CDC25A phosphatase promotes cell cycle progression by activating G 1 cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts.In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening.ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH 2 -terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses.Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm.The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A.This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1.Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli.Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression.These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses.
Phosphatidylserine (PS)-targeting monoclonal Abs (mAbs) that directly target PS and target PS via β2-gp1 (β2GP1) have been in preclinical and clinical development for over 10 y for the treatment of infectious diseases and cancer. Although the intended targets of PS-binding mAbs have traditionally included pathogens as well as stressed tumor cells and its associated vasculature in oncology, the effects of PS-targeting mAbs on activated immune cells, notably T cells, which externalize PS upon Ag stimulation, is not well understood. Using human T cells from healthy donor PBMCs activated with an anti-CD3 + anti-CD28 Ab mixture (anti-CD3/CD28) as a model for TCR-mediated PS externalization and T cell stimulation, we investigated effects of two different PS-targeting mAbs, 11.31 and bavituximab (Bavi), on TCR activation and TCR-mediated cytokine production in an ex vivo paradigm. Although 11.31 and Bavi bind selectivity to anti-CD3/28 activated T cells in a PS-dependent manner, surprisingly, they display distinct functional activities in their effect on IFN-γ and TNF-ɑ production, whereby 11.31, but not Bavi, suppressed cytokine production. This inhibitory effect on anti-CD3/28 activated T cells was observed on both CD4+ and CD8+ cells and independently of monocytes, suggesting the effects of 11.31 were directly mediated by binding to externalized PS on activated T cells. Imaging showed 11.31 and Bavi bind at distinct focal depots on the cell membrane. Collectively, our findings indicate that PS-targeting mAb 11.31 suppresses cytokine production by anti-CD3/28 activated T cells.
The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity. The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of "self" versus "other" considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity. The process of physiological cell death assures the elimination of functionally inappropriate cells in a manner that does not elicit inflammation (1.Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-256Crossref PubMed Scopus (12819) Google Scholar, 2.Savill J. Dransfield I. Gregory C. Haslett C. Nat. Rev. Immunol. 2002; 2: 965-975Crossref PubMed Scopus (1325) Google Scholar). Professional phagocytes, including resident macrophages and dendritic cells, participate in the efficient clearance of apoptotic corpses in vivo (3.Savill J.S. Wyllie A.H. 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Studies of neutrophil death and resulting phagocytosis by macrophages provided the first experimental evidence that a variety of cytokines and chemokines associated with inflammation, including interleukin (IL) 4The abbreviations used are: IL, interleukin; EGF, epidermal growth factor; PI, propidium iodide; PS, phosphatidylserine; LPS, lipopolysaccharide; RLU, relative light units; CFDA, 5,6-carboxyfluorescein diacetate succinimidyl ester; PBS, phosphate-buffered saline; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethyl rhodamine; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; NF-κB, nuclear factor κB; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; TGRβ, transforming growth factor β; TLR, toll-like receptor.4The abbreviations used are: IL, interleukin; EGF, epidermal growth factor; PI, propidium iodide; PS, phosphatidylserine; LPS, lipopolysaccharide; RLU, relative light units; CFDA, 5,6-carboxyfluorescein diacetate succinimidyl ester; PBS, phosphate-buffered saline; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethyl rhodamine; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; NF-κB, nuclear factor κB; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; TGRβ, transforming growth factor β; TLR, toll-like receptor.-6 and IL-8, are not secreted from phagocytes that engulf apoptotic targets (3.Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1347) Google Scholar, 7.Meagher L.C. Savill J.S. Baker A. Fuller R.W. Haslett C. J. Leukocyte Biol. 1992; 52: 269-272Crossref PubMed Scopus (247) Google Scholar, 8.Hughes J. Liu Y. Damme J.V. Savill J. J. Immunol. 1997; 158: 4389-4397PubMed Google Scholar, 9.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2552) Google Scholar, 10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). More significantly, the lack of inflammatory cytokine release reflects an affirmative inhibitory response. For example, whereas stimulation of macrophages via the Toll-like receptor (TLR) 4 signaling complex (11.Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar) upon engagement with bacterial lipopolysaccharide (LPS) triggers significant cytokine secretion, the additional ingestion of apoptotic cells attenuates this response potently (9.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2552) Google Scholar, 10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 12.Voll R.E. Herrmann M. Roth E.A. Stach C. Kalden J.R. Girkontaite I. Nature. 1997; 390: 350-351Crossref PubMed Scopus (1505) Google Scholar). The ability of apoptotic cells to be cleared in a noninflammatory manner by professional phagocytes, such as macrophages, is a consequence of their specific expression of determinants for recognition and modulation of proinflammatory responses. The acquisition of these apoptotic determinants represents a gain of function and is common to all physiological cell deaths, without regard to suicidal stimulus (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Cells that die necrotically also are recognized by professional phagocytes; in contrast, however, necrotic corpses do not down-regulate inflammatory responses. Discrimination between apoptotic and necrotic corpses occurs on the level of binding and not engulfment and involves distinct and noncompeting mechanisms of recognition (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). The modulatory activity of the apoptotic corpse is manifest as an immediate-early inhibition of macrophage proinflammatory cytokine gene transcription and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Apoptotic cells target the proinflammatory transcriptional machinery of macrophages, with which they interact through a novel regulatory pathway. Inhibition appears to involve sequestration of critical transcriptional co-activator molecules without effect on proximal signaling events induced by inflammatory receptors, including innate immune receptors of the TLR family (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The ubiquity of apoptotic cells in all tissues throughout organismal life prompted us to ask whether this innate discrimination of apoptotic cells is limited to professional phagocytes. Certainly, normal homeostatic cell turnover in vivo, and especially in solid tissues and intact cellular strata, results in apoptotic cells that are in immediate contact with their neighbors independent of (and before the arrival of) mobile phagocytes (6.Wood W. Turmaine M. Weber R. Camp V. Maki R.A. McKercher S.R. Martin P. Development. 2000; 127: 5245-5252Crossref PubMed Google Scholar, 14.Saunders Jr., J.W. Science. 1966; 154: 604-612Crossref PubMed Scopus (720) Google Scholar, 15.Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6720) Google Scholar, 16.Parnaik R. Raff M.C. Scholes J. Curr. Biol. 2000; 10: 857-860Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17.Monks J. Rosner D. Geske F.J. Lehman L. Hanson L. Neville M.C. Fadok V.A. Cell Death Differ. 2005; 12: 107-114Crossref PubMed Scopus (178) Google Scholar). We asked whether nonprofessional phagocytes discriminate and respond specifically to apoptotic cells in an anti-inflammatory manner. Here we describe studies that reveal that they do. Remarkably, this profound innate immune function is manifest fully and ubiquitously among professional and nonprofessional phagocytes and even nonphagocytic cell types. Cells and Death Induction—RAW 264.7 murine macrophages, DO11.10 murine T hybridoma cells, Jurkat human acute T leukemia cells, Ramos (RA-1) human Burkitt's B lymphoma cells, and PLB-985 human myelomonoblastic leukemia cells (generously provided by Dr. Peter Henson, National Jewish Medical and Research Center) were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with heat-inactivated fetal bovine serum (10% v/v), 2 mm l-glutamine, and 50 μm 2-mercaptoethanol. HeLa human cervical carcinoma cells and 293T human transformed kidney epithelial cells were grown in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose (Mediatech) supplemented with fetal bovine serum (10% (v/v); HyClone Laboratories, Logan, UT) and 2 mm l-glutamine. Chinese hamster ovary cells were grown in α-minimal essential medium (Invitrogen) supplemented only with fetal bovine serum (10%, v/v). Human umbilical vein endothelial cells were grown in supplemented endothelial growth medium (Cambrex Bio Science, East Rutherford, NJ) on gelatin-coated plates. Immortalized murine 3T3 fibroblast cell lines were derived from mouse embryo fibroblasts following the 3T3 protocol of Todaro and Green (18.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2003) Google Scholar). Briefly, the embryo fibroblasts were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose (Mediatech) supplemented with fetal bovine serum (10%, v/v; HyClone Laboratories), 2 mml-glutamine, and 50 μm 2-mercaptoethanol, replating at 3 × 105/60-mm diameter dish every 3 days. Immortalized cell lines were established from cells that grew from cultures that had become senescent. Physiological cell death (apoptosis) was induced by treatment of target cells with the macromolecular synthesis inhibitors actinomycin D (200 ng/ml, 12 h) or cycloheximide (1 μg/ml, 12 h) (19.Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar). Cells were killed pathologically (necrotic death) by incubation in phosphate-buffered saline (PBS) at 55 °C for 20 min (until trypan blue uptake indicated compromise of membrane integrity) (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). In all cases, target cells (viable, apoptotic, and necrotic) were washed twice and resuspended in the medium of the responder cells to be tested. In some experiments, apoptotic and viable target cells were fixed by incubation with formaldehyde (125 mm in PBS, 25 °C, 20 min; Polysciences, Inc., Warrington, PA) or were cycled through three rounds of freezing and thawing. Target preparations then were washed twice and resuspended in the medium of the responder cells to be tested. Plasma membrane vesicles were prepared from HeLa cells following the approach of Baumann et al. (20.Baumann N.A. Vidugiriene J. Machamer C.E. Menon A.K. J. Biol. Chem. 2000; 275: 7378-7389Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Monolayers of cells, either untreated or induced to die with actinomycin D (and still adherent), were stimulated to vesiculate by incubation at 37 °C in Vesiculation Buffer (10 mm Hepes (pH 7.4), 150 mm NaCl, 2 mm CaCl2, 2 mm dithiothreitol, and 25 mm formaldehyde). Supernatants were collected after ∼2.5 h (when abundant small membrane vesicles were apparent in the culture fluid). Nonadherent cells were removed (1,000 × g for 10 min), and vesicles were pelleted from the cleared supernatant by centrifugation (30,000 × g, 60 min, 4 °C). Cytofluorimetric analysis indicated that vesicles were ∼0.8 μm in diameter and free of contaminating intact cells. Phagocytosis Assay and Other Cytofluorimetric Analyses—Phagocytosis was assessed as previously described for macrophages (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Target cells were labeled green with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA; 0.2 μm; Molecular Probes, Inc., Eugene, OR) and were then induced to undergo apoptotic cell death, killed pathologically by heat treatment, or left untreated. Phagocytes (or cells being tested for phagocytic activity) were labeled red with 5-(and 6)-(((4-chloromethyl)benzoyl)-amino) tetramethyl rhodamine (CMTMR; 10 μm; Molecular Probes). In all cases, cells were labeled on the day preceding the experiment and cultured in serum-containing medium overnight to eliminate unbound label. Labeled phagocytes were co-cultured with the apoptotic, necrotic, or viable target cells for 30 min at 37 °C. Cells were harvested with PBS supplemented with 0.4 mm Na2EDTA and analyzed cytofluorimetrically on a FACSCaliber instrument (BD Biosciences). Cytofluorimetric data were processed with WinMDI software (Joe Trotter, Scripps Research Institute, La Jolla, CA). Cells that were both CMTMR-positive (Exλ = 488 nm, Emλ = 610 ± 15 nm) and CFDA-positive (Exλ = 488 nm; Emλ = 530 ± 15 nm) and that had scatter properties of the phagocyte population represented phagocytes that had engulfed targets. Engulfment is calculated as the fraction of double-positive phagocytes (all CMTMR-positive cells that also are CFDA-positive). Most targets that are bound but not engulfed are disrupted and do not remain adherent during the analysis, although they could be enumerated under static microscopic examination (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). The accessibility of phosphatidylserine was revealed by the binding of fluorescein isothiocyanate-conjugated annexin V (Pharmingen; San Diego, CA; Exλ = 488 nm, Emλ = 525 nm). Cells were harvested and washed twice with cold PBS. Cells were resuspended in 100 μl of binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) and incubated with 5 μl of fluorescein isothiocyanate-conjugated annexin V for 15 min in the dark at 25 °C. After incubation, 400 μl of binding buffer was added per sample, and cells were analyzed cytofluorimetrically. Propidium iodide (PI) was employed to assess plasma membrane integrity. PI was added to cells at 1 μg/ml immediately before cytofluorimetric analysis (Exλ = 488 nm, Emλ = 610 nm). Transfections and Luciferase Assays—Apoptotic modulation of specific transcription (e.g. dependent on nuclear factor κB (NF-κB) or the IL-8 promoter) was assessed in various cell types following transfection of relevant transcriptional reporter constructs, using a dual luciferase strategy, as described previously (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The efficiencies of transfection were measured in parallel with farnesylated green fluorescent protein as a transfection marker (21.Harvey K.J. Lukovic D. Ucker D.S. Cytometry. 2001; 43: 273-278Crossref PubMed Scopus (27) Google Scholar). Cells were co-transfected with pNF-κB-Luc, a plasmid containing the firefly (Photinus pyralis) luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif (Clontech), together with pRL-SV40, a Renilla (sea pansy; R. reniformis) luciferase control vector, the constant expression of which is dependent on the SV40 early enhancer/promoter region (Promega, Madison, WI). RAW 264.7 macrophages (5.0 × 106 cells/60-mm diameter dish) and HeLa, 293T, and Chinese hamster ovary cells, at ∼75% confluence, were transfected using Effectene Transfection Reagent (Qiagen, Valencia, CA). 3T3 cells were transfected using the MEF1 Nucleofector Kit (AMAXA Biosystems; Gaithersburg, MD), with MEF Nucleofector Solution 1 and a machine setting of A-23. Jurkat, Ramos RA-1, and human umbilical vein endothelial cells were transfected using GenePORTER2 Transfection Reagent (Gene Therapy Systems, San Diego, CA). The next day, the cells were replated in 24-well plates (1.0 × 105 cells/well) and incubated without or with the indicated target cells (at a target cell/macrophage ratio of 10:1) and/or a proinflammatory stimulus in a final volume of 2 ml. The proinflammatory stimuli used included LPS (100 ng/ml; Escherichia coli O111:B4; Sigma), tumor necrosis factor-α (TNFα; 10 ng/ml; R&D Systems; Minneapolis, MN), IL-1β (5 ng/ml; R&D Systems), and phorbol 12-myristate 13-acetate (PMA; 1.3 ng/ml; EMD Biosciences, San Diego, CA) alone or with ionomycin (200 ng/ml; Molecular Probes). Cell extracts were prepared after further incubation as indicated, and luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega) in an FB12 luminometer (Zylux; Oak Ridge, TN). Each condition was repeated in triplicate wells, and the luciferase activities in cells from each well were determined independently. Within any experiment, Renilla luciferase activities among samples varied less than 6%. The firefly luciferase activity in each sample was normalized with respect to the internal Renilla luciferase activity, and the relative level of normalized firefly luciferase activity compared with the activity in an untreated population was taken as a measure of specific (e.g. NF-κB-dependent) transcriptional activity. Stably transfected reporter cells were generated by transfection of 293T cells with another NF-κB-Luc reporter construct, 4×NF-κB(HIV)tkluc (22.Wissink S. van de Stolpe A. Caldenhoven E. Koenderman L. van der Saag P.T. Immunobiology. 1997; 198: 50-64Crossref PubMed Scopus (40) Google Scholar), and an unlinked vector conferring hygromycin resistance. Cells resistant to hygromycin (50 μg/ml) were selected, cloned at limiting dilution, and tested for NF-κB-dependent responsiveness. Extracts were prepared and analyzed as above, except that the luciferase assay system (Promega) was used. Data with one clone, B2, are described here. Quantification of Cytokine Release—Cytokine production was assessed following incubation of responder cells with target cells. Where indicated, proinflammatory stimuli were added simultaneously with the addition of targets. Culture supernatants were withdrawn from wells at the indicated times and frozen at –20 °C until analysis. Secreted cytokines were quantified by ELISA, using matched pair cytokine-specific capture and biotinylated reporter antibodies for murine IL-6 (eBiosciences; San Diego, CA) or human IL-8 (BIOSOURCE, Camarillo, CA). The reporter reactions were developed with horseradish peroxidase-conjugated streptavidin (R&D Systems) and measured spectrophotometrically at 450 nm (corrected for turbidity at 550 nm; Microplate Autoreader model EL311; Bio-Tek Instruments, Winooski, VT). Cellular Extract Preparation and Immunoblot Analysis—Activation of Akt and inhibition of ERK1/2 were assessed in 3T3 cells cultured overnight in serum-free medium and left unstimulated or stimulated for 15 min with a 5-fold excess of apoptotic DO11.10 cells (the apoptotic cells, which had been cultured under serum-free conditions, were centrifuged briefly onto the adherent 3T3 cells to initiate the interaction) and/or subsequent stimulation with epidermal growth factor (EGF; 10 nm; Calbiochem). After washing, cell extracts were prepared from the adherent 3T3 cells. Cells were lysed in lysis buffer (150 mm NaCl, 50 mm HEPES (pH 7.5), 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 200 μm orthovanadate). Lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatants were stored at –70 °C. Protein samples (20 μg each, determined by the bicinchoninic acid protein assay; Pierce) were boiled in 5× sample buffer, run on 12% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA). Blots were blocked with 5% dry milk in 150 mm PBS, 20 mm Tris HCl, pH 7.5, before probing with a phospho-Akt(Thr308)-specific rabbit anti-serum (Cell Signaling, Beverly, MA) or an affinity-purified phospho-ERK1/2 (Thr183/Tyr185)-specific polyclonal rabbit IgG (Promega, Madison, WI). Following incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase, immunoreactive bands were visualized by the luminol reaction (ECLplus; Amersham Biosciences). Equivalent loading of protein samples was monitored by Ponceau S staining (0.25% (w/v) (Sigma) in 0.1% acetic acid; 5 min) of blotted proteins. Specific Recognition and Response to Apoptotic Cells Is Not Limited to Macrophages—The anti-inflammatory response triggered in macrophages by their specific recognition of apoptotic cells is exerted on the level of cytokine gene transcription (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Although key transcriptional activators of cytokine gene expression, such as NF-κB (23.Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (733) Google Scholar, 24.Collart M.A. Baeuerle P. Vassalli P. Mol. Cell. Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar), are not the molecular targets of apoptotic modulation, modulation is evident on the level of NF-κB-dependent transcription, and an NF-κB-dependent transcriptional reporter serves as a sensitive, reliable, and convenient readout for the modulatory effect exerted by apoptotic targets (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The experiment in Fig. 1A exemplifies this analysis. We transiently transfected RAW 264.7 macrophages with pNF-κB-Luc, a plasmid containing the firefly luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif. Macrophages were co-transfected with a constitutive (NF-κB-independent) Renilla luciferase control vector, which served as an internal normalization control for transfection efficiency and cell viability. Following transfection, macrophages were incubated with different target cell populations and/or bacterial LPS, a potent proinflammatory agonist. Firefly and Renilla luciferase activities then were measured. As indicated by these luciferase reporters, LPS-activated NF-κB-dependent transcription (but not global transcription) in macrophages is inhibited specifically following their interaction with apoptotic cells; necrotic and viable cells do not exert this effect (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). This response is elicited by apoptotic cells generally, regardless of species, cell type, or suicidal stimulus. Here, murine DO11.10 T cells and human HeLa epithelial carcinoma cells, triggered to die with different suicidal stimuli (inhibitors of translation and transcription, respectively), were equally effective at triggering modulation in these murine macrophages. As a first test of the ability of nonprofessional phagocytes to recognize and respond to apoptotic cells, we examined the responsiveness of HeLa cells, the same cells used as targets in Fig. 1A, utilizing the identical transcriptional reporter strategy. The transfected HeLa cells were incubated with apoptotic, necrotic, or viable populations of target cells and/or the inflammatory cytokine TNFα as a stimulus of an NF-κB-dependent transcriptional response. Significantly, HeLa cells do not die in response to TNFα unless the NF-κB-dependent response is attenuated (e.g. by an inhibitor of macromolecular synthesis) (25.Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2933) Google Scholar, 26.Wang C.-Y. Mayo M.W. Baldwin A.S.J. Science. 1996; 274: 784-787Crossref PubMed Scopus (2509) Google Scholar, 27.Harvey K.J. Lukovic D. Ucker D.S. J. Cell Biol. 2000; 148: 59-72Crossref PubMed Scopus (83) Google Scholar). Just as with LPS-stimulated macrophage responsiveness, robust TNFα-activated NF-κB-dependent transcription in HeLa cells was inhibited specifically and profoundly following the interaction of those cells with apoptotic, but not necrotic or viable, targets (Fig. 1B). It is notable that the ranges of absolute values of NF-κB-dependent luciferase activities were quite different in HeLa cells and macrophages at comparably early times (as much as 300-fold; see Fig. 1), reflecting differences in the efficiencies of transfection and transgene expression. Still, expressed relative to basal luciferase levels, these data present a consistent pattern of modulation and reveal an identical response to apoptotic targets in different responder cell populations. Again, this selective response to apoptotic cells occurred without species restriction. Most dramatically, HeLa cells even were able to recognize and respond specifically to homotypic apoptotic cells. The Characteristic Repertoire of Anti-inflammatory Responses Elicited Specifically upon Apoptotic Cell Recognition Is Evident in Murine Fibroblasts—To begin a more comprehensive exploration of the recognition and response to apoptotic targets by nonprofessional phagocytes, we examined nontransformed murine fibroblasts. Immortalized murine embryo fibroblast cells, derived by the 3T3 protocol of Todaro and Green (18.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2003) Google Scholar), were highly phagocytic for dead cells (Fig. 2A; for consistency, we used DO11.10 cells as targets in this and the following experiments). Apoptotic and necrotic cell targets were engulfed rapidly and to equal extents by 3T3 fibroblasts, whereas viable cells were not ingested (Fig. 2A; we take the low level of engulfment of "viable" cells to reflect the small fraction of dead and dying apoptotic cells present in any cell culture). These 3T3 fibroblasts secrete the inflammatory cytokine IL-6 in response to a variety of proinflammatory stimuli, including IL-1β, TNFα, and, to a lesser degree, bacterial LPS (Fig. 2B and data not shown) (28.Kurt-Jones E.A. Sandor F. Ortiz Y. Bowen G.N. Counter S.L. Wang T.C. Finberg R.W. J. Endotoxin Res. 2004; 10: 419-424Crossref PubMed Google Scholar). We characterized the release of IL-6 from fibroblasts following their interaction with target cells as one indication of inflammatory responsiveness; IL-6 secretion in macrophages reflects inflammatory responsiveness generally (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). IL-1β-stimulated IL-6 secretion was potently attenuated when fibroblasts interacted with apoptotic targets, but not with necrotic or viable targets (Fig. 2B). Apoptotic target cell contact was necessary for this response, since supernatants from apoptotic cell cultures could not substitute for the target cells themselves to modulate IL-6 secretion (data not shown). The ability of apoptotic cells to block IL-6 secretion by IL-1β-stimulated fibroblasts parallels their ability to abrogate the secretion of IL-6 and other inflammatory cytokines by LPS-stimulated macrophages (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163
Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.
Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: 1) attraction of phagocytes via soluble "find me" signals, 2) recognition and phagocytosis via cell surface-presenting "eat me" signals, and 3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of proinflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the current study, using wild-type and adenosine A(2A) receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, as a result of possible activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase/protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether, our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.
The role of the target cell in its own death mediated by cytotoxic T lymphocytes (CTL) has been controversial. The ability of the pore-forming granule components of CTL to induce target cell death directly has been taken to suggest an essentially passive role for the target. This view of CTL-mediated killing ascribes to the target the single role of providing an antigenic stimulus to the CTL; this signal results in the vectoral degranulation and secretion of pore-forming elements onto the target. On the other hand, by a number of criteria, target cell death triggered by CTL appears fundamentally different from death resulting from membrane damage and osmotic lysis. CTL-triggered target cell death involves primary internal lesions of the target cell that reflect a physiological cell death process. Orderly nuclear disintegration, including lamin phosphorylation and solubilization, chromatin condensation, and genome digestion, are among the earliest events, preceding the loss of plasma membrane integrity. We have tested directly the involvement of the target cell in its own death by examining whether we could isolate mutants of target cells that have retained the ability to be recognized by and provide an antigenic stimulus to CTL while having lost the capacity to respond by dying. Here, we describe one such mutant, BW87. We have used this CTL-resistant mutant to analyze the mechanisms of CTL-triggered target cell death under a variety of conditions. The identification of a mutable target cell element essential for the cell death response to CTL provides genetic evidence that target cell death reflects an active cell suicide process similar to other physiological cell deaths.
The intriguing cell biology of apoptotic cell death results in the externalization of numerous autoantigens on the apoptotic cell surface, including protein determinants for specific recognition, linked to immune responses. Apoptotic cells are recognized by phagocytes and trigger an active immunosuppressive response ("innate apoptotic immunity" (IAI)) even in the absence of engulfment. IAI is responsible for the lack of inflammation associated normally with the clearance of apoptotic cells; its failure also has been linked to inflammatory and autoimmune pathology, including systemic lupus erythematosus and rheumatic diseases. Apoptotic recognition determinants underlying IAI have yet to be identified definitively; we argue that these molecules are surface-exposed (during apoptotic cell death), ubiquitously expressed, protease-sensitive, evolutionarily conserved, and resident normally in viable cells (SUPER). Using independent and unbiased quantitative proteomic approaches to characterize apoptotic cell surface proteins and identify candidate SUPER determinants, we made the surprising discovery that components of the glycolytic pathway are enriched on the apoptotic cell surface. Our data demonstrate that glycolytic enzyme externalization is a common and early aspect of cell death in different cell types triggered to die with distinct suicidal stimuli. Exposed glycolytic enzyme molecules meet the criteria for IAI-associated SUPER determinants. In addition, our characterization of the apoptosis-specific externalization of glycolytic enzyme molecules may provide insight into the significance of previously reported cases of plasminogen binding to α-enolase on mammalian cells, as well as mechanisms by which commensal bacteria and pathogens maintain immune privilege.
The failure of Thy-1 and Ly-6 to trigger interleukin-2 production in the absence of surface T-cell antigen receptor complex (TCR) expression has been interpreted to suggest that functional signalling via these phosphatidylinositol-linked alternative activation molecules is dependent on the TCR. We find, in contrast, that stimulation of T cells via Thy-1 or Ly-6 in the absence of TCR expression does trigger a biological response, the cell suicide process of activation-driven cell death. Activation-driven cell death is a process of physiological cell death that likely represents the mechanism of negative selection of T cells. The absence of the TCR further reveals that signalling leading to activation-driven cell death and to lymphokine production are distinct and dissociable. In turn, the ability of alternative activation molecules to function in the absence of the TCR raises another issue: why immature T cells, thymomas, and hybrids fail to undergo activation-driven cell death in response to stimulation via Thy-1 and Ly-6. One possibility is that these activation molecules on immature T cells are defective. Alternatively, susceptibility to activation-driven cell death may be developmentally regulated by TCR-independent factors. We have explored these possibilities with somatic cell hybrids between mature and immature T cells, in which Thy-1 and Ly-6 are contributed exclusively by the immature partner. The hybrid cells exhibit sensitivity to activation-driven cell death triggered via Thy-1 and Ly-6. Thus, the Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 molecules of the immature T cells can function in a permissive environment. Moreover, with regard to susceptibility to Thy-1 and Ly-6 triggering, the mature phenotype of sensitivity to cell death is genetically dominant.
