Abstract
Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.
Similar content being viewed by others
Abbreviations
- ADORA2A:
-
Adenosine A2A receptor
- APC:
-
Antigen presenting cell
- Ct:
-
Comparative threshold
- DC-SIGN:
-
Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin
- FC:
-
Fold change
- FPRL1:
-
Formyl peptide receptor-like 1
- HMDM:
-
Human monocyte derived macrophage
- ICAM3:
-
Intercellular adhesion molecule 3
- ITC:
-
Isotype control
- ITGAL:
-
Integrin alphaL
- ITGB2:
-
Integrin beta2
- KD:
-
Knock-down
- LFA-1:
-
Lymphocyte function-associated antigen 1
- LPS:
-
Lipopolysaccharides
- M-CSF:
-
Macrophage colony-stimulating factor
- PCD:
-
Programmed cell death
- Scr:
-
Scrambled
- siRNA:
-
Small interfering ribonucleic acid
- THBS1:
-
Thrombospondin 1
- TLDA:
-
TaqMan low-density array
References
Fesus L (1993) Biochemical events in naturally occurring forms of cell death. FEBS Lett 328:1–5
Assunção Guimarães C, Linden R (2004) Programmed cell deaths. Apoptosis and alternative deathstyles. Eur J Biochem 271:1638–1650
Bursch W, Ellinger A, Kienzl H, Török L, Pandey S, Sikorska M, Walker R, Hermann RS (1996) Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy. Carcinogenesis 17:1595–1607
Golstein P, Kroemer G (2007) Cell death by necrosis: towards a molecular definition. Trends Biochem Sci 32:37–43
Remijsen Q, Kuijpers TW, Wirawan E, Lippens S, Vandenabeele P, Vanden Berghe T (2011) Dying for a cause: NETosis, mechanisms behind an antimicrobial cell death modality. Cell Death Differ 18:581–588. doi:10.1038/cdd.2011.1
Henson PM, Hume DA (2006) Apoptotic cell removal in development and tissue homeostasis. Trends Immunol 27:244–250
Hume DA (2006) The mononuclear phagocyte system. Curr Opin Immunol 18:49–53
Dini L, Pagliara P, Carlà EC (2002) Phagocytosis of apoptotic cells by liver: a morphological study. Microsc Res Tech 57:530–540
Gardai SJ, Bratton DL, Ogden CA, Henson PM (2006) Recognition ligands on apoptotic cells: a perspective. J Leukoc Biol 79:896–903
Ravichandran KS (2010) Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums. J Exp Med 207:1807–1817. doi:10.1084/jem.20101157
Ravichandran KS (2011) Beginnings of a good apoptotic meal: the find-me and eat-me signaling pathways. Immunity 35:445–455. doi:10.1016/j.immuni.2011.09.004
Nagata S, Hanayama R, Kawane K (2010) Autoimmunity and the clearance of dead cells. Cell 140:619–630. doi:10.1016/j.cell.2010.02.014
Nathan C, Ding A (2010) Nonresolving inflammation. Cell 140:871–882. doi:10.1016/j.cell.2010.02.029
Rovere-Querini P, Brunelli S, Clementi E, Manfredi AA (2008) Cell death: tipping the balance of autoimmunity and tissue repair. Curr Pharm Des 14:269–277
Mevorach D (2010) Clearance of dying cells and systemic lupus erythematosus: the role of C1q and the complement system. Apoptosis 15:1114–1123. doi:10.1007/s10495-010-0530-8
Juan M, Vilella R, Mila J, Yagüe J, Miralles A, Campbell KS, Friedrich RJ, Cambier J, Vives J, De Fougerolles AR, Springer TA (1993) CDw50 and ICAM-3: two names for the same molecule. Eur J Immunol 23:1508–1512
Fawcett J, Holness CL, Needham LA, Turley H, Gatter KC, Mason DY, Simmons DL (1992) Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 360:481–484
Vazeux R, Hoffman PA, Tomita JK, Dickinson ES, Jasman RL, St John T, Gallatin WM (1992) Cloning and characterization of a new intercellular adhesion molecule ICAM-R. Nature 360:485–488
Montoya MC, Sancho D, Bonello G, Collette Y, Langlet C, He HT, Aparicio P, Alcover A, Olive D, Sánchez-Madrid F (2002) Role of ICAM-3 in the initial interaction of T lymphocytes and APCs. Nat Immunol 3:159–168
van Buul JD, Mul FP, van der Schoot CE, Hordijk PL (2004) ICAM-3 activation modulates cell–cell contacts of human bone marrow endothelial cells. J Vasc Res 41:28–37
Douglas IS, Leff AR, Sperling AI (2000) CD4+ T cell and eosinophil adhesion is mediated by specific ICAM-3 ligation and results in eosinophil activation. J Immunol 164:3385–3391
Moffatt OD, Devitt A, Bell ED, Simmons DL, Gregory CD (1999) Macrophage recognition of ICAM-3 on apoptotic leukocytes. J Immunol 162:6800–6810
Torr EE, Gardner DH, Thomas L, Goodall DM, Bielemeier A, Willetts R, Griffiths HR, Marshall LJ, Devitt A (2012) Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells. Cell Death Differ 19:671–679. doi:10.1038/cdd.2011.167
Corbi AL, Larson RS, Kishimoto TK, Springer TA, Morton CC (1988) Chromosomal location of the genes encoding the leukocyte adhesion receptors LFA-1, Mac-1 and p150,95. Identification of a gene cluster involved in cell adhesion. J Exp Med 167:1597–1607
Hogg N, Patzak I, Willenbrock F (2011) The insider’s guide to leukocyte integrin signaling and function. Nat Rev Immunol 11:416–426. doi:10.1038/nri2986
Marazuela M, Postigo AA, Acevedo A, Díaz-González F, Sanchez-Madrid F, de Landázuri MO (1994) Adhesion molecules from the LFA-1/ICAM-1,3 and VLA-4/VCAM-1 pathways on T lymphocytes and vascular endothelium in Graves’ and Hashimoto’s thyroid glands. Eur J Immunol 24:2483–2490
Perez OD, Mitchell D, Jager GC, South S, Murriel C, McBride J, Herzenberg LA, Kinoshita S, Nolan GP (2003) Leukocyte functional antigen 1 lowers T cell activation thresholds and signaling through cytohesin-1 and Jun-activating binding protein 1. Nat Immunol 4:1083–1092
Zahuczky G, Kristóf E, Majai G, Fésüs L (2011) Differentiation and glucocorticoid regulated apopto-phagocytic gene expression patterns in human macrophages. Role of Mertk in enhanced phagocytosis. PLoS ONE 6:e21349. doi:10.1371/journal.pone.0021349
Petrovski G, Zahuczky G, Katona K, Vereb G, Martinet W, Nemes Z, Bursch W, Fésüs L (2007) Clearance of dying autophagic cells of different origin by professional and non-professional phagocytes. Cell Death Differ 14:1117–1128
Miksa M, Komura H, Wu R, Shah KG, Wang P (2009) A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester. J Immunol Methods 342:71–77. doi:10.1016/j.jim.2008.11.019
Toda S, Hanayama R, Nagata S (2012) Two-step engulfment of apoptotic cells. Mol Cell Biol 32:118–125. doi:10.1128/MCB.05993-11
Aziz M, Yang WL, Wang P (2013) Measurement of phagocytic engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester. Curr Protoc Immunol Chapter 14: Unit 14.31. doi:10.1002/0471142735.im1431s100
Park D, Han CZ, Elliott MR, Kinchen JM, Trampont PC, Das S, Collins S, Lysiak JJ, Hoehn KL, Ravichandran KS (2011) Continued clearance of apoptotic cells critically depends on the phagocyte Ucp2 protein. Nature 477:220–224. doi:10.1038/nature10340
McColl A, Bournazos S, Franz S, Perretti M, Morgan BP, Haslett C, Dransfield I (2009) Glucocorticoids induce protein S-dependent phagocytosis of apoptotic neutrophils by human macrophages. J Immunol 183:2167–2175. doi:10.4049/jimmunol.0803503
Sun J, Williams J, Yan HC, Amin KM, Albelda SM, DeLisser HM (1996) Platelet endothelial cell adhesion molecule-1 (PECAM-1) homophilic adhesion is mediated by immunoglobulin-like domains 1 and 2 and depends on the cytoplasmic domain and the level of surface expression. J Biol Chem 271:18561–18570
Bogoevska V, Nollau P, Lucka L, Grunow D, Klampe B, Uotila LM, Samsen A, Gahmberg CG, Wagener C (2007) DC-SIGN binds ICAM-3 isolated from peripheral human leukocytes through Lewis x residues. Glycobiology 17:324–333
Dransfield I, Stocks SC, Haslett C (1995) Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis. Blood 85:3264–3273
Jersmann HP, Ross KA, Vivers S, Brown SB, Haslett C, Dransfield I (2003) Phagocytosis of apoptotic cells by human macrophages: analysis by multiparameter flow cytometry. Cytometry A 51:7–15
Hart SP, Ross JA, Ross K, Haslett C, Dransfield I (2000) Molecular characterization of the surface of apoptotic neutrophils: implications for functional downregulation and recognition by phagocytes. Cell Death Differ 7:493–503
Kasperkovitz PV, Khan NS, Tam JM, Mansour MK, Davids PJ, Vyas JM (2011) Toll-like receptor 9 modulates macrophage antifungal effector function during innate recognition of Candida albicans and Saccharomyces cerevisiae. Infect Immun 79:4858–4867. doi:10.1128/IAI.01135-10
Shen Y, Kawamura I, Nomura T, Tsuchiya K, Hara H, Dewamitta SR, Sakai S, Qu H, Daim S, Yamamoto T, Mitsuyama M (2010) Toll-like receptor 2- and MyD88-dependent phosphatidylinositol 3-kinase and Rac1 activation facilitates the phagocytosis of Listeria monocytogenes by murine macrophages. Infect Immun 78:2857–2867. doi:10.1128/IAI.01138-09
Gumienny TL, Brugnera E, Tosello-Trampont AC, Kinchen JM, Haney LB, Nishiwaki K, Walk SF, Nemergut ME, Macara IG, Francis R, Schedl T, Qin Y, Van Aelst L, Hengartner MO, Ravichandran KS (2001) CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 107:27–41
Tosello-Trampont AC, Nakada-Tsukui K, Ravichandran KS (2003) Engulfment of apoptotic cells is negatively regulated by Rho-mediated signaling. J Biol Chem 278:49911–49919
Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 148:2207–2216
Nakaya M, Kitano M, Matsuda M, Nagata S (2008) Spatiotemporal activation of Rac1 for engulfment of apoptotic cells. Proc Natl Acad Sci USA 105:9198–9203. doi:10.1073/pnas.0803677105
Tóth B, Garabuczi E, Sarang Z, Vereb G, Vámosi G, Aeschlimann D, Blaskó B, Bécsi B, Erdõdi F, Lacy-Hulbert A, Zhang A, Falasca L, Birge RB, Balajthy Z, Melino G, Fésüs L, Szondy Z (2009) Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells. J Immunol 182:2084–2092. doi:10.4049/jimmunol.0803444
Voll RE, Herrmann M, Roth EA, Stach C, Kalden JR, Girkontaite I (1997) Immunosuppressive effects of apoptotic cells. Nature 390:350–351
Stucki A, Hayflick JS, Sandmaier BM (2000) Antibody engagement of intercellular adhesion molecule 3 triggers apoptosis of normal and leukaemic myeloid marrow cells. Br J Haematol 108:157–166
Park JK, Park SH, So K, Bae IH, Yoo YD, Um HD (2010) ICAM-3 enhances the migratory and invasive potential of human non-small cell lung cancer cells by inducing MMP-2 and MMP-9 via Akt and CREB. Int J Oncol 36:181–192
Serrador JM, Vicente-Manzanares M, Calvo J, Barreiro O, Montoya MC, Schwartz-Albiez R, Furthmayr H, Lozano F, Sánchez-Madrid F (2002) A novel serine-rich motif in the intercellular adhesion molecule 3 is critical for its ezrin/radixin/moesin-directed subcellular targeting. J Biol Chem 277:10400–10409
Arroyo AG, Campanero MR, Sánchez-Mateos P, Zapata JM, Ursa MA, del Pozo MA, Sánchez-Madrid F (1994) Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase. J Cell Biol 126:1277–1286
Skubitz KM, Ahmed K, Campbell KD, Skubitz AP (1995) CD50 (ICAM-3) is phosphorylated on tyrosine and is associated with tyrosine kinase activity in human neutrophils. J Immunol 154:2888–2895
Serrador JM, Alonso-Lebrero JL, del Pozo MA, Furthmayr H, Schwartz-Albiez R, Calvo J, Lozano F, Sánchez-Madrid F (1997) Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. J Cell Biol 138:1409–1423
Curtis BM, Scharnowske S, Watson AJ (1992) Sequence and expression of a membrane-associated C-type lectin that exhibits CD4-independent binding of human immunodeficiency virus envelope glycoprotein gp120. Proc Natl Acad Sci USA 89:8356–8360
Khoo US, Chan KY, Chan VS, Lin CL (2008) DC-SIGN and L-SIGN: the SIGNs for infection. J Mol Med 86:861–874. doi:10.1007/s00109-008-0350-2
McGreal EP, Miller JL, Gordon S (2005) Ligand recognition by antigen-presenting cell C-type lectin receptors. Curr Opin Immunol 17:18–24
Miyanishi M, Tada K, Koike M, Uchiyama Y, Kitamura T, Nagata S (2007) Identification of Tim4 as a phosphatidylserine receptor. Nature 450:435–439
Park D, Tosello-Trampont AC, Elliott MR, Lu M, Haney LB, Ma Z, Klibanov AL, Mandell JW, Ravichandran KS (2007) BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module. Nature 450:430–434
Park SY, Jung MY, Kim HJ, Lee SJ, Kim SY, Lee BH, Kwon TH, Park RW, Kim IS (2008) Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor. Cell Death Differ 15:192–201
Hanayama R, Tanaka M, Miwa K, Shinohara A, Iwamatsu A, Nagata S (2002) Identification of a factor that links apoptotic cells to phagocytes. Nature 417:182–187
Lemke G, Rothlin CV (2008) Immunobiology of the TAM receptors. Nat Rev Immunol 8:327–336. doi:10.1038/nri2303
Scannell M, Flanagan MB, deStefani A, Wynne KJ, Cagney G, Godson C, Maderna P (2007) Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages. J Immunol 178:4595–4605
Scott RS, McMahon EJ, Pop SM, Reap EA, Caricchio R, Cohen PL, Earp HS, Matsushima GK (2001) Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411:207–211
Kinchen JM, Cabello J, Klingele D, Wong K, Feichtinger R, Schnabel H, Schnabel R, Hengartner MO (2005) Two pathways converge at CED-10 to mediate actin rearrangement and corpse removal in C. elegans. Nature 434:93–99
Bustelo XR, Sauzeau V, Berenjeno IM (2007) GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. BioEssays 29:356–370
Janiak A, Zemskov EA, Belkin AM (2006) Cell surface transglutaminase promotes RhoA activation via integrin clustering and suppression of the Src-p190RhoGAP signaling pathway. Mol Biol Cell 17:1606–1619
Grimsley C, Ravichandran KS (2003) Cues for apoptotic cell engulfment: eat-me, don’t eat-me and come-get-me signals. Trends Cell Biol 13:648–656
Savill J, Dransfield I, Gregory C, Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat Rev Immunol 2:965–975
Cvetanovic M, Ucker DS (2004) Innate immune discrimination of apoptotic cells: repression of proinflammatory macrophage transcription is coupled directly to specific recognition. J Immunol 172:880–889
Cvetanovic M, Mitchell JE, Patel V, Avner BS, Su Y, van der Saag PT, Witte PL, Fiore S, Levine JS, Ucker DS (2006) Specific recognition of apoptotic cells reveals a ubiquitous and unconventional innate immunity. J Biol Chem 281:20055–20067
Rovere-Querini P, Dumitriu IE (2003) Corpse disposal after apoptosis. Apoptosis 8:469–479
Han CZ, Ravichandran KS (2011) Metabolic connections during apoptotic cell engulfment. Cell 23:1442–1445. doi:10.1016/j.cell.2011.12.006
Fadok VA, Bratton DL, Konowal A, Freed PW, Westcott JY, Henson PM (1998) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest 101:890–898
Han C, Jin J, Xu S, Liu H, Li N, Cao X (2010) Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b. Nat Immunol 11:734–742. doi:10.1038/ni.1908
Pathak SK, Sköld AE, Mohanram V, Persson C, Johansson U, Spetz AL (2012) Activated apoptotic cells induce dendritic cell maturation via engagement of Toll-like receptor 4 (TLR4), DC-SIGN and beta-2 integrins. J Biol Chem 287:13731–13742. doi:10.1074/jbc.M111.336545
Mukundan L, Odegaard JI, Morel CR, Heredia JE, Mwangi JW, Ricardo-Gonzalez RR, Goh YP, Eagle AR, Dunn SE, Awakuni JU, Nguyen KD, Steinman L, Michie SA, Chawla A (2009) PPAR-delta senses and orchestrates clearance of apoptotic cells to promote tolerance. Nat Med 15:1266–1272. doi:10.1038/nm.2048
A-Gonzalez N, Bensinger SJ, Hong C, Beceiro S, Bradley MN, Zelcer N, Deniz J, Ramirez C, Díaz M, Gallardo G, de Galarreta CR, Salazar J, Lopez F, Edwards P, Parks J, Andujar M, Tontonoz P, Castrillo A (2009) Apoptotic cells promote their own clearance and immune tolerance through activation of the nuclear receptor LXR. Immunity 31:245–258. doi:10.1016/j.immuni.2009.06.018
Acknowledgments
This work has been supported by Grants from the Hungarian Scientific Research Fund (OTKA NK 105046, K 61868), TÁMOP 4.2.1./B-09/1/KONV-2010-0007 and TÁMOP-4.2.2/B-10/1-2010-0024 projects implemented through the New Hungary Development Plan, co-financed by the European Social Fund, EU FP7 TRANSCOM IAPP 251506, TRANSPATH ITN 289964 and NKTH NTP Schizo08. We thank Dr. Zsuzsa Szondy for her consultation and for reviewing the manuscript. We acknowledge Jennifer Nagy and Szilvia Szalóki for excellent technical assistance.
Conflict of interest
The authors declare that they have no conflict of interest.
Author information
Authors and Affiliations
Corresponding author
Additional information
E. Kristóf and G. Zahuczky contributed equally to this study.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kristóf, E., Zahuczky, G., Katona, K. et al. Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages. Apoptosis 18, 1235–1251 (2013). https://doi.org/10.1007/s10495-013-0873-z
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10495-013-0873-z