The Presumptive Phosphatidylserine Receptor Is Dispensable for Innate Anti-inflammatory Recognition and Clearance of Apoptotic Cells
2005; Elsevier BV; Volume: 281; Issue: 9 Linguagem: Inglês
10.1074/jbc.m509775200
ISSN1083-351X
AutoresJustin E. Mitchell, Marija Cvetanović, Nitu Tibrewal, Vimal Patel, Oscar R. Colamonici, Ming O. Li, Richard A. Flavell, Jerrold S. Levine, Raymond B. Birge, David S. Ucker,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR+/+, PSR+/-, and PSR-/- fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells. The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR+/+, PSR+/-, and PSR-/- fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells. Physiological cell death is a process whose purpose is the elimination of functionally inappropriate cells in a manner that does not elicit inflammation. The ability of apoptotic corpses to be cleared in a noninflammatory manner by phagocytes is a consequence of their specific expression of determinants for recognition and modulation of pro-inflammatory responses. The acquisition of these apoptotic determinants is a gain-of-function common to all physiological cell deaths, without regard to suicidal stimulus, and conserved widely across species (1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar, 2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar).Numerous cellular alterations associated with apoptotic cell death have been described, including plasma membrane reorganization associated with blebbing (3Hogue M.J. J. Exp. Med. 1919; 30: 617-647Crossref PubMed Scopus (24) Google Scholar), shrinkage, and the loss of membrane phospholipid asymmetry (4Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-256Crossref PubMed Scopus (12723) Google Scholar, 5Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar). In particular, phosphatidylserine (PS), 4The abbreviations used are: PS, phosphatidylserine; CFDA, 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester; CMTMR, 5 (and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; GFP, green fluorescent protein; GST, glutathione S-transferase; MFG-E8, milk fat globule factor-E8; PSR, presumptive PS-specific receptor; RT, reverse transcriptase; IL-1β, interleukin 1β; IL-6, interleukin 6; TGFβ, transforming growth factor-β; TNFα, tumor necrosis factor-α; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; mAb, monoclonal antibody; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; CRIB, Cdc42/Rac interactive binding. 4The abbreviations used are: PS, phosphatidylserine; CFDA, 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester; CMTMR, 5 (and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; GFP, green fluorescent protein; GST, glutathione S-transferase; MFG-E8, milk fat globule factor-E8; PSR, presumptive PS-specific receptor; RT, reverse transcriptase; IL-1β, interleukin 1β; IL-6, interleukin 6; TGFβ, transforming growth factor-β; TNFα, tumor necrosis factor-α; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; mAb, monoclonal antibody; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; CRIB, Cdc42/Rac interactive binding. an anionic phospholipid normally cloistered in the inner leaflet of the plasma membrane, is externalized during physiological cell death (5Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar). It still remains to be determined what specific molecular events are responsible for the recognition of the effete cell.The view that externalized PS serves as a ligand for macrophage recognition of apoptotic cells followed from studies demonstrating that similar changes target aged erythrocytes for clearance (6Schroit A.J. Madsen J.W. Tanaka Y. J. Biol. Chem. 1985; 260: 5131-5138Abstract Full Text PDF PubMed Google Scholar, 7McEvoy L. Williamson P. Schlegel R.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3311-3315Crossref PubMed Scopus (191) Google Scholar) and gained support from observations that phospho-l-serine and PS vesicles could inhibit partially the interaction of dying nucleated cells with macrophages (5Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar, 8Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (126) Google Scholar, 9Fadok V.A. Warner M.L. Bratton D.L. Henson P.M. J. Immunol. 1998; 161: 6250-6257PubMed Google Scholar).A presumptive cell surface PS-specific receptor (PSR) was identified molecularly following a screen for monoclonal antibodies whose binding to human macrophages was inhibited by PS-containing liposomes (10Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekowitz R.A.B. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1247) Google Scholar). The product of that screen, mAb 217, bound to cell surface determinants on macrophages and other cell types, notably excluding lymphoid cells. Significantly, mAb 217 triggered macrophages to release the anti-inflammatory cytokine TGFβ, further suggesting that mAb 217 engaged an apoptotic-like recognition mechanism (10Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekowitz R.A.B. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1247) Google Scholar).Controversy regarding this presumptive receptor arose, however, when PSR was observed to localize to the nucleus in mammalian cells (11Cui P. Qin B. Liu N. Pan G. Pei D. Exp. Cell Res. 2004; 293: 154-163Crossref PubMed Scopus (95) Google Scholar) as well as in Hydra (12Cikala M. Alexandrova O. David C.N. Pröschel M. Stiening B. Cramer P. Böttger A. BMC Cell Biol. 2004; 5: 26Crossref PubMed Scopus (81) Google Scholar). The role of PSR has been further clouded by the disparate results of three groups of investigators who independently generated mice with targeted disruptions of the PSR locus (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar, 14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar, 15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar). While homozygous PSR disruptions result in perinatal lethality in each case, different effects on the phagocytosis of apoptotic cells have been reported for the three PSR deficiencies.Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) described a disruption beginning upstream of exon 1 (which includes the translational start site of the PSR gene product) and extending through exon 2, in a mixed 129 × C57BL/6 background, which leads to severe lung defects as well as less penetrant brain aberrations. Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar) generated a disruption, also starting upstream of exon 1 and extending into exon 3, in a chimeric 129 × C57BL/6 background, which results in defective erythroid differentiation and severe anemia. The disruption generated by Böse et al. (15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar) in a pure C57BL/6 background is limited to exons 1 and 2 and leads to growth retardation, defects in embryonic lung, kidney, gut, and erythroid development, and, at low frequency, aberrant eye and brain development. Hemizygous PSR deficiency has no phenotype in any of these cases.An increased number of apoptotic cells (with digested genomic DNA, identified by TUNEL (terminal deoxyribonucleotidyltransferase end labeling) staining) was observed by Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) in the affected tissues of their PSR-deficient mice and attributed to the diminished engulfment of dead cells. Interestingly, Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar) also suggested that phagocytosis of apoptotic cells by macrophages was diminished during embryogenesis, based on immunohistochemical of staining fetal liver and thymus sections their PSR-/- of mice. Their data are confounded, however, by an apparent reduction in the number of tissue-resident macrophages (stained with F4/80 antibody; Ref. 16Austyn J.M. Gordon S. Eur. J. Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1278) Google Scholar), hinting at another possible developmental impairment related to PSR deficiency. Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) further explored engulfment in vitro with PSR-deficient macrophages generated by adoptive transfer of PSR-/- fetal liver into wild-type hosts. They reported a 50% overall reduction and a complete absence of PS-inhibitable engulfment by PSR-deficient elicited macrophages. In light of the observations of Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar), it is important to note that the differentiation and activation states of these PSR-/- and PSR+/+ macrophages were not compared; previous work has suggested that PS-inhibitable engulfment pertains particularly to activated macrophages (8Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (126) Google Scholar, 9Fadok V.A. Warner M.L. Bratton D.L. Henson P.M. J. Immunol. 1998; 161: 6250-6257PubMed Google Scholar). In marked contrast to the results of Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) and Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar), extensive histological analysis of numerous tissues by Böse et al. (15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar) revealed no defects in the phagocytosis of apoptotic (TUNEL+) cells. Their in vitro phagocytosis studies with fetal liver-derived macrophages (differentiated in culture) also showed no engulfment defect of PSR-deficient macrophages. Of great significance, Böse et al. (15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar) reported that the ability of apoptotic cells to trigger an antiinflammatory response in engulfing macrophages (both the inhibition of secretion of pro-inflammatory cytokines, such as TNFα, and the induction of anti-inflammatory cytokines, such as TGFβ and IL-10) was unimpaired in the absence of PSR expression.Our studies of the process of apoptotic cell clearance have demonstrated that the ability of apoptotic corpses to be engulfed specifically and in a non-inflammatory manner by macrophages and other phagocytes is a consequence of a process of specific recognition and modulation of pro-inflammatory phagocyte responses (1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar). The modulatory effect of the apoptotic corpse is manifest as an immediate-early inhibition of pro-inflammatory cytokine gene transcription within the responding phagocyte with which it interacts, and is exerted upon binding, independent of subsequent engulfment or soluble factor involvement (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Cells that die pathologically (that is, necrotic corpses) also are recognized by phagocytes but do not down-regulate inflammatory responses. The recognition of these two classes of native dying cells occurs via distinct and non-competing mechanisms (1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar).Importantly, non-professional phagocytes are fully capable of noninflammatory recognition and clearance of apoptotic cells. 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. Indeed, we have found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including non-phagocytic lymphocytes, and reflects an innate immunity that discriminates live from effete cells without regard to self (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. In particular, the ability of fibroblastic cells to respond to apoptotic corpses permits mouse embryo fibroblasts established from animals with targeted disruptions of genes of interest (including essential genes) to be used in the evaluation of genetic contributions to apoptotic recognition and response without the generation or selection of particular cell populations.We have applied this analysis to embryo fibroblasts that harbor a disruption of the PSR locus. Our data, which demonstrate that PSR-deficient cells are fully competent to recognize apoptotic cells, definitively exclude a general role for PSR in apoptotic recognition and inflammatory modulation.EXPERIMENTAL PROCEDURESCells and Death Induction—Immortalized murine fibroblast cell lines were derived from mouse embryo fibroblasts (MEFs) following the 3T3 protocol of Todaro and Green (17Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1993) Google Scholar). Briefly, MEFs 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, Herndon, VA) supplemented with fetal bovine serum (10% v/v; HyClone Laboratories, Logan, UT), 2mml-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.NIH 3T3 cells were maintained similarly. 293T human transformed kidney epithelial cells were grown in the same medium without 2-mercaptoethanol, and DO11.10 murine T hybridoma and CEM human T lymphoblastoid cells were grown in RPMI 1640 medium (Mediatech) supplemented with heat inactivated fetal bovine serum (10% v/v), 2 mml-glutamine, and 50 μm 2-mercaptoethanol.Physiological cell death (apoptosis) was induced by treatment of target cells with the macromolecular synthesis inhibitor actinomycin D (200 ng/ml, 12 h; Ref. 18Chang 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) or with UV-B irradiation (20 mJ/cm2). Cells were killed pathologically (necrotic death) by incubation at 55 °C for 20 min (until trypan blue uptake indicated compromise of membrane integrity; Ref. 1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar). In all cases, target cells (viable, apoptotic, and necrotic) were washed twice in PBS or complete medium before addition to experimental cultures.Reverse Transcriptase (RT)-PCR Analysis—PSR expression on the level of transcripts was evaluated by RT-PCR analysis. Total cellular RNA was isolated using TRIzol reagent (Invitrogen). cDNA synthesis and PCR were performed sequentially using the SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen). For the PSR-specific reaction, the forward primer was 5′-CAAGACGGTAAGAGGGAGACC-3′ (nucleotides 1086-1106, within exon 4), and the reverse primer was 5′-GTCACCTGGAGGAGCTGCG-3′ (complementary to nucleotides 1402-1384, within exon 6), yielding a product of 276 bp. As a control for RNA integrity, transcripts of constitutively expressed glyceraldehyde-3-phosphate dehydrogenase were assessed in parallel, using forward (5′-CCATGGAGAAGGCTGGGG-3′) and reverse (5′-CAAAGTTGTCATGGATGACC-3′) primers, generating a 188-bp product from 5′ end of the mRNA.Phagocytosis Assay—Phagocytosis by 3T3 fibroblasts was assessed as previously described for macrophages (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Target cells were labeled green with 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFDA; 0.2 μm; Molecular Probes, Eugene, OR) and were then induced to undergo cell death, killed by heat treatment, or left untreated. 3T3 cells were labeled red with 5 (and 6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine (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 3T3 cells 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). Cells with 3T3-like scatter properties that were both CMTMR-positive (λEx = 488 nm, λEm = 610 nm ± 15 nm) and CFDA-positive (λEx = 488 nm; λEm = 530 ± 15 nm) represented 3T3 cells that had engulfed targets. Engulfment is calculated as the fraction of double-positive 3T3 fibroblasts (all CMTMR-positive cells that also are CFDA-positive). Targets that are bound but not engulfed are disrupted and do not remain adherent during the analysis.Phagocytosis by transfected 293T cells was assayed similarly, employing a green fluorescent protein (GFP) marker expressed from a bicistronic vector to label the phagocytes green in place of CFDA. 293T cells were transfected with pIRES-2-PSR, pCX-β5 (which includes the same cytomegalovirus promoter/internal ribosome entry site (IRES) structure), or empty pIRES-2 vector (19Albert M.L. Kim J.I. Birge R.B. Nat. Cell Biol. 2000; 2: 899-905Crossref PubMed Scopus (324) Google Scholar). Target cells were labeled red with PKH26-GL red fluorescent cell linker kit (Sigma). 48 h after transfection, the 293T cells were co-cultured with labeled apoptotic cells for 2 h at 37 °C and analyzed as above.Transfections and Luciferase Assays—Apoptoticmodulation of NFκB-dependent transcription was assessed using a dual luciferase strategy, as described previously (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). We found that routine transfection protocols for 3T3 cells triggered high levels of cell death and NFκB activation (“transfection stress”). A transfection protocol that was reasonably efficient (∼40% viable cell transfection, as measured in parallel with farnesylated GFP as a transfection marker; Ref. 20Harvey K.J. Lukovic D. Ucker D.S. Cytometry. 2001; 43: 273-278Crossref PubMed Scopus (27) Google Scholar) and minimally stressful (i.e. low spontaneous NFκB activation) was selected.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.” 2 × 106 3T3 fibroblasts were transfected with 4.5 μg of pNFκB-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 Laboratories; Palo Alto, CA), together with 0.5 μg of pRL-SV40, a Renilla (sea pansy; Renilla reniformis) luciferase control vector, the constant expression of which is dependent on the SV40 early enhancer/promoter region (Promega, Madison, WI). Transfected cells were cultured overnight in 100-mm diameter dishes and replated the following day into 24-well plates at 1 × 105 cells/2 ml/well. After culturing a further 24 h, transfected cells were incubated without or with the indicated target cells (at a target cell:fibroblast ratio of 10:1) and/or IL-1β (5 ng/ml; R&D Systems; Minneapolis, MN) or TNFα (10 ng/ml; R&D Systems) for 12 h.Cell extracts were prepared, 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. 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 NFκB-dependent transcriptional activity.For other studies involving transfection, routine methods were employed. NIH 3T3 cells were transfected with Lipofectamine 2000 Transfection Reagent (Qiagen, Valencia, CA), and 293T cells were transfected using Effectene Transfection Reagent (Qiagen).Intracellular Immunostaining—5 × 104 NIH 3T3 cells were plated on poly-d-lysine-coated cover slips and transfected with epitope-tagged PSR constructs (either N-terminal HA-PSR in pRK or C-terminal PSR-V5-His in pcDNA3.1). 24 h post-transfection, the cells were fixed with paraformaldehyde (5% in PBS). The cells then were permeabilized with Triton X-100 (0.1% in PBS) for 5 min and incubated for 1 h with anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-V5 (Invitrogen) antibodies (diluted in PBS + 0.1% gelatin). After four washes (5 min each) in PBS + 0.1% gelatin, the cells were stained with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody and rhodamine-conjugated phalloidin (Molecular Probes). After several further washes, the coverslips were dried and mounted with mounting medium (ProLong Antifade Kit; Molecular Probes). The cells were visualized by epifluorescence microscopy.Cellular Extract Preparation and Immunoblot Analysis—PSR expression was assessed in unmanipulated 3T3 cells by immunoblotting. Activation of Akt and inhibition of extracellular signal-regulated kinases 1 and 2 (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 (10 nm; Calbiochem). After washing, cell extracts were prepared from the adherent 3T3 cells. Cells were lysed in lysis buffer 1 (150 mm NaCl, 50 mm HEPES (pH 7.5), 1.5 mm MgCl2,1mm 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 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, Billerica, MA). Blots were blocked with 5% dry milk in PBS before probing with a phospho-Akt(Thr308)-specific rabbit antiserum (Cell Signaling, Beverly,MA), an affinity-Purified phospho-ERK1/2 (Thr183/Tyr185)-specific rabbit antibody (Promega), or an affininty-purified PSR-specific rabbit antibody (specific for amino acids 363-381; Abcam, Cambridge, MA). 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.Rac1 Pull-down Assay—The level of GTP-bound Rac1 was determined by the GST-PAK CRIB “pull-down” assay as described previously (21Akakura S. Singh S. Spataro M. Akakura R. Kim J.I. Albert M.L. Birge R.B. Exp. Cell Res. 2004; 292: 403-416Crossref PubMed Scopus (177) Google Scholar). In brief, 5 × 105 3T3 fibroblasts were plated for 2 h on 60-mm diameter dishes that had been coated previously with MFG-E8 or bovine serum albumin (10 μg/ml). The cells then were lysed for 10 min in lysis buffer 2 (50 mm Tris (pH 7.2), 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 500 mm NaCl, and 10 mm MgCl2, plus protease inhibitors). 500 μg of each cell lysate was incubated for 1 h with glutathione-agarose beads coated with a bacterially expressed fusion protein of glutathione S-transferase (GST) with the GTPase binding domain (CRIB domain; amino acid residue 56-272) of the human PAK kinase, a downstream effector molecule for Rac1, which specifically binds the activated, GTP-loaded form of Rac1. The beads then were washed four times in wash buffer (50 mm Tris (pH 7.2), 1% (v/v) Triton X-100, 150 mm NaCl, and 10 mm MgCl2, plus protease inhibitors). Active Rac1, precipitating with beads, and total Rac1, in the starting lysates, were quantified by densitometry following immunoblotting with a Rac1 antibody (Upstate Biotechnology, Waltham, MA).RESULTSImmortalized 3T3 Cell Lines from PSR+/+, PSR+/-, and PSR-/- Mouse Embryo Fibroblasts—The role of PSR in the non-inflammatory clearance of apoptotic cells remains unresolved. Taking advantage of the ability of non-professional phagocytes to recognize, engulf, and respond to apoptotic cells specifically, 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. we established immortalized 3T3 fibroblast cells lines from PSR+/+, PSR+/-, and PSR-/- mouse embryo fibroblasts to test the role of PSR in these processes. The immortalized cell lines were derived from (129 × C57BL/6) embryo fibroblasts taken at day E14.5, prior to manifestations of lethality associated with PSR deficiency (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar), following the 3T3 protocol of Todaro and Green (17Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1993) Google Scholar). We chose to examine cells with the targeted PSR locus of Li et al. (Fig. 1A; Ref. 13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar), because the best genetic evidence implicating PSR in apoptotic clearance is derived from studies of mice harboring that disruption.PSR expression in these cells was evaluated by RT-PCR analysis of PSR transcripts (Fig. 1B) and by immunoblotting with a PSR-specific antibody (Fig. 1C). These tests confirmed both the presence of PSR expression in wild-type cells and in PSR+/- heterozygotes, at roughly equivalent levels, and its absence in PSR-/- fibroblasts. These results a
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