Mobilization of sialidase from intracellular stores to the surface of human neutrophils and its role in stimulated adhesion responses of these cells. A S Cross, D G Wright J Clin Invest. 1991;88(6):2067-2076. https://doi.org/10.1172/JCI115536. Research Article Desialation of cell surfaces has been associated with the initiation or modification of diverse cellular functions. In these studies we have examined the subcellular distribution of sialidase (SE) in human neutrophils as well as the mobilization of this enzyme following neutrophil activation. Separation of subcellular fractions by density gradient centrifugation showed that SE is present not only in neutrophil primary and secondary granule populations, like lysozyme, but also in plasma membrane fractions. Neutrophil activation was associated with a redistribution of SE from secondary granule-enriched fractions to the plasma membrane. Furthermore, SE activity detected on the surface of intact neutrophils with a fluorescent SE substrate increased rapidly after activation with kinetics that matched both the loss of total cell-associated sialic acid and release of free sialic acid from the cells. These activation-dependent events were in each case blocked by incubation of neutrophils with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Aggregation responses of neutrophils as well as adhesion responses to nylon and plastic surfaces were also inhibited by 2-deoxyNANA. Our findings indicate that the activation-dependent desialation of the neutrophil surface is associated with mobilization of an endogenous SE to the plasma membrane and has a role in stimulated adhesion responses of these cells. Find the latest version: https://jci.me/115536/pdf Mobilization of Sialidase from Intracellular Stores to the Surface of Human Neutrophils and Its Role in Stimulated Adhesion Responses of These Cells Alan S. Cross and Daniel G. Wright Departments ofBacterial Diseases and Hematology, Walter Reed Army Institute ofResearch, Washington, D.C. 20307-5100 Abstract charged, more deformable and more adhesive, coincident with a progressive loss of cell surface sialic acid (6). Desialation of cell surfaces has been associated with the initia- Cells may be desialated by sialidases (or neuraminidases) tion or modification of diverse cellular functions. In these stud- which cleave n-glycosidic-linked sialic acids in a one-step reac- ies we have examined the subcellular distribution of sialidase tion (9). These enzymes, like sialic acids, are widely distributed (SE) in human neutrophils as well as the mobilization of this in nature and exist in a variety offorms that may differ substan- enzyme following neutrophil activation. Separation of subcellu- tially in electrophysical properties and also in their ability to lar fractions by density gradient centrifugation showed that SE cleave distinct glycosidic linkages (10-12). Sialidases of mam- is present not only in neutrophil primary and secondary granule malian cells are generally membrane bound and have been less populations, like lysozyme, but also in plasma membrane frac- well characterized than microbial neuraminidases (13). None- tions. Neutrophil activation was associated with a redistribu- theless, it is evident that these enzymes may provide a mecha- tion of SE from secondary granule-enriched fractions to the nism for rapidly modulating cell function by modifying glyco- plasma membrane. Furthermore, SE activity detected on the sylated cell surface structures ( 14). surface of intact neutrophils with a fluorescent SE substrate Human neutrophils have been shown by several laborato- increased rapidly after activation with kinetics that matched ries to contain sialidase activity (15, 16). Moreover, studies of both the loss of total cell-associated sialic acid and release of the appearance of sialidase activity during induced maturation free sialic acid from the cells. These activation-dependent of the myeloid leukemia cell line, HL60, have suggested that events were in each case blocked by incubation of neutrophils neutrophil sialidase is acquired during the latter phases of neu- with the SE inhibitor, 2-deoxy-N-acetyl-neuraminic acid. Ag- trophilic maturation (15). Because desialation of cells has been gregation responses of neutrophils as well as adhesion re- associated with changes in various cell functions that are sponses to nylon and plastic surfaces were also inhibited by known to occur with neutrophil activation (e.g., increased adhe- 2-deoxyNANA. Our findings indicate that the activation-de- siveness [17] and decreased surface charge [18, 19]), we were pendent desialation of the neutrophil surface is associated with attracted to the possibility that mobilization of an endogenous mobilization of an endogenous SE to the plasma membrane and sialidase in neutrophils might have a role in the functional has a role in stimulated adhesion responses of these cells. (J. activation of these cells. To explore this possibility, we exam- Clin. Invest. 1991.88:2067-2076.) Keywords: sialicacideneur- ined the intracellular compartmentalization of sialidase in hu- aminidase * cell activation * granulocytes * aggregation man neutrophils, together with the mobilization and redistri- bution of this enzyme during cell activation. Introduction Methods The presence of sialic acids on cell surfaces, as terminal sugars of glycosylated cell surface molecules, has been highly con- Isolation of human blood neutrophils and preparation of subcellular served in evolution (1). Furthermore, there is substantial evi- fractions. Neutrophils were isolated from 50-200-ml samples of hepa- rinized venous blood obtained from healthy volunteers dence that sialic acids are structural determinants for a by combining variety Hypaque-Ficoll of important cell-cell interactions and cellular such density gradient centrifugation and dextran sedimenta- functions, tion procedures, followed by hypotonic lysis ofresidual erythrocytes, as as the adhesiveness and metastatic potential of neoplastic cells described previously (20, 21). For certain studies that required large (2-4) and the phenomenon of contact inhibition in tissue cul- numbers of cells, up to 5 x 1O' neutrophils were isolated by the same ture (5). methods from 200 to 400-ml leukocyte concentrates obtained by con- Immature myeloid cells have been shown to be rich in cell tinuous flow, centrifugation leukapharesis of normal volunteers (2997 surface sialic acid (6). Moreover, there is evidence that this high continuous flow centrifuge; IBM Corp., Danbury, CT). In both cases, degree of sialation contributes to the negative surface charge, final leukocyte preparations contained > 95% neutrophils and were rigidity, and lack of adhesiveness of these cells (7, 8), for as > 99% viable as determined by trypan blue dye exclusion. myeloid precursor cells mature, they become less negatively For preparation of subcellular fractions, neutrophils were sus- pended in calcium-free 10 mM piperazine-N,NT-bis(2-ethane sulfonic acid (Pipes) buffer with 1.0 mM ATP and 3.5 mM MgCl2 at a cell concentration of 5 X 107 neutrophils/ml. Cells were then disrupted by Address correspondence to Dr. Alan S. Cross, Department of Bacterial nitrogen cavitation at 40C using a precooled pressure homogenizer Diseases, Walter Reed Army Institute of Research, Washington, DC (Parr Instrument Co., Moline, IL) as described previously (21). Cavi- 20307-5100. tates were then centrifuged at 1,400 g for 10 min to remove nuclei and Received for publication 7 December 1989 and in revised form 6 unbroken cells, and supernatants were applied to continuous sucrose August 1991. gradients (21, 22) or to discontinuous PercollR (Pharmacia, Uppsala, Sweden) gradients (21, 23) and centrifuged in a refrigerated (40C) ultra- The Journal of Clinical Investigation, Inc. centrifuge at 95,000 g for 4 h or at 48,000 g for 30 min, respectively. Volume 88, December 1991, 2067-2076 Stepwise elution of gradients from the bottom of centrifuge tubes was Neutrophil Sialidase 2067 then carried out. Granule-rich and plasma membrane vesicle-rich frac- extracts. For some studies, serine esterase inhibitors (PMSF [from tions were identified by direct turbidometric measurements of frac- Sigma], TPCK [N-tosyl-L-phenylalanine chloromethyl ketone] or tions at 450 nm (21, 22) and by the measurement of previously charac- TLCK [N-tosyl-L-lysine chloromethyl ketone], obtained from Calbio- terized granule and plasma membrane markers: lysozyme (24), myelo- chem Corp., La Jolla, CA) were added to reaction mixtures. peroxidase (25), vitamin B12 binding protein (26), and alkaline To examine the possibility that neutrophils contained or generated phosphatase (27), after the addition of 0.1% Triton X-100 (vol/vol, an inhibitor of sialidase activity, we carried out mixing experiments to final concentration) to fractions. For certain studies that addressed the determine whether intact neutrophils or neutrophil extracts modified subcellular distribution of neutrophil sialidase with and without prior the ability of microbial neuraminidase to release sialic acid from neura- activation of the cells, fractions separated on discontinuous PercollR minyllactose. Release of sialic acid from neuraminyllactose mediated gradients were collected by aspiration of the gradients from the top by neuraminidase and by neutrophils or neutrophil extracts were mea- using a fine-tipped Pasteur pipette; aspirated fractions that were col- sured both separately and together to determine whether the sialidase lected at the separate visible