Proc. Natl. Acad. Sci. USA Vol. 95, pp. 6290–6295, May 1998 Immunology A cell surface receptor defined by a mAb mediates a unique type of cell death similar to oncosis CHONGHUI ZHANG*†‡,YUHUI XU§,JIJIE GU*†, AND STUART F. SCHLOSSMAN*† *Tumor Immunology and §Structural Molecular Biology, Dana–Farber Cancer Institute; and †Department of Medicine, Harvard Medical School, Boston, MA 02115 Contributed by Stuart F. Schlossman, March 18, 1998 ABSTRACT Cell death is mediated by distinct pathways conditions that are distinct from necrosis, the pathological end including apoptosis and oncosis in response to various death result of a number of noxious stimuli (14–16). Relying on the signals. To characterize molecules involved in cell death, a morphologic appearance of dying cells, Majno and Joris (17) panel of mAbs was raised by immunizing mice with apoptotic have found that oncosis and apoptosis define two distinct cells. One of these antibodies, designated anti-Porimin (for pathways of cell death. The term oncosis (from o´nkos, meaning pro-oncosis receptor inducing membrane injury), was found swelling) was initially coined to characterize ischemic cell to directly induce a unique type of cell death in Jurkat cells. death in osteocytes and recently reintroduced to define the cell Anti-Porimin defines a 110-kDa cell surface receptor on death lacking apoptotic characteristics. In contrast to apopto- Jurkat cells. Functionally, anti-Porimin alone rapidly medi- sis with cellular shrinkage and nuclear disruption, oncosis is ates pore formation on the plasma membrane and induces cell defined in the literature as a form of cell death characterized death without participation of complement. Both the cellular by cellular swelling, membrane blebbing, and an increase in the expression and functional characteristics of the Porimin membrane permeability. Morphologically, cell death by onco- y antigen indicate that it is distinct from the CD95 (Fas Apo-1) sis leads to necrosis with karyolysis whereas apoptotic cell and other cell receptors known to induce apoptosis. Anti- death leads to necrosis with karyorrhexis, a degenerative Porimin-mediated cell death was preceded by cell aggregation, change or fragmentation of the cell nucleus (17). formation of plasma membrane pores, and the appearance of In an effort to investigate the molecular mechanisms of membrane blebs. More important, these cells show neither programmed cell death in human T cells, we have character- DNA fragmentation nor apoptotic bodies, but display lethal ized molecules involved in cell death by developing mAbs to damage of the cell membrane. Cell death by anti-Porimin is dying cells. Anti-Porimin, one of these mAbs, efficiently distinct from complement-dependent cytolysis or comple- induces a unique type of cell death in Jurkat cells. Cell death ment-independent apoptosis but is similar to that described mediated by anti-Porimin is distinct from complement- for oncosis, a form of cell death accompanied by the mem- dependent cytolysis or complement-independent apoptosis, brane damage followed by karyolysis. The induction of cell but is very similar to the cell death pathway defined for oncosis. death by anti-Porimin may represent a unique cell surface The molecular and biochemical characterization of this cell receptor-mediated pathway of cell death in the human lym- surface receptor and its possible role in the cell death pathways phoid system. are described. Programmed cell death is an essential process in normal development and the regulation of tissue homeostasis whereas MATERIALS AND METHODS a failure of the cell death machinery contributes to the possible Anti-Porimin mAb. Monoclonal anti-Porimin was generated pathogenesis of cancer or other diseases (1). One major focus and characterized as described previously (18). Briefly, of cell death research has been the characterization of the role BALByc mice were immunized with Jurkat cells that had been of cell surface receptors and their ligands in mediating signals induced to undergo cell death by 1-b-D-arabinofuranosylcy- leading to the elimination of potentially harmful cells from the tosine. Splenocytes from the immunized mouse were fused body. Two well characterized death receptors, the CD95 y with P3-X63-Ag8.653 cells. Anti-Porimin was screened and (Fas Apo-1) and tumor necrosis factor receptor 1 (TNFR-1), selected by its reactivity with cell surface antigen and its ability are known to induce apoptosis in lymphoid cells upon binding to induce cell death. The mAb was determined to be mouse to their ligands (2–4). These receptors, as well as several IgM subclass by using commercial Ig-isotyping reagents (Am- recently identified cell death receptors (5–10), have been ersham). Ascites for anti-Porimin was produced in mice, and shown to engage the apoptotic pathway through their inter- the mAb was purified from the ascites fluid by using a IgM action with a family of death domain-containing homologous purification kit from Pierce. proteins. After engagement of these receptors by specific Other Antibodies and Reagents. Monoclonal anti-CD95 antibody or their ligands, the death domain orchestrates the (IgM) (19), anti-CD6 (IgM), and mouse IgM (MsIgM) were assembly of a signaling complex to recruit a series of proapo- used as controls in this study and were obtained from Coulter. ptotic proteases that cleave structural proteins and interfere Affinity-purified goat anti-mouse Ig-fluorescein isothiocya- with critical repair processes leading to cell death (11). nate (FITC) conjugate for flow cytometry and goat anti-mouse Cell death by apoptosis has been investigated extensively and Ig-peroxidase conjugate for immunoblotting assays were pur- shown to be a major mediator of programmed cell death. chased from Jackson ImmunoResearch. Propidium iodide and Nevertheless, other cell death mechanisms have been de- all other chemicals were obtained from Sigma unless otherwise scribed during development (12, 13) and under experimental indicated. The publication costs of this article were defrayed in part by page charge Abbreviations: Porimin, pro-oncosis receptor-inducing membrane in- payment. This article must therefore be hereby marked ‘‘advertisement’’ in jury; MFI, mean fluorescence intensity. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: Tumor Immunol- © 1998 by The National Academy of Sciences 0027-8424y98y956290-6$2.00y0 ogy, Dana–Farber Cancer Institute, 44 Binney Street, Boston, MA PNAS is available online at http:yywww.pnas.org. 02115. e-mail: [email protected]. 6290 Downloaded by guest on September 23, 2021 Immunology: Zhang et al. Proc. Natl. Acad. Sci. USA 95 (1998) 6291 Cell Culture and Induction of Cell Death by Anti-Porimin. extraction in which DNA fragmentation was detected by Jurkat cells and all other human cell lines used in this study agarose gel electrophoresis as described previously (22). were obtained from the American Type Culture Collection Electron Microscopy. To examine the surface structural (Manassas, VA) and were maintained in RPMI 1640 medium changes of cells by scanning electron microscopy, Jurkat cells (BioWhittaker) supplemented with 10% fetal bovine serum, were treated with anti-Porimin or control antibodies for1hat and2mML-glutamine, penicillin (50 unitsyml), and strepto- 37°C and fixed in 1.5% glutaraldehyde in 0.1 M cacodylate mycin (50 mgyml). For induction of cell death, cells were buffer. The cells were dehydrated in an ascending series of suspended in the culture medium at a density of 5 3 105 cells alcohol followed by drying in a Ladd critical point dryer. The per ml and were incubated with anti-Porimin in an atmosphere samples were coated with palladium coating system and ex- of 5% CO2 at 37°C. For some experiments, the induction of cell amined by a JEOL JSM-35CF scanning electron microscope. death by anti-Porimin was performed in serum-free culture For transmission electron microscopy, cells were fixed by medium PFHM-II (GIBCOyBRL) or in PBS alone. 1.25% glutaraldehyde in cacodylate buffer containing 1% Cell Staining and Flow Cytometric Analysis. To test the CaCl2 and postfixed in 1% OsO4. After dehydration and reactivity of anti-Porimin with Jurkat cells and other human embedding, the samples were sectioned, stained, and examined cell types, cells were labeled for flow cytometry by indirect with a transmission electron microscope. immunofluorescent staining and analyzed by using an Epics Elite flow cytometer (Coulter). RESULTS Quantification of Cell Death in Suspension Culture. The dead cells in suspension culture after exposure to cytotoxic Anti-Porimin Rapidly Mediates Membrane Blebbing, Pore- mAb were quantified by flow cytometry and by cell prolifer- Formation, and Increased Permeability of the Plasma Mem- ation inhibition assays. For flow cytometry, cells (1 3 106 cells brane Followed by Cell Death. In an effort to characterize cell per sample) were washed once with PBS after induction of cell surface molecules involved in the cell death of human lym- death by mAb and suspended in 1 ml of cold PBS containing phoid cells, a panel of mAbs has been raised in the present propidium iodide at a concentration of 5 mgyml. Cell death was study by immunizing mice with apoptotic Jurkat cells. One of assessed by determining the intracellular propidium iodide these antibodies, designated anti-Porimin, was found to react (red) fluorescence with a flow cytometer as described previ- with Jurkat cells and was shown to be cytotoxic for these cells. ously (14, 20). When Jurkat cells were incubated with anti-Porimin, the cells For cell proliferation inhibition assay, Jurkat cells were aggregated in suspension culture within minutes and under- cultured in 96-well microtiter plates and incubated in the went morphologic changes with damages to the plasma mem- absence or presence of anti-Porimin antibody. After pulsing brane, which was then followed by cell death. Anti-Porimin with 1 mCi (1 mCi 5 37 kBq) of [3H]thymidine (NEN) per well mediated cell death was complement-independent and was for 4 h, cells were harvested and [3H]thymidine incorporation observed after incubation at either 37°C or room temperature.