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Abstract

CD81 is an integral membrane protein belonging to the tetraspanin superfamily. It has two extracellular domains that interact with cell surface proteins and two intracellular tails that contribute to cellular processes. Although there are considerable data about how CD81 affects T- and B-cell function, not much is known about how it impacts macrophages. To address this, we established four cell lines from mouse bone marrow in the presence of macrophage colony-stimulating factor and transfection with SV40 large T antigen. Two were CD81−/− (ASD1 and ASD2) and two were CD81+/− (2ASD1.10 and 2BSD1.10). Cells were Mac-2-, PU.1-, and c-fms-positive and all the cell lines were phagocytic indicating that they were macrophage-like. In mixtures of the two cell types in tissue culture, CD81−/− cells out competed CD81+/− cells with CD81-bearing cells being undetectable after 50 cell culture passages. Although cell divisions during log-phase growth were not significantly different between CD81+/− macrophage cells and CD81−/− macrophage cells, we found that CD81−/− macrophage cells reached a higher density at confluency than CD81+/− macrophage cells. CD81 transcript levels increased as cultures became confluent, but transcript levels of other tetraspanin-related molecules remained relatively constant. Transfection of CD81 into ASD1 (CD81−/−) cells reduced the density of confluent cultures of transformants compared to cells transfected with vector alone. These data suggest that CD81 potentially plays a role in macrophage cell line growth regulation.

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References

  • Armstrong, J. W.; Kirby-Dobbels, K.; Chapes, S. K. The effects of rM-CSF and rIL-6 therapy on immunosuppressed antiorthostatically suspended mice. J. Appl. Physiol. 78: 968–975; 1995.

    PubMed  CAS  Google Scholar 

  • Armstrong, J. W.; Nelson, K. A.; Simske, S. J.; Luttges, M. W.; Iandolo, J. J.; Chapes, S. K. Skeletal unloading causes organ-specific changes in immune cell responses. J. Appl. Physiol. 75: 2734–2739; 1993.

    PubMed  CAS  Google Scholar 

  • Beharka, A. A.; Armstrong, J. W.; Chapes, S. K. Macrophage cell lines derived from major histocompatibility complex II-negative mice. In Vitro Cell. Dev. Biol. Anim. 34: 499–507; 1998. doi:10.1007/s11626-998-0085-y.

    Article  PubMed  CAS  Google Scholar 

  • Chan, J.; Leenen, P. J.; Bertoncello, I.; Nishikawa, S. I.; Hamilton, J. A. Macrophage lineage cells in inflammation: characterization by colony-stimulating factor-1 (CSF-1) receptor (c-Fms), ER-MP58, and ER-MP20 (Ly-6C) expression. Blood 92: 1423–1431; 1998.

    PubMed  CAS  Google Scholar 

  • Chapes, S. K.; Didier, E. S.; Tompkins, W. A. Macrophage cell line B6MP102 resembles peritoneal macrophages in tumor cell recognition and killing. J. Leukoc. Biol. 43: 28–35; 1988.

    PubMed  CAS  Google Scholar 

  • Chapes, S. K.; O’Neill, A. E.; Flaherty, L.; Gooding, L. R. Macrophage-resistant murine simian virus 40 tumors express a retroviral type-specific gp70. J. Virol. 61: 928–932; 1987.

    PubMed  CAS  Google Scholar 

  • Charrin, S.; Le Naour, F.; Labas, V.; Billard, M.; Le Caer, J. P.; Emile, J. F.; Petit, M. A.; Boucheix, C.; Rubinstein, E. EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells. Biochem. J. 373: 409–421; 2003. doi:10.1042/BJ20030343.

    Article  PubMed  CAS  Google Scholar 

  • Claas, C.; Seiter, S.; Claas, A.; Savelyeva, L.; Schwab, M.; Zoller, M. Association between the rat homologue of CO-029, a metastasis-associated tetraspanin molecule and consumption coagulopathy. J. Cell Biol. 141: 267–280; 1998. doi:10.1083/jcb.141.1.267.

    Article  PubMed  CAS  Google Scholar 

  • Clark, K. L.; Zeng, Z.; Langford, A. L.; Bowen, S. M.; Todd, S. C. PGRL is a major CD81-associated protein on lymphocytes and distinguishes a new family of cell surface proteins. J. Immunol. 167: 5115–5121; 2001.

    PubMed  CAS  Google Scholar 

  • Cocquerel, L.; Kuo, C. C.; Dubuisson, J.; Levy, S. CD81-dependent binding of hepatitis C virus E1E2 heterodimers. J. Virol. 77: 10677–10683; 2003. doi:10.1128/JVI.77.19.10677-10683.2003.

    Article  PubMed  CAS  Google Scholar 

  • Das, S. K.; Stanley, E. R.; Guilbert, L. J.; Forman, L. W. Human colony-stimulating factor (CSF-1) radioimmunoassay: resolution of three subclasses of human colony-stimulating factors. Blood 58: 630–641; 1981.

    PubMed  CAS  Google Scholar 

  • DeKoter, R. P.; Walsh, J. C.; Singh, H. PU.1 regulates both cytokine-dependent proliferation and differentiation of granulocyte/macrophage progenitors. EMBO J. 17: 4456–4468; 1998. doi:10.1093/emboj/17.15.4456.

    Article  PubMed  CAS  Google Scholar 

  • Dijkstra, S.; Geisert, E. E. Jr.; Dijkstra, C. D.; Bar, P. R.; Joosten, E. A. CD81 and microglial activation in vitro: proliferation, phagocytosis and nitric oxide production. J. Neuroimmunol. 114: 151–159; 2001. doi:10.1016/S0165-5728(01)00240-5.

    Article  PubMed  CAS  Google Scholar 

  • Esashi, E.; Sekiguchi, T.; Ito, H.; Koyasu, S.; Miyajima, A. Cutting edge: a possible role for CD4 + thymic macrophages as professional scavengers of apoptotic thymocytes. J. Immunol. 171: 2773–2777; 2003.

    PubMed  CAS  Google Scholar 

  • Fearon, D. T.; Carter, R. H. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13: 127–149; 1995. doi:10.1146/annurev.iy.13.040195.001015.

    Article  PubMed  CAS  Google Scholar 

  • Fernandez-Sesma, A.; Peluso, R. W.; Bai, X.; Schulman, J. L.; Levy, D. E.; Moran, T. M. Superantigen-activated T cells redirected by a bispecific antibody inhibit vesicular stomatitis virus replication in vitro and in vivo. J. Immunol. 160: 1841–1849; 1998.

    PubMed  CAS  Google Scholar 

  • Ferret-Bernard, S.; Sai, P.; Bach, J. M. In vitro induction of inhibitory macrophage differentiation by granulocyte-macrophage colony-stimulating factor, stem cell factor and interferon-gamma from lineage phenotypes-negative c-kit-positive murine hematopoietic progenitor cells. Immunol. Lett. 91: 221–227; 2004. doi:10.1016/j.imlet.2003.12.008.

    Article  PubMed  CAS  Google Scholar 

  • Geisert, E. E. Jr.; Yang, L.; Irwin, M. H. Astrocyte growth, reactivity, and the target of the antiproliferative antibody, TAPA. J. Neurosci. 16: 5478–5487; 1996.

    PubMed  CAS  Google Scholar 

  • Gensert, J. M.; Baranova, O. V.; Weinstein, D. E.; Ratan, R. R. CD81, a cell cycle regulator, is a novel target for histone deacetylase inhibition in glioma cells. Neurobiol. Dis. 26: 671–680; 2007. doi:10.1016/j.nbd.2007.03.008.

    Article  PubMed  CAS  Google Scholar 

  • Hanisch, U. K. Microglia as a source and target of cytokines. Glia 40: 140–155; 2002. doi:10.1002/glia.10161.

    Article  PubMed  Google Scholar 

  • Hanisch, U. K.; van Rossum, D.; Xie, Y.; Gast, K.; Misselwitz, R.; Auriola, S.; Goldsteins, G.; Koistinaho, J.; Kettenmann, H.; Moller, T. The microglia-activating potential of thrombin: the protease is not involved in the induction of proinflammatory cytokines and chemokines. J. Biol. Chem. 279: 51880–51887; 2004. doi:10.1074/jbc.M408318200.

    Article  PubMed  CAS  Google Scholar 

  • Huang, S.; Yuan, S.; Dong, M.; Su, J.; Yu, C.; Shen, Y.; Xie, X.; Yu, Y.; Yu, X.; Chen, S.; Zhang, S.; Pontarotti, P.; Xu, A. The phylogenetic analysis of tetraspanins projects the evolution of cell-cell interactions from unicellular to multicellular organisms. Genomics 86: 674–684; 2005. doi:10.1016/j.ygeno.2005.08.004.

    Article  PubMed  CAS  Google Scholar 

  • Janabi, N.; Peudenier, S.; Heron, B.; Ng, K. H.; Tardieu, M. Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci. Lett. 195: 105–108; 1995. doi:10.1016/0304-3940(94)11792-H.

    Article  PubMed  CAS  Google Scholar 

  • Kelic, S.; Levy, S.; Suarez, C.; Weinstein, D. E. CD81 regulates neuron-induced astrocyte cell-cycle exit. Mol. Cell. Neurosci. 17: 551–560; 2001. doi:10.1006/mcne.2000.0955.

    Article  PubMed  CAS  Google Scholar 

  • Kool, J.; van Zaane, W.; van der, Eb, A. J.; Terleth, C. Down-regulation of T-STAR, a growth inhibitory protein, after SV40-mediated immortalization. Cell. Growth. Differ. 12: 535–541; 2001.

    PubMed  CAS  Google Scholar 

  • Kuchler, R. J.; Merchant, D. J. Propagation of strain L (earle) cells in agitated fluid suspension cultures. Proc. Soc. Exp. Biol. Med. 92: 803–806; 1956.

    PubMed  CAS  Google Scholar 

  • Leenen, P. J.; de Bruijn, M. F.; Voerman, J. S.; Campbell, P. A.; van Ewijk, W. Markers of mouse macrophage development detected by monoclonal antibodies. J. Immunol. Methods. 174: 5–19; 1994. doi:10.1016/0022-1759(94)90005-1.

    Article  PubMed  CAS  Google Scholar 

  • Leenen, P. J.; Melis, M.; Slieker, W. A.; Van Ewijk, W. Murine macrophage precursor characterization. II. Monoclonal antibodies against macrophage precursor antigens. Eur. J. Immunol. 20: 27–34; 1990. doi:10.1002/eji.1830200105.

    Article  PubMed  CAS  Google Scholar 

  • Levy, S.; Todd, S. C.; Maecker, H. T. CD81 (TAPA-1): a molecule involved in signal transduction and cell adhesion in the immune system. Annu. Rev. Immunol. 16: 89–109; 1998. doi:10.1146/annurev.immunol.16.1.89.

    Article  PubMed  CAS  Google Scholar 

  • Ludlow, J. Interactions between SV40 large-tumor antigen and the growth suppressor proteins pRB and p53. FASEB J. 7: 866–871; 1993.

    PubMed  CAS  Google Scholar 

  • Maecker, H. T.; Levy, S. Normal lymphocyte development but delayed humoral immune response in CD81-null mice. J. Exp. Med. 185: 1505–1510; 1997. doi:10.1084/jem.185.8.1505.

    Article  PubMed  CAS  Google Scholar 

  • Maecker, H. T.; Todd, S. C.; Kim, E. C.; Levy, S. Differential expression of murine CD81 highlighted by new anti-mouse CD81 monoclonal antibodies. Hybridoma 19: 15–22; 2000. doi:10.1089/027245700315752.

    Article  PubMed  CAS  Google Scholar 

  • Melnicoff, M. J.; Horan, P. K.; Morahan, P. S. Kinetics of changes in peritoneal cell populations following acute inflammation. Cell Immunol. 118: 178–191; 1989. doi:10.1016/0008-8749(89)90367-5.

    Article  PubMed  CAS  Google Scholar 

  • Metcalf, D. The molecular contol of cell division, differentiation commitment and maturation in haemopoietic cells. Nature 339: 27–30; 1989. doi:10.1038/339027a0.

    Article  PubMed  CAS  Google Scholar 

  • Mittelbrunn, M.; Yanez-Mo, M.; Sancho, D.; Ursa, A.; Sanchez-Madrid, F. Cutting edge: dynamic redistribution of tetraspanin CD81 at the central zone of the immune synapse in both T lymphocytes and APC. J. Immunol. 169: 6691–6695; 2002.

    PubMed  CAS  Google Scholar 

  • Oren, R.; Takahashi, S.; Doss, C.; Levy, R.; Levy, S. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol. Cell Biol. 10: 4007–4015; 1990.

    PubMed  CAS  Google Scholar 

  • Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: e45; 2001. doi:10.1093/nar/29.9.e45.

    Article  PubMed  CAS  Google Scholar 

  • Potts, B. E.; Hart, M. L.; Snyder, L. L.; Boyle, D.; Mosier, D. A.; Chapes, S. K. Differentiation of C2D macrophage cells after adoptive transfer. Clin. Vaccine Immunol. 15: 243–252; 2008. doi:10.1128/CVI.00328-07.

    Article  PubMed  CAS  Google Scholar 

  • Rhoads, D.; Roufa, D. Emetine resistance of Chinese hamster cells: Structures of wild-type and mutant ribosomal protein S14 mRNAs. Mol. Cell. Biol. 5: 1655–1659; 1985.

    PubMed  CAS  Google Scholar 

  • Rubinstein, E.; Ziyyat, A.; Prenant, M.; Wrobel, E.; Wolf, J. P.; Levy, S.; Le Naour, F.; Boucheix, C. Reduced fertility of female mice lacking CD81. Dev. Biol. 290: 351–358; 2006. doi:10.1016/j.ydbio.2005.11.031.

    Article  PubMed  CAS  Google Scholar 

  • Shoham, T.; Rajapaksa, R.; Boucheix, C.; Rubinstein, E.; Poe, J. C.; Tedder, T. F.; Levy, S. The tetraspanin CD81 regulates the expression of CD19 during B cell development in a postendoplasmic reticulum compartment. J. Immunol. 171: 4062–4072; 2003.

    PubMed  CAS  Google Scholar 

  • Sullivan, C. D.; Geisert, E. E. Jr. Expression of rat target of the antiproliferative antibody (TAPA) in the developing brain. J. Comp. Neurol. 396: 366–380; 1998. doi:10.1002/(SICI)1096-9861(19980706)396:3<366::AID-CNE7>3.0.CO;2-0.

    Article  PubMed  CAS  Google Scholar 

  • Sundstrom, C.; Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int. J. Cancer 17: 565–577; 1976. doi:10.1002/ijc.2910170504.

    Article  PubMed  CAS  Google Scholar 

  • Takeda, Y.; Tachibana, I.; Miyado, K.; Kobayashi, M.; Miyazaki, T.; Funakoshi, T.; Kimura, H.; Yamane, H.; Saito, Y.; Goto, H.; Yoneda, T.; Yoshida, M.; Kumagai, T.; Osaki, T.; Hayashi, S.; Kawase, I.; Mekada, E. Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes. J. Cell Biol. 161: 945–956; 2003. doi:10.1083/jcb.200212031.

    Article  PubMed  CAS  Google Scholar 

  • Testa, J. E.; Brooks, P. C.; Lin, J. M.; Quigley, J. P. Eukaryotic expression cloning with an antimetastatic monoclonal antibody identifies a tetraspanin (PETA-3/CD151) as an effector of human tumor cell migration and metastasis. Cancer Res. 59: 3812–3820; 1999.

    PubMed  CAS  Google Scholar 

  • Todd, S. C.; Lipps, S. G.; Crisa, L.; Salomon, D. R.; Tsoukas, C. D. CD81 expressed on human thymocytes mediates integrin activation and interleukin 2-dependent proliferation. J. Exp. Med. 184: 2055–2060; 1996. doi:10.1084/jem.184.5.2055.

    Article  PubMed  CAS  Google Scholar 

  • Toledo, M. S.; Suzuki, E.; Handa, K.; Hakomori, S. Cell growth regulation through GM3-enriched microdomain (glycosynapse) in human lung embryonal fibroblast WI38 and its oncogenic transformant VA13. J. Biol. Chem. 279: 34655–34664; 2004. doi:10.1074/jbc.M403857200.

    Article  PubMed  CAS  Google Scholar 

  • Toledo, M. S.; Suzuki, E.; Handa, K.; Hakomori, S. Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor. J. Biol. Chem. 280: 16227–16234; 2005. doi:10.1074/jbc.M413713200.

    Article  PubMed  CAS  Google Scholar 

  • Yanez-Mo, M.; Alfranca, A.; Cabanas, C.; Marazuela, M.; Tejedor, R.; Ursa, M. A.; Ashman, L. K.; de Landazuri, M. O.; Sanchez-Madrid, F. Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions. J. Cell Biol. 141: 791–804; 1998. doi:10.1083/jcb.141.3.791.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

We would like to thank Jenna Kennedy, Ryan Gallagher, and David Amrine for their assistance in the maintenance and genotyping of the mice used and Betsey Potts for her help and information on macrophage phenotyping. We also thank Elizabeth Miller, Regina Fleming, Alison Fedrow, and Taryn Penabaz for laboratory assistance and Caroline Delandre for participating in our group discussions about tetraspanins. We thank Drs. Carol Mckown (US Meat Animal Research Center, Hastings, NE) and Sherry Fleming (Kansas State University) for reviewing the manuscript. This project has been supported by NIH grants AI55052, AI052206, RR16475, and RR17686; NASA grants NAG2-1274 and NCC8-242; the Kansas Agriculture Experiment Station and the Terry C. Johnson Center for Basic Cancer Research. This is Kansas Agriculture Experiment Station publication 08-128-J.

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Correspondence to Stephen K. Chapes.

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Mordica, W.J., Woods, K.M., Clem, R.J. et al. Macrophage cell lines use CD81 in cell growth regulation. In Vitro Cell.Dev.Biol.-Animal 45, 213–225 (2009). https://doi.org/10.1007/s11626-008-9167-0

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