Activation of Protein Kinase C βII by the Stereo-specific Phosphatidylserine Receptor Is Required for Phagocytosis of Apoptotic Thymocytes by Resident Murine Tissue Macrophages
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m202967200
ISSN1083-351X
AutoresJill C. Todt, Bin Hu, Antonello Punturieri, Joanne Sonstein, Timothy Polak, Jeffrey L. Curtis,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoWe showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AMø) and peritoneal macrophages (PMø) and that such phagocytosis is markedly lower in AMø compared with PMø. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two Mø populations. By immunoblotting, AMø expressed equivalent PKC η but lower amounts of other isoforms (α, βI, βII, δ, ε, μ, and ζ), with the greatest difference in βII expression. A requirement for PKC βII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC βII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC βII myristoylated peptide as a blocker. Exposure of PMø to phosphatidylserine (PS) liposomes specifically induced translocation of PKC βII and other isoforms to membranes and cytoskeleton. Both AMø and PMø expressed functional PS receptor, blockade of which inhibited PKC βII translocation. Our results indicate that murine tissue Mø require PKC βII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in Mø phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC βII activation. We showed previously that protein kinase C (PKC) is required for phagocytosis of apoptotic leukocytes by murine alveolar (AMø) and peritoneal macrophages (PMø) and that such phagocytosis is markedly lower in AMø compared with PMø. In this study, we examined the roles of individual PKC isoforms in phagocytosis of apoptotic thymocytes by these two Mø populations. By immunoblotting, AMø expressed equivalent PKC η but lower amounts of other isoforms (α, βI, βII, δ, ε, μ, and ζ), with the greatest difference in βII expression. A requirement for PKC βII for phagocytosis was demonstrated collectively by phorbol 12-myristate 13-acetate-induced depletion of PKC βII, by dose-response to PKC inhibitor Ro-32-0432, and by use of PKC βII myristoylated peptide as a blocker. Exposure of PMø to phosphatidylserine (PS) liposomes specifically induced translocation of PKC βII and other isoforms to membranes and cytoskeleton. Both AMø and PMø expressed functional PS receptor, blockade of which inhibited PKC βII translocation. Our results indicate that murine tissue Mø require PKC βII for phagocytosis of apoptotic cells, which differs from the PKC isoform requirement previously described in Mø phagocytosis of other particles, and imply that a crucial action of the PS receptor in this process is PKC βII activation. alveolar macrophage(s) atypical PKC isoform conventional PKC isoform diacylglycerol monoclonal antibody macrophage(s) novel PKC isoform phosphatidylinositol protein kinase C peritoneal macrophages phorbol 12-myristate 13-acetate phosphatidylserine phosphate-buffered saline receptor for activated C kinase Phagocytosis, the uptake of large particles (>0.5 μm) via actin-dependent mechanisms (1Aderem A. Underhill D.M. Annu. Rev. Immunol. 1999; 17: 593-623Crossref PubMed Scopus (2061) Google Scholar), is the obligatory means of clearing apoptotic cells during development and in resolving inflammation (2Fadok V.A. Chimini G. Semin. Immunol. 2001; 13: 365-372Crossref PubMed Scopus (136) Google Scholar). Only macrophages can efficiently clear the large numbers of apoptotic leukocytes produced during waning immune responses (3Savill 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 (1343) Google Scholar, 4Akbar A.N. Savill J. Gombert W. Bofill M. Borthwick N.J. Whitelaw F. Grundy J. Janossy G. Salmon M. J. Exp. Med. 1994; 180: 1943-1947Crossref PubMed Scopus (72) Google Scholar, 5Stern M. Savill J. Haslett C. Am. J. Pathol. 1996; 149: 911-921PubMed Google Scholar, 6Haslett C. Am. J. Respir. Crit. Care Med. 1999; 160: S5-S11Crossref PubMed Scopus (442) Google Scholar). Indeed, the efficiency of this process is evidenced by the fact that apoptotic cells are rarely observed in vivo (7Surh C.D. Sprent J. Nature. 1994; 372: 100-103Crossref PubMed Scopus (931) Google Scholar); one exception is in the lungs of mice, where apoptotic lymphocytes are easily demonstrable both in health and inflammation (8Milik A.M. Buechner-Maxwell V.A. Sonstein J. Kim S. Seitzman G.D. Beals T.F. Curtis J.L. J. Clin. Invest. 1997; 99: 1082-1091Crossref PubMed Scopus (52) Google Scholar). This defect in clearance is consistent with the finding that the principal resident lung phagocytes, alveolar macrophages (AMø),1 exhibit markedly lower capacity for phagocytosis of apoptotic leukocytes, either compared with inflammatory lung Mø (in rabbits) (9Newman S.L. Henson J.E. Henson P.M. J. Exp. Med. 1982; 156: 430-442Crossref PubMed Scopus (256) Google Scholar) or with resident peritoneal Mø (PMø) (in mice) (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar). In the latter system, no disparity between AMø and PMø was detected using three other particle types (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar, 11Hu B. Punturieri A. Todt J. Sonstein J. Polak T. Curtis J.L. J. Leukocyte Biol. 2002; 71: 881-889PubMed Google Scholar). Murine AMø also exhibited a relative deficit in phagocytosis of apoptotic cells in vivo (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar). We have recently found that human AMø also show much lower phagocytosis of apoptotic cells than of other particles in vitro. 2J. C. Todt, B. Hu, A. Punturieri, J. Sonstein, T. Polak, and J. L. Curtis, unpublished observation. 2J. C. Todt, B. Hu, A. Punturieri, J. Sonstein, T. Polak, and J. L. Curtis, unpublished observation. Contrasting the properties of these two types of resident tissue Mø could aid in defining the molecular basis of apoptotic cell recognition, which is poorly understood. Recognition of apoptotic cells is initiated through at least two pathways. Using a 70-kDa glycosylated type II transmembrane protein called PS-R′ (12Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekewitz R.A. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1249) Google Scholar), Mø and other cell types recognize externalized phosphatidylserine (PS), which translocates from the inner to the outer leaflet of the cell membrane early in apoptosis (13Verhoven B. Krahling S. Schlegel R.A. Williamson P. Cell Death Differ. 1999; 6: 262-270Crossref PubMed Scopus (114) Google Scholar, 14Schlegel R.A. Stevens M. Lumley-Sapanski K. Williamson P. Immunol. Lett. 1993; 36: 283-288Crossref PubMed Scopus (77) Google Scholar, 15Koopman G. Reutelingsperger C.P. Kuijten G.A. Keehnen R.M. Pals S.T. van Oers M.H. Blood. 1994; 84: 1415-1420Crossref PubMed Google Scholar, 16Homburg C.H. de Haas M. von dem Borne A.E. Verhoeven A.J. Reutelingsperger C.P. Roos D. Blood. 1995; 85: 532-540Crossref PubMed Google Scholar, 17Martin S.J. Reutelingsperger C.P. McGahon A.J. Rader J.A. van Schie R.C. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2556) Google Scholar). Recognition of externalized PS has been suggested to be both necessary and sufficient to generate a signal for ingestion (13Verhoven B. Krahling S. Schlegel R.A. Williamson P. Cell Death Differ. 1999; 6: 262-270Crossref PubMed Scopus (114) Google Scholar, 18Fadok V.A. de Cathelineau A. Daleke D.L. Henson P.M. Bratton D.L. J. Biol. Chem. 2001; 276: 1071-1077Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). More recently, the Mø-specific receptor tyrosine kinase Mer has been identified as critical for the phagocytosis of apoptotic cells by murine Mø (19Scott R.S. McMahon E.J. Pop S.M. Reap E.A. Caricchio R. Cohen P.L. Earp H.S. Matsushima G.K. Nature. 2001; 411: 207-211Crossref PubMed Scopus (923) Google Scholar). How signaling from these two receptors leads to apoptotic cell phagocytosis is undefined. A host of other Mø cell surface receptors (reviewed in Ref. 20Gregory C.D. Curr. Opin. Immunol. 2000; 12: 27-34Crossref PubMed Scopus (162) Google Scholar) have also been implicated in clearance of apoptotic cells, but they appear to be involved principally in adhesion rather than in recognition of cell death (21Hoffmann P.R. deCathelineau A.M. Ogden C.A. Leverrier Y. Bratton D.L. Daleke D.L. Ridley A.J. Fadok V.A. Henson P.M. J. Cell Biol. 2001; 155: 649-659Crossref PubMed Scopus (44) Google Scholar). Moreover, although we (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar) and others (22Platt N. da Silva R.P. Gordon S. Immunol. Lett. 1999; 65: 15-19Crossref PubMed Scopus (38) Google Scholar) have identified a number of differences in expression of adhesion molecules between murine AMø and PMø, blocking experiments using monoclonal antibodies (mAbs) or the arginine-glycine-aspartic acid-serine (RGDS) tetrapeptide have not supported any identified adhesion receptor, including several integrins, as being responsible for the functional difference in phagocytosis (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar). An alternative explanation for disparity between Mø types in apoptotic cell phagocytosis would be differences in postreceptor signal transduction. A logical candidate for such a difference is protein kinase C (PKC), because we and others have shown that it is required for apoptotic cell clearance (11Hu B. Punturieri A. Todt J. Sonstein J. Polak T. Curtis J.L. J. Leukocyte Biol. 2002; 71: 881-889PubMed Google Scholar, 23Marguet D. Luciani M.F. Moynault A. Williamson P. Chimini G. Nat. Cell Biol. 1999; 1: 454-456Crossref PubMed Scopus (146) Google Scholar). PKC comprises a family of related serine/threonine kinases divided into three groups on the basis of structure and cofactor requirements (24Webb B.L. Hirst S.J. Giembycz M.A. Br. J. Pharmacol. 2000; 130: 1433-1452Crossref PubMed Scopus (138) Google Scholar). Activation of PKC requires phosphorylation on serines/threonines, displacement of its autoinhibitory pseudosubstrate domain, and translocation to specific cytoskeletal and intracellular membrane sites of action (25Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1468) Google Scholar). Activation of the conventional group (cPKC) (α, βI, βII, and γ) is calcium- and diacylglycerol (DAG)-dependent. Activation of the novel group (nPKC) (ε, δ, η, and θ) also depends on binding of DAG, but it is calcium-independent. The atypical group (aPKC) (ι/λ and ζ) cannot be activated by calcium or DAG. All PKC family members bind PS on the cytosolic leaflet of the cell membrane, but aPKCs require additional incompletely defined lipid activators (24Webb B.L. Hirst S.J. Giembycz M.A. Br. J. Pharmacol. 2000; 130: 1433-1452Crossref PubMed Scopus (138) Google Scholar). Another isoform, PKC μ (often called PKD in the mouse), does not fit into any of the major groups. PKC μ contains two unique hydrophobic domains in its amino terminus and is phospholipid-dependent but calcium-insensitive (26Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (509) Google Scholar). Individual cell types usually express several PKC isoforms, each of which appears to mediate unique functions (27Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar). Even the 50-amino acid difference in the alternatively spliced forms of PKC β (βI and βII) appears to be responsible for the unique role of each PKC β isozyme (28Chalfant C.E. Ohno S. Konno Y. Fisher A.A. Bisnauth L.D. Watson J.E. Cooper D.R. Mol. Endocrinol. 1996; 10: 1273-1281Crossref PubMed Scopus (62) Google Scholar, 29Gokmen-Polar Y. Fields A.P. J. Biol. Chem. 1998; 273: 20261-20266Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Thus, differences between murine AMø and PMø in PKC isoform expression or function could explain the functional difference between these two cell types in apoptotic cell phagocytosis. We recently showed (11Hu B. Punturieri A. Todt J. Sonstein J. Polak T. Curtis J.L. J. Leukocyte Biol. 2002; 71: 881-889PubMed Google Scholar) that phagocytosis of apoptotic thymocytes by murine AMø and PMø was reduced by the nonspecific PKC inhibitor staurosporine and by Gö 6976 but only incompletely by calphostin C. Gö 6976 has been reported to act as a partially selective inhibitor of the cPKC α and βI isoforms (30Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), whereas calphostin C has greater activity against nPKCs than cPKCs (31Keenan C. Goode N. Pears C. FEBS Lett. 1997; 415: 101-108Crossref PubMed Scopus (68) Google Scholar). However, current data on the specificity of these inhibitors are too inconclusive (32Way K.J. Chou E. King G.L. Trends Pharmacol. Sci. 2000; 21: 181-187Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar) to allow us to predict with certainty which isoforms are involved. Therefore, in this study, we used six approaches to further define the PKC isoform(s) involved in Mø phagocytosis of apoptotic thymocytes. First, because the pattern of PKC isoforms in primary murine tissue Mø has not been described, we analyzed PKC isoform expression using isotype-specific antibodies and Western blotting. Second, we tested the effect of overnight exposure of Mø to phorbol 12-myristate 13-acetate (PMA), which depletes cPKC and nPKC isoforms by interacting with their DAG-binding sites, on AMø and PMø phagocytosis of apoptotic thymocytes. Third, we employed the isoform-selective inhibitors, rottlerin and Ro-32-0432. Fourth, we tested the effect of myristoylated blocking peptides against PKC βI, PKC βII, and PKC α on phagocytosis of apoptotic thymocytes by AMø and PMø. Fifth, we examined the effect of PS liposomes, as models for apoptotic thymocytes, on translocation of PKC isoforms to cell membranes and cytoskeleton; apoptotic thymocytes could not be used, because they themselves express multiple PKC isoforms. Finally, we studied Mø expression of PS-R′ and the relationship between PS-R′ stimulation and PKC translocation. Collectively, our results indicate a requirement for PKC βII in Mø phagocytosis of apoptotic thymocytes, show that an antibody against PS-R′ blocks translocation of PKC βII in response to PS liposomes, and suggest that relative deficiency of PKC βII and possibly other PKC isoforms may partially explain the functional difference in apoptotic cell clearance by AMø. Rottlerin and Ro-32-0432 were purchased fromCalbiochem. PMA, PBS, RPMI 1640, fetal bovine serum, HEPES, pyruvate, 1 kb Plus brand up markers and penicillin/streptomycin were obtained from Invitrogen. Dimethyl sulfoxide, dexamethasone, 2-mercaptoethanol, sodium deoxycholate, glycerol, NaCl, Tris-HCl, Triton X-100, Tween 20, 1 kb Plus brand up markers,l-α-phosphatidylinositol (PI), l-α-PS, and phosphatase inhibitor mixture II were purchased from Sigma. Antibodies and blocking peptides for PKC isoforms and horseradish peroxidase-conjugated anti-rabbit IgG were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Complete mini-protease inhibitor tablets and the lactate dehydrogenase cytotoxicity detection kit were purchased from Roche Molecular Biochemicals. SDS, 0.2-μm polyvinylidene difluoride membrane, nonfat dry milk blocker, and 10% Ready Acrylamide gels were obtained from Bio-Rad. Supersignal West Femto Maximum Sensitivity substrate was obtained from Pierce. Eastman Kodak Co. X-Omat AR film and eight-well Lab-Tek slides were obtained from Fisher. mAb 217 (anti-murine PS-R; rat IgM) was generously provided by Dr. Valerie Fadok (National Jewish Medical Center, Denver, CO) as a culture supernatant; in selected experiments, a commercial preparation of this mAb (Cascade BioScience, Winchester, MA) was used. Myristoylated PKC peptides were synthesized by BIOSOURCE Quality Controlled Biochemicals (Hopkinton, MA) at 90% purity, as confirmed by high pressure liquid chromatography and mass spectroscopy performed by the manufacturer. All experiments were performed using pathogen-free C57BL/6 female mice purchased from Charles River Laboratories Inc. (Wilmington, MA) at 7–8 weeks of age and used at 8–14 weeks of age. Mice were housed in the Animal Care Facility at the Ann Arbor VA Medical Center, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. This study complied with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (Department of Health, Education, and Welfare Publication No. 80-32) and followed a protocol approved by the Animal Care Subcommittee of the local institutional review board. Mice were euthanized by asphyxia in a high CO2 environment, which we have previously shown does not impair the capacity of AMø to ingest apoptotic thymocytes (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar). Resident AMø and PMø were harvested and cultured as previously described (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar). Mø were isolated by adherence onto tissue culture plates (for protein isolation) or eight-well Lab-Tek slides (for phagocytosis assay) for 2–4 h at 37 °C in 5% CO2 in complete medium (RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 25 mm HEPES, 2 mml-glutamine, 1 mm pyruvate, 100 units/ml penicillin/streptomycin, 55 μm2-mercaptoethanol). Nonadherent cells were removed by gentle washing. In experiments in which PKC localization was analyzed by Western blotting, complete medium was replaced with serum-free medium for 1 h. Thymuses were harvested from normal mice and minced to yield a single cell suspension. To induce apoptosis, thymocytes were resuspended in complete medium to a concentration of 1 × 106 cells/ml and incubated for 6 h in complete medium containing 1 μmdexamethasone. This treatment yields a population with a low percentage (mean 13.4%) contamination of late apoptotic or necrotic cells (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar,11Hu B. Punturieri A. Todt J. Sonstein J. Polak T. Curtis J.L. J. Leukocyte Biol. 2002; 71: 881-889PubMed Google Scholar). Resident AMø and PMø from normal mice were isolated and plated at 1.0 × 106 cells/ml on 100 × 15-mm plates in medium containing 10% serum and incubated for 2 h at 37 °C and 5% CO2. Next, Mø were washed and solubilized in ice-cold lysis buffer consisting of 1.0% Triton X-100, 20 mmTris-HCl, pH 8.0, 150 mm NaCl, 0.1% SDS, 0.5% sodium deoxycholate, and 10% glycerol with protease inhibitors (complete minitablet) and phosphatase inhibitor mixture II (1:100) for 30 min on ice. After sonicating for 3 s and centrifuging at 13,800 ×g for 3 min, 7 μg of protein/sample was run on a 10% acrylamide gel under reducing conditions and transferred to a polyvinylidene difluoride membrane using 25 mm Tris, 192 mm glycine, 20% methanol. Blots were blocked with 5% nonfat dry milk in PBS (blocker), incubated with the appropriate anti-PKC isoform antibody (1:1000 dilution in blocker), and washed five times for 5 min each with PBS containing 0.1% Tween 20. Blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:10,000 in PBS containing 5% nonfat dry milk), and the chemiluminescence signal was developed by adding a peroxidase/luminol-based substrate (Supersignal Femto reagent; Pierce). The identity of PKC isoforms on Western blots was verified using isoform-specific blocking peptides. No bands were seen when blots were stained using horseradish peroxidase-conjugated goat anti-rabbit IgG alone. To analyze the subcellular distribution of PKC isozymes, Mø were isolated and plated at 1.0 × 106 cells/ml on 30 × 15-mm plates in medium containing 10% serum and incubated for 2 h at 37 °C and 5% CO2. The medium was then removed and replaced with serum-free medium for 1 h followed by washing and scraping in cold PBS. Next, Mø were sonicated for 10 s in 50 mm Tris, pH 8.0, 9 mm EDTA and then centrifuged at 100,000 × g for 45 min. The supernatant was collected and defined as the cytosolic fraction. The pellet, defined as the membrane and cytoskeleton fractions, was solubilized in ice-cold lysis buffer (defined above) for 30 min on ice. This solubilized pellet fraction was sonicated for 3 s and then centrifuged at 13,800 × g for 3 min. Equal amounts of protein (3–7 μg) were run on a 10% SDS-PAGE gel under reducing conditions, transferred to the polyvinylidene difluoride membrane, and stained as described above. To produce liposomes, PS or the negatively charged control lipid PI was dried under N2 and resuspended in serum-free medium by vortexing. Liposome size was determined by Coulter counter analysis to be in a similar range as apoptotic thymocytes, 2–3.2 μm. In these experiments, Mø were incubated for 1 h in serum-free medium, liposomes in serum-free medium were added to Mø monolayers in a final PS or PI concentration of 0.11 mm, and Mø and liposomes were co-incubated for the indicated time at 37 °C and 5% CO2. Cells were washed and scraped in cold PBS; sonicated for 10 s in 50 mm Tris, pH 8.0, 9 mm EDTA; and centrifuged at 100,000 × g for 45 min. Total RNA was isolated from adherent AMø and PMø using TRIzol (Invitrogen). Reverse transcriptase-PCRs were performed using kits from Invitrogen. The primer sets used were the following: for mouse PS-R′ (GenBankTM accession number AF304118), forward CTC ACG ATG AAC CAC AAG AGC and reverse GGA CCA GCC CTC TTG TGC ATT; for mouse glyceraldehyde-3-phosphate dehydrogenase (GenBankTMaccession number M32599), forward GGT CGG TGT GAA CGG ATT TGG and reverse ATG AGG TCC ACC ACC CTG TTG. The expected PCR product sizes are 245 bp for PS-R′ and 968 bp for glyceraldehyde-3-phosphate dehydrogenase. PCR products were analyzed on a 2% agarose gel and stained with ethidium bromide. The identity of the target products was confirmed by sequencing. Apoptosis was measured by simultaneous annexin V and propidium iodide staining (apoptosis detection kit; R & D Systems, Minneapolis, MN) according to the manufacturer's protocol. Cells were analyzed without fixation by flow cytometry within 1 h of staining. Staining of surface receptors and flow cytometry were performed as previously described in detail (33Curtis J.L. Kim S. Scott P.J. Buechner-Maxwell V.A. Am. J. Respir. Cell Mol. Biol. 1995; 12: 520-530Crossref PubMed Scopus (29) Google Scholar) using a FACScan cytometer (BD PharMingen) running Cell Quest software on a PowerPC microcomputer (Apple, Cupertino, CA) for data collection and analysis. A minimum of 10,000 cells were analyzed. Phagocytosis of apoptotic thymocytesin vitro was assayed by co-incubation of 0.5–2.0 × 105 adherent Mø with 2.0 × 106 apoptotic thymocytes for 90 min at 37 °C in 5% CO2 as previously described (10Hu B. Sonstein J. Christensen P.J. Punturieri A. Curtis J.L. J. Immunol. 2000; 165: 2124-2133Crossref PubMed Scopus (62) Google Scholar). Results are expressed as the percentage of Mø containing at least one ingested thymocyte (percentage of phagocytosis) and as the phagocytic index, which was generated by multiplying the percentage of phagocytosis by the mean number of ingested cells per Mø. Cell-permeable PKC inhibitors were added either 30 min (rottlerin) or 18 h (Ro-32-0432 and PMA) before the addition of apoptotic thymocytes. Myristoylated PKC peptides from the carboxyl terminus of the V5 region of PKC α (myr-PQFVHPILQSAV-amide), PKC βI (myr-DQNEFAGFSYTNPEFVINV-amide), or PKC βII (myr-SFVNSEFLKPEVKS-amide) were added to a final concentration of 100 μm 30 min before the addition of apoptotic thymocytes. The myristate moieties coupled to the amino terminus of these peptides allow membrane permeability, permitting their use in primary cells (34Stebbins E.G. Mochly-Rosen D. J. Biol. Chem. 2001; 276: 29644-29650Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). All inhibitors were nontoxic at the times and concentrations utilized, as determined by the lactate dehydrogenase cytotoxicity detection kit.2 Adherence of apoptotic thymocytes to Møin vitro was assayed in the same fashion as phagocytosis, except that 2 × 107 apoptotic thymocytes suspended in 400 μl of complete medium were added to each well, yielding a 100:1 ratio of thymocytes to Mø. The slides were incubated for various times at 37 °C and then washed in a standardized fashion by dipping individual slides five times in each of two Wheaton jars filled with ice-cold PBS. Slides were then stained using hematoxylin-eosin Y (Richard-Allan; Kalamazoo, MI) and coverslipped. Adhesion was evaluated by counting 200–300 mø per well at ×1000 magnification under oil immersion and scoring for bound thymocytes. Results are expressed as the percentage of Mø binding at least one thymocyte (percentage of adhesive Mø) and as the adhesion index, which was generated by multiplying the percentage of adherence-positive Mø by the mean number of adherent thymocytes per Mø. In blocking experiments, mAbs were used at concentrations previously determined to be saturating by flow cytometry, and culture supernatant containing mAb 217 was concentrated 10-fold using Centriprep tubes (Amicon, Bedford, MA). Data are expressed as mean ± S.E. Statistical calculations were performed using Statview on a Macintosh Power PC G3 computer. Continuous ratio scale data were evaluated by unpaired Student's t test (for two samples) or analysis of variance (for multiple comparisons) withpost hoc analysis by the Tukey-Kramer test or by the two-tailed Dunnett test, which specifically compares treatment groups with a control group (35Zar J.H. Biostatistical Analysis. 2nd Ed. Prentice-Hall, Englewood Cliffs, NJ1974: 122-165Google Scholar). Use of these parametric statistics was deemed appropriate, since phagocytosis of apoptotic thymocytes by PMø has been shown to follow a Gaussian distribution (36Licht R. Jacobs C.W. Tax W.J. Berden J.H. J. Immunol. Methods. 1999; 223: 237-248Crossref PubMed Scopus (44) Google Scholar). Significant differences were defined as p < 0.05. Western blot analysis using PKC isoform-specific antibodies demonstrated markedly lower expression of PKC α, βI, βII, δ, ε, μ, and ξ in resident murine AMø in comparison with resident murine PMø (Fig. 1). By contrast, AMø had slightly higher expression of PKC η. Staining did not detect expression of PKC γ or PKC λ in either murine AMø or PMø,2 in agreement with previous analyses of human tissue Mø (24Webb B.L. Hirst S.J. Giembycz M.A. Br. J. Pharmacol. 2000; 130: 1433-1452Crossref PubMed Scopus (138) Google Scholar, 37Monick M.M. Carter A.B. Gudmundsson G. Geist L.J. Hunninghake G.W. Am. J. Physiol. 1998; 275: L389-L397Crossref PubMed Google Scholar), and we did not test expression of the lymphocyte-specific isoform PKC θ. Thus, the lower expression of several PKC isoforms by AMø is a potential explanation for the previously observed decreased phagocytosis of apoptotic cells by this cell type. Chronic (18-h) PMA treatment depletes cPKCs and nPKCs but not aPKCs, with the degree to which individual isoforms are affected depending on the cell type and the PMA concentration (27Dempsey E.C. Newton A.C. Mochly-Rosen D. Fields A.P. Reyland M.E. Insel P.A. Messing R.O. Am. J. Physiol. 2000; 279: L429-L438Crossref PubMed Google Scholar,37Monick M.M. Carter A.B. Gudmundsson G. Geist L.J. Hunninghake G.W. Am. J. Physiol. 1998; 275: L389-L397Crossref PubMed Google Scholar, 38Nixon J.B. McPhail L.C. J. Immunol. 1999; 163: 4574-4582PubMed Google Scholar, 39Ahnadi C.E. Giguere P. Gravel S. Gagne D. Goulet A.C. Fulop Jr., T. Payet M.D. Dupuis G. J. Leukocyte Biol. 2000; 68: 293-300PubMed Google Scholar). In preliminary experiments, the concentration and time of PMA addition were optimized to achieve maximal inhibition of apoptotic cell phagocytosis. We determined that the conditions used were nontoxic, as indicated by assay of lactate dehydrogenase release.2Western blot analysis on AMø and PMø after overnight treatment using 8.1 μm PMA confirmed that PKC α, βI, βII, and δ were significantly depleted (p < 0.05, unpairedt test) by PMA treatment in either of the two types of Mø (Fig. 2). As anticipated, PKC ε, η, μ, and ζ were not depleted by overnight PMA treatment in either Mø type. In the functional assay, the same overnight PMA treatment significantly decreased phagocytosis by AMø and PMø (p < 0.05, unpaired Student t test) (Fig.3). Control experiments showed that overnight PMA treatment did not influence adhesion of thymocytes to PMø (percentage of adhesive Mø, 86.1 ± 1.7% (control)versus 82.0 ± 2.0% (PMA-treated), p = 0.16; adhesion index, 2.6 ± 0.2 (control) versus2.7 ± 0.1 (PMA-treated); mean ± S.E., n = 4, p = 0.47, both comparisons made by unpairedt test) and actually slightly increased adhesion to AMø (adhesive Mø, 60.5 ± 1.5% (control) versus 71.5 ± 2.2% (PMA-treated); mean S.E., n = 4;p < 0.001; adhesion index, 1.2 ± 0.1 (control)versus 1.7 ± 0.0 (PMA-treated); p < 0.001, unpaired t test). Collectively, these data indicate that the nPKC isoforms ε and η, the aPKC isoform ζ, and PKC μ/PKD are not essential for Mø phagocytosis of apoptotic cells but leave open the question of which cPKC or other nPKC isoforms are required.Fi
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