Proc. Natl. Acad. Sci. USA Vol. 92, pp. 5037-5041, May 1995 Immunology A disaccharide that inhibits tumor necrosis factor a is formed from the extracellular matrix by the enzyme heparanase (cytokines/extracellular matrix/heparan sulfate/delayed-type hypersensitivity) OFER LIDER*, LioRA CAHALON*, DALiA GILAT*, RAMI HERSHKOVIZ*, DANIEL SIEGELt, RAANAN MARGALIT*, ODED SHOSEYOVt, and IRUN R. COHEN*t *Department of Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel; and tThe Kennedy-Leigh Center for Horticultural Research, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel Communicated by Michael Sela, The Weizmann Institute of Science, Rehovot, Israel, January 11, 1995 (received for review December 1, 1994) ABSTRACT The activation of T cells by antigens or gram amounts of ECM disaccharide suppresses T-cell medi- mitogens leads to the secretion of cytokines and enzymes that ated reactivity in vivo. Incubation of T cells with the ECM shape the inflammatory response. Among these molecular disaccharide inhibits T-cell production of the active form of mediators of inflammation is a heparanase enzyme that tumor necrosis factor (TNF)-a, a major inflammatory cytokine. degrades the heparan sulfate scaffold of the extracellular matrix (ECM). Activated T cells use heparanase to penetrate the ECM and gain access to the tissues. We now report that MATERIALS AND METHODS among the breakdown products of the ECM generated by Heparanase. Mammalian heparanases have not yet been heparanase is a trisulfated disaccharide that can inhibit cloned; because T-cell heparanase was not available in quan- delayed-type hypersensitivity (DTH) in mice. This inhibition tity, we used heparanase obtained from human placentas. The ofT-cell mediated inflammation in vivo was associated with an heparanase enzyme was prepared and purified to >3.4 x inhibitory effect of the disaccharide on the production of 105-fold by ammonium sulfate precipitation followed by se- biologically active tumor necrosis factor a (TNF-a) by acti- quential chromatographies on carboxymethyl-, heparin-, and vated T cells in vitro; the trisulfated disaccharide did not affect Con A-Sepharose columns (RAD Chemicals, Ness Ziona, or T-cell viability responsiveness generally. Both the in vivo Israel) by modifying the technique of Oosta et at (7). The and in vitro effects of the disaccharide a manifested bell- specific activity of the heparanase was determined, as de- shaped dose-response curve. The inhibitory effects of the scribed, by its ability to release 35S04-labeled fragments of trisulfated disaccharide were lost if the sulfate groups were heparan sulfate from removed. Thus, the disaccharide, which may be a natural the ECM (3, 4). product of inflammation, can regulate the functional nature Preparation of ECM Disaccharide. ECM-coated plates of the response by the T cell to activation. Such a feedback were prepared from bovine corneal endothelial cells as de- control mechanism could enable the T cell to assess the extent scribed (5). Each ECM-coated plate was incubated for 48 h at of tissue degradation and adjust its behavior accordingly. 37°C with 20 ,ul of mammalian heparanase (0.5 mg/ml) in 1 ml of phosphate citrate buffer (18 mM citric acid/64 mM Na2HPO4/50 mM Some years ago, we reported that activated T cells secreted NaCl/1 mM CaCl2/1 mM dithiothreitol, pH heparanase, an enzyme that degrades the heparan-sulfate 6.2). The culture medium containing the ECM heparan sulfate component of the extracellular matrix (ECM; refS. 1 and 2). degradation products was then collected and applied to a The expression of heparanase by T cells, unlike that by tumor Sepharose 4B column (0.7 x 35 cm). The mobile phase was cells, was found to be tightly regulated by contact with specific phosphate citrate buffer at a flow rate of 5 ml/h. Fractions of antigen or mitogen. Naive T cells responded to activation by 1.6 ml were collected and monitored at 206 nm to detect synthesizing heparanase de novo but memory T cells were able oligosaccharides (Fig. 1A) or at 232 nm to detect unsaturated to release heparanase from preformed stores within minutes of disaccharides. Quantitation of the uronic acid containing contact with antigen (2). disaccharides was done by using the carbazole assay (8). The In addition to the positive induction of heparanase expres- lower molecular weight fractions, fractions 5 and 6, of the sion by T-cell activators, heparanase expression was also found Sepharose 4B peak were combined and freeze-dried, and the to be inhibitable by heparin (3-6). Heparin is very similar powder was resuspended in 10% of the initial volume. The chemically to heparan sulfate, the natural substrate of hepara- disaccharide was then isolated from the elution profile by using nase, and it was proposed that heparin, by occupying the HPLC gel-filtration chromatography followed by HPLC ion- binding site of the enzyme, may act as a competitive inhibitor exchange chromatography. Samples of 0.1 ml were injected of heparanase activity (3, 5, 6). However, we found that low into an HPLC column [Toyo Soda (Tokyo) TSK-Gel G3000 doses of heparin appeared to act directly on T cells to inhibit SW; 7.5 mm x 50 cm; and G2000 SW; 7.5 mm x 50 cm; in their release of heparanase (4, 5). Heparin is composed of series with a 7.5 mm x 10 cm guard column from Phenomenex different sulfated sugar molecules, and the saccharide moieties (Belmont, CA)]. The mobile phase was 0.5 M NaCl at a flow that inhibit T-cell heparanase release differ from those that rate of 1 ml/min. Fractions (1 ml) were collected and moni- inhibit blood coagulation (4). We reasoned that the T-cell tored at 206 and 232 nm. The column was calibrated with inhibitory molecules in heparin might actually mimic mole- heparin-oligosaccharide standards (Seikagaku Kogyo, Tokyo). cules produced by the action of heparanase on its natural The peak labeled pl in Fig. 1B, having the retention time of a substrate heparan sulfate. We report here the isolation of a disaccharide, was collected from nine identical runs. The trisulfated disaccharide generated by the action of heparanase substantially homogeneous fractions were combined and on the ECM (ECM disaccharide). Administration of nano- freeze-dried. The publication costs of this article were defrayed in part by page charge Abbreviations: DTH, delayed-type hypersensitivity; ECM, extracellu- payment. This article must therefore be hereby marked "advertisement" in lar matrix; TNF-a, tumor necrosis factor a; PHA, phytohemagglutinin. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 5037 Downloaded by guest on September 29, 2021 5038 Immunology: Lider et al Proc. Nati Acad Sci. USA 92 (1995) A injected into an analytical Sax-HPLC column (4.6 x 250 mm; packed with Spherisorb; Phase Separation, Inc., Norwalk, CT; -F5 5-gm particle size). The column flow rate was 1.5 ml/min and an NaCl linear gradient program was employed as shown in Table 1. The column eluent was monitored at 206 nm (Fig. 1 C) and the peak labeled A23/4 was collected and quantified by the carbazole assay. To characterize the isolated disaccharide molecule, we compared its retention time to various known heparin disaccharide standards with different levels of sulfa- tion (Sigma) that were injected into the Sax-HPLC column under identical conditions. The retention time of these stan- dards indicated that the ECM disaccharide was probably trisulfated. A trisulfated disaccharide standard (a-A&UA-2S- 1--4-GlcNS, 6S; Sigma; H 9267) had a very similar retention time to that obtained for peakA23/4: 23.07 min and 23.10 min, Fraction no. respectively. We were able to isolate a radiolabeled trisulfated disaccharide when the heparanase was used to degrade an B ECM metabolically labeled with Na235SO4 (ref. 2; data not shown). This indicates that the isolated disaccharide did in fact originate from the ECM. 0.03 Fast atom bombardment-mass spectrometry (FAB-MS) of methylated, partially desulfated A23/4 was consistent with a molecular weight of about 530 for a methylated derivative. The 0.02 proton NMR spectrum of the trisulfated disaccharide was recorded in 2H20 at 500 MHz. Typical sugar signals were 9 (4 evident between 3 and 5.5 ppm. A doublet signal for the '11. anomeric proton was found at 5.39 ppm having a coupling 0.01 factor of 3 Hz, suggesting the presence of a glucosamine sugar unit in an a configuration. The chemical shift of the anomeric proton, together with what is believed to be the 13-glucuronide 0* specificity of placental heparanase, leads to the tentative conclusion that the ECM disaccharide has a glucosamine at the nonreducing end in an a configuration attached 1-4 to a -0.01 glucuronide acid residue at the reducing end. The complete 40 50 60 identification and the organic synthesis of the proposed mol- Retention time, min ecule will be published elsewhere. A desulfated disaccharide was obtained by incubating some 0.03 C of the disaccharide of peak A23/4 for 4 days at room tem- perature at pH 3.5. The nonsulfated disaccharide was isolated 0.026 A23/4 by its shorter retention time on the Sax-HPLC column, which was very similar to the retention time of a monosulfated 0.022- disaccharide standard (a-AUA-2S-[1-+4]-GlcNAc; Sigma; H 8767). 0.018- Delayed-Type Hypersensitivity (DTH) Assay. Groups of 10 A female inbred BALB/c mice (The Jackson Laboratory) were 0.014 sensitized on the shaved abdominal skin with 100 ,ul of 2% 0.01 oxazalone dissolved in acetone/olive oil [4:1 (vol/vol)] applied topically (5). DTH sensitivity was elicited 5 days later by 0.006 challenging the mice with 20 ,ul of 0.5% oxazalone in acetone/ olive oil, 10 ,ul administered topically to each side of the ear.