Histochemistry and Cell Biology

, Volume 147, Issue 3, pp 317–339 | Cite as

Truncated EphA2 likely potentiates cell adhesion via integrins as well as infiltration and/or lodgment of a monocyte/macrophage cell line in the red pulp and marginal zone of the mouse spleen, where ephrin-A1 is prominently expressed in the vasculature

  • Naoko Konda
  • Noritaka Saeki
  • Shingo Nishino
  • Kazushige Ogawa
Original Paper


We previously established a J774.1 monocyte/macrophage subline expressing a truncated EphA2 construct lacking the kinase domain. We demonstrated that following ephrin-A1 stimulation, endogenous EphA2 promotes cell adhesion through interaction with integrins and integrin ligands such as ICAM1 and that truncated EphA2 potentiates the adhesion and becomes associated with the integrin/integrin ligand complex. Based on these findings, we hypothesized that the EphA/ephrin-A system, particularly EphA2/ephrin-A1, regulates transendothelial migration/tissue infiltration of monocytes/macrophages, because ephrin-A1 is widely recognized to be upregulated in inflammatory vasculatures. To evaluate whether this hypothesis is applicable in the spleen, we screened for EphA2/ephrin-A1 expression and reexamined the cellular properties of the J774.1 subline. We found that ephrin-A1 was expressed in the vasculature of the marginal zone and the red pulp and that its expression was upregulated in response to phagocyte depletion; further, CD115, F4/80, and CXCR4 were expressed in J774.1 cells, which serve as a usable substitute for monocytes/macrophages. Moreover, following ephrin-A1 stimulation, truncated EphA2 did not detectably interfere with the phosphorylation of endogenous EphA2, and it potentiated cell adhesion possibly through modulation of integrin avidity. Accordingly, by intravenously injecting mice with equal numbers of J774.1 and the subline cells labeled with distinct fluorochromes, we determined that truncated EphA2 markedly potentiated preferential cell infiltration into the red pulp and the marginal zone. Thus, modulation of EphA2 signaling might contribute to effective transplantation of tissue-specific resident macrophages and/or monocytes.


EphA2 Ephrin-A1 Spleen Infiltration Adhesion 



Human EphA2 with a portion of the cytoplasmic domain replaced with EGFP


Intercellular adhesion molecule 1


Mucosal vascular addressin cell adhesion molecule 1


Reverse-transcription PCR


Vascular cell adhesion molecule



This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to K.O.; Nos. 24580429, 15K07769).

Compliance with ethical standards

Conflict of interest

No potential conflicts of interest were disclosed.


  1. Aichele P, Zinke J, Grode L, Schwendener RA, Kaufmann SH, Seiler P (2003) Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses. J Immunol 171:1148–1155CrossRefPubMedGoogle Scholar
  2. Ansel KM et al (2000) A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 406:309–314. doi: 10.1038/35018581 CrossRefPubMedGoogle Scholar
  3. Birjandi SZ, Ippolito JA, Ramadorai AK, Witte PL (2011) Alterations in marginal zone macrophages and marginal zone B cells in old mice. J Immunol 186:3441–3451. doi: 10.4049/jimmunol.1001271 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bratosin D et al (1998) Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A Rev Biochim 80:173–195CrossRefGoogle Scholar
  5. Coulthard MG et al (2012) Eph/Ephrin signaling in injury and inflammation. Am J Pathol 181:1493–1503. doi: 10.1016/j.ajpath.2012.06.043 CrossRefPubMedGoogle Scholar
  6. Davies LC, Jenkins SJ, Allen JE, Taylor PR (2013) Tissue-resident macrophages. Nat Immunol 14:986–995. doi: 10.1038/ni.2705 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Ellyard JI, Avery DT, Mackay CR, Tangye SG (2005) Contribution of stromal cells to the migration, function and retention of plasma cells in human spleen: potential roles of CXCL12, IL-6 and CD54. Eur J Immunol 35:699–708CrossRefPubMedGoogle Scholar
  8. Forster R, Schubel A, Breitfeld D, Kremmer E, Renner-Muller I, Wolf E, Lipp M (1999) CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 99:23–33CrossRefPubMedGoogle Scholar
  9. Garcia-Ceca J, Alfaro D, Montero-Herradon S, Tobajas E, Munoz JJ, Zapata AG (2015) Eph/ephrins-mediated thymocyte-thymic epithelial cell interactions control numerous processes of thymus biology. Front Immunol 6:333. doi: 10.3389/fimmu.2015.00333 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Geissmann F, Manz MG, Jung S, Sieweke MH, Merad M, Ley K (2010) Development of monocytes, macrophages, and dendritic cells. Science 327:656–661. doi: 10.1126/science.1178331 CrossRefPubMedPubMedCentralGoogle Scholar
  11. Gucciardo E, Sugiyama N, Lehti K (2014) Eph- and ephrin-dependent mechanisms in tumor and stem cell dynamics. Cell Mol Life Sci 71:3685–3710. doi: 10.1007/s00018-014-1633-0 CrossRefPubMedGoogle Scholar
  12. Gunn MD, Kyuwa S, Tam C, Kakiuchi T, Matsuzawa A, Williams LT, Nakano H (1999) Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med 189:451–460CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hargreaves DC et al (2001) A coordinated change in chemokine responsiveness guides plasma cell movements. J Exp Med 194:45–56CrossRefPubMedPubMedCentralGoogle Scholar
  14. Hogg N, Patzak I, Willenbrock F (2011) The insider’s guide to leukocyte integrin signalling and function. Nat Rev Immunol 11:416–426. doi: 10.1038/nri2986 CrossRefPubMedGoogle Scholar
  15. Inra CN, Zhou BO, Acar M, Murphy MM, Richardson J, Zhao Z, Morrison SJ (2015) A perisinusoidal niche for extramedullary haematopoiesis in the spleen. Nature 527:466–471. doi: 10.1038/nature15530 CrossRefPubMedPubMedCentralGoogle Scholar
  16. Kania A, Klein R (2016) Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol. doi: 10.1038/nrm.2015.16 PubMedGoogle Scholar
  17. Kurotaki D, Uede T, Tamura T (2015) Functions and development of red pulp macrophages. Microbiol Immunol 59:55–62. doi: 10.1111/1348-0421.12228 CrossRefPubMedGoogle Scholar
  18. Ley K, Laudanna C, Cybulsky MI, Nourshargh S (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7:678–689. doi: 10.1038/nri2156 CrossRefPubMedGoogle Scholar
  19. Luo BH, Carman CV, Springer TA (2007) Structural basis of integrin regulation and signaling. Annu Rev Immunol 25:619–647. doi: 10.1146/annurev.immunol.25.022106.141618 CrossRefPubMedPubMedCentralGoogle Scholar
  20. McGaha TL, Chen Y, Ravishankar B, van Rooijen N, Karlsson MC (2011) Marginal zone macrophages suppress innate and adaptive immunity to apoptotic cells in the spleen. Blood 117:5403–5412. doi: 10.1182/blood-2010-11-320028 Epub 322011 Mar 320028 CrossRefPubMedGoogle Scholar
  21. Mebius RE, Kraal G (2005) Structure and function of the spleen. Nat Rev Immunol 5:606–616. doi: 10.1038/nri1669 CrossRefPubMedGoogle Scholar
  22. Miwa Y et al (2013) Up-regulated expression of CXCL12 in human spleens with extramedullary haematopoiesis. Pathology 45:408–416. doi: 10.1097/PAT.0b013e3283613dbf CrossRefPubMedGoogle Scholar
  23. Mueller SN, Germain RN (2009) Stromal cell contributions to the homeostasis and functionality of the immune system. Nat Rev Immunol 9:618–629. doi: 10.1038/nri2588 PubMedPubMedCentralGoogle Scholar
  24. Ngo VN et al (1999) Lymphotoxin alpha/beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J Exp Med 189:403–412CrossRefPubMedPubMedCentralGoogle Scholar
  25. Nguyen TM, Arthur A, Gronthos S (2015) The role of Eph/ephrin molecules in stromal-hematopoietic interactions. Int J Hematol. doi: 10.1007/s12185-015-1886-x Google Scholar
  26. Nourshargh S, Alon R (2014) Leukocyte migration into inflamed tissues. Immunity 41:694–707. doi: 10.1016/j.immuni.2014.10.008 CrossRefPubMedGoogle Scholar
  27. Ogawa K, Pasqualini R, Lindberg RA, Kain R, Freeman AL, Pasquale EB (2000) The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization. Oncogene 19:6043–6052. doi: 10.1038/sj.onc.1204004 CrossRefPubMedGoogle Scholar
  28. Ogawa K, Wada H, Okada N, Harada I, Nakajima T, Pasquale EB, Tsuyama S (2006) EphB2 and ephrin-B1 expressed in the adult kidney regulate the cytoarchitecture of medullary tubule cells through Rho family GTPases. J Cell Sci 119:559–570. doi: 10.1242/jcs.02777 CrossRefPubMedGoogle Scholar
  29. Ogawa K, Takemoto N, Ishii M, Pasquale EB, Nakajima T (2011) Complementary expression and repulsive signaling suggest that EphB receptors and ephrin-B ligands control cell positioning in the gastric epithelium. Histochem Cell Biol 136:617–636. doi: 10.1007/s00418-011-0867-2 CrossRefPubMedGoogle Scholar
  30. Ogawa K, Saeki N, Igura Y, Hayashi Y (2013) Complementary expression and repulsive signaling suggest that EphB2 and ephrin-B1 are possibly involved in epithelial boundary formation at the squamocolumnar junction in the rodent stomach. Histochem Cell Biol 140:659–675. doi: 10.1007/s00418-013-1129-2 CrossRefPubMedGoogle Scholar
  31. Pandey A, Shao H, Marks RM, Polverini PJ, Dixit VM (1995) Role of B61, the ligand for the Eck receptor tyrosine kinase, in TNF-alpha-induced angiogenesis. Science 268:567–569CrossRefPubMedGoogle Scholar
  32. Pasquale EB (2005) Eph receptor signalling casts a wide net on cell behaviour. Nat Rev Mol Cell Biol 6:462–475CrossRefPubMedGoogle Scholar
  33. Pasquale EB (2008) Eph-ephrin bidirectional signaling in physiology and disease. Cell 133:38–52. doi: 10.1016/j.cell.2008.03.011 CrossRefPubMedGoogle Scholar
  34. Pasquale EB (2010) Eph receptors and ephrins in cancer: bidirectional signalling and beyond. Nat Rev Cancer 10:165–180. doi: 10.1038/nrc2806 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Saeki N, Nishino S, Shimizu T, Ogawa K (2015) EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces. Cell Adhes Migr 9:469–482. doi: 10.1080/19336918.2015.1107693 CrossRefGoogle Scholar
  36. Sakamoto A et al (2011) Expression profiling of the ephrin (EFN) and Eph receptor (EPH) family of genes in atherosclerosis-related human cells. J Int Med Res 39:522–527CrossRefPubMedGoogle Scholar
  37. Sharfe N, Nikolic M, Cimpeon L, Van De Kratts A, Freywald A, Roifman CM (2008) EphA and ephrin-A proteins regulate integrin-mediated T lymphocyte interactions. Mol Immunol 45:1208–1220 Epub 2007 Nov 1205 CrossRefPubMedGoogle Scholar
  38. Shireman PK (2007) The chemokine system in arteriogenesis and hind limb ischemia. J Vasc Surg 45(Suppl A):A48–A56. doi: 10.1016/j.jvs.2007.02.030 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Sieweke MH, Allen JE (2013) Beyond stem cells: self-renewal of differentiated macrophages. Science 342:1242974. doi: 10.1126/science.1242974 CrossRefPubMedGoogle Scholar
  40. Suzuki T et al (2014) Pulmonary macrophage transplantation therapy. Nature 514:450–454. doi: 10.1038/nature13807 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Swirski FK et al (2009) Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 325:612–616. doi: 10.1126/science.1175202 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Takada Y, Ye X, Simon S (2007) The integrins. Genome Biol 8:215. doi: 10.1186/gb-2007-8-5-215 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Welford AF et al (2011) TIE2-expressing macrophages limit the therapeutic efficacy of the vascular-disrupting agent combretastatin A4 phosphate in mice. J Clin Investig 121:1969–1973. doi: 10.1172/jci44562 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Laboratory of Veterinary Anatomy, Graduate School of Life and Environmental SciencesOsaka Prefecture UniversityIzumisanoJapan

Personalised recommendations