadex same buffer at 40C with 6000-8000 Mr cut-off dialysis mem- G-100 in 0.05 M Tris.HCI,