Attack of sea urchin eggs by dogfish phagocytes: Model of phagocyte-mediated cellular cytotoxicity

Abstract

To test whether lysosomal degranulation of phagocytes is associated with antibody-dependent cytotoxicity, eggs of Arbacia punctulata were used as targets for blood phagocytes of Mustelus canis. Eggs were coated with heataggregated dogfish IgM and exposed to phagocytes, and cytolysis of eggs was observed by Nomarski optics. Phagocytes adhered, degranulated, and raised fertilization membranes resembling those induced by sperm or ionophore A23187. Lysis was then observed as damage radiating from the point of phagocyte-egg contact. By 4 hr. coated eggs exposed to phagocytes released 8.9, 12.3, and 7.4% of total catalase (EC 1.11.1.6), f-glucuronidase (EC 3.2.1.31), and superoxide dismutase (EC 1.15.1.1) into the medium. Cytotoxic enzyme release significantly exceeded that from uncoated eggs incubated with phagocytes or eggs alone (uncoated or coated). Because activated eggs release a neutral protease, it was considered possible that this enzyme might be responsible for autolysis of eggs. This possibility was excluded because (i) lysis of eggs was not inhibited by soybean trypsin inhibitor (SBTI) whereas the egg protease was sensitive to SBTI, and (ii) the major try sin-li e activity of phagocytes was not inhibited by SBTI. These experiments demonstrate that Ig-coated cells are first activated, and then killed, when exposed to degranulating phagocytes and suggest that enzymes from attacking phagocytes, and not target cells, are responsible for cell death. Phagocytes, such as polymorphonuclear leukocytes (PMNs) or macrophages, degranulate and secrete lysosomal enzymes when exposed to immune complexes or aggregated immunoglobulins (1-3). Lysosomal, but not cytoplasmic, enzymes appear in the surrounding medium after the cells' Fc receptors engage immunoglobulins, either in the bulk phase or dispersed on nonphagocytosable surfaces (4). The latter process, called "reverse endocytosis" (5) or "frustrated phagocytosis" (6), is launched when phagocytes encounter aggregated immunoglobulins or immune complexes adsorbed onto large surfaces that the cell cannot ingest. Proteolytic enzymes released from phagocytes by immune stimulation can hydrolyze defined substrates of the extracellular milieu, such as proteoglycans (7,8), collagen (9), and elastin (10). It is less clear whether phagocytes release materials capable of affecting living target cells, although lysosomal secretion may mediate macrophage-dependent cellular cytotoxicity (11).To elucidate mechanisms whereby reverse endocytosis brings about injury to living cells coated with antibody, oocytes of sea urchins (Arbacia punctulata) have been coated with aggregated immunoglobulin of the dogfish (Mustelus canis). The target (Me2SO), cytochrome c, hypoxanthine, and Triton X-100 were supplied by Sigma (St. Louis, MO), soybean trypsin inhibitor (SBTI) by Worthington, and xanthine oxidase by Boehringer Mannheim. Antipain and leupeptin were the generous gifts of the U.S.-Japan Cooperative Cancer Program; ionophore A23187, of R. Hersley (Eli Lilly); Cbz-Gly-GlyArg-amido-...

Inhibitors of membrane transport reduce lysosomal enzyme secretion from dogfish phagocytes and their killing of sea urchin eggs

“…This enzyme is undetectable in dogfish phagocytes (2). Percentage inhibition of catalase release from cytotoxic mixtures paralleled the inhibition of ,B-glucuronidase release shown in Fig.…”

“…We therefore studied whether these agents can inhibit the postphagocytic secretion of lysosomal enzymes from blood phagocytes of M. canis and whether their order of activity in this regard parallels their order of activity in inhibiting phagocyte-mediated cytotoxicity of sea urchin eggs. Because dogfish phagocytes resemble the PMNs of mammals with respect to enzymic equipment (2, 12, 13), response to immunologic (2,12) or nonimmunologic (13) stimulation, and capacity to provoke lysis of appropriate target cells (2), these studies should be relevant to modification of phagocyte-mediated injury in inflammatory states (14).…”

“…Phagocytes of M. canis and eggs from A. punctulata were obtained as described (2,12). Phagocytes were suspended in elasmobranch Ringer's solution (ER) containing heparin (500 units/ml) and were distinguished by uptake of neutral red (1 These experiments were carried out as described (2); release of f-glucuronidase and catalase was measured.…”

Comparative phagocytic and pinocytic activities of leucocytes from peripheral blood and lymphomyeloid tissues of the nurse shark (Ginglymostoma cirratumBonaterre) and the clearnose skate (Raja eglanteriaBosc)

“…In contrast, in the Port Jackson shark (Heterodontus portusjacksoni), Stokes & Firkin (1971) found that a greater number of yeast were ingested by neutrophils than by eosinophils, with an average of six and two per cell, respectively. Phagocytic cells of the smooth dogfish (Mustelus canis) were found to resemble polyorphonuclear leucocytes (Weissman et al, 1978). In other studies with nurse shark cells, only the neutrophils, not the eosinophils, were reported to be phagocytic (Hyder et al, 1983).…”

“…Cells produced by the epigonal and/or Leydig organs may be important in immune responses and in inflammatory processes of elasmobranchs (Fänge & Mattisson, 1981). Studies on phagocytosis by elasmobranch immune cells include both in vivo (Fänge, 1968;Parish et al, 1986b) and in vitro (Weissman et al, 1978;Hyder et al, 1983;Parish et al, 1986a;McKinney & Flajnik, 1997) investigations, but the involvement of cells from the Leydig and epigonal organs has not been examined.…”