Abstract
CD40 ligand (CD40L) acts as an immune modulator in activated T cells, and mutations in the extracellular domain are associated to X-linked hyper IgM syndrome. A role for platelet CD40L in mediating thrombotic and inflammatory processes in atherosclerosis has also been reported. Using the Cre/loxP recombination technology we generated four knockout lines of mice with deletion of the Cd40lg gene restricted to the hematopoietic system. Mouse lines with expression of Cre recombinase driven by the Tie2, Vav1, or CD4 promoters showed in vivo ablation of CD40L in leukocytes and platelets. In contrast, in mice with Cre expression driven by the megakaryocyte lineage-restricted Pf4 promoter, abolition of CD40L expression was observed in megakaryocytes cultured in vitro, but not in circulating platelets. Characterization of these animals revealed reduced in vivo thrombogenesis and defective activation of washed CD40L-deficient platelets, suggesting that membrane-bound CD40L is involved in the control of haemostasis acting as a platelet co-activator. In addition, we report the practically absence of CD40L in mouse and human endothelial cells, as well as the detection of an exon 3-deleted CD40L transcript in both platelets and leukocytes of mouse and human origin. Finally, compared with their corresponding littermate floxed controls, Cre+ mice carrying CD40-deficient leukocytes did not exhibit increased IgM levels, and reduction of IgA and IgG levels was statistically significant only in Tie2-Cre+ mice, suggesting that expression of CD40L in an earlier developmental step may be determinant in the regulation of the class switch recombination process.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR (2002a) Platelet-derived CD40L: the switch-hitting player of cardiovascular disease. Circulation 106(8):896–899
Andre P, Prasad KS, Denis CV, He M, Papalia JM, Hynes RO, Phillips DR, Wagner DD (2002b) CD40L stabilizes arterial thrombi by a beta3 integrin-dependent mechanism. Nat Med 8(3):247–252
Aruffo A, Farrington M, Hollenbaugh D, Li X, Milatovich A, Nonoyama S, Bajorath J, Grosmaire LS, Stenkamp R, Neubauer M et al (1993) The CD40 ligand, gp39, is defective in activated T cells from patients with X-linked hyper-IgM syndrome. Cell 72(2):291–300
Aversa G, Punnonen J, Carballido JM, Cocks BG, de Vries JE (1994) CD40 ligand-CD40 interaction in Ig isotype switching in mature and immature human B cells. Semin Immunol 6(5):295–301
Basch RS, Dolzhanskiy A, Zhang XM, Karpatkin S (1996) The development of human megakaryocytes. II. CD4 expression occurs during haemopoietic differentiation and is an early step in megakaryocyte maturation. Br J Haematol 94(3):433–442
de Boer J, Williams A, Skavdis G, Harker N, Coles M, Tolaini M, Norton T, Williams K, Roderick K, Potocnik AJ, Kioussis D (2003) Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur J Immunol 33(2):314–325
Elgueta R, Benson MJ, de Vries VC, Wasiuk A, Guo Y, Noelle RJ (2009) Molecular mechanism and function of CD40/CD40L engagement in the immune system. Immunol Rev 229(1):152–172
Elzey BD, Ratliff TL, Sowa JM, Crist SA (2011) Platelet CD40L at the interface of adaptive immunity. Thromb Res 127(3):180–183
Geraldes P, Gagnon S, Hadjadj S, Merhi Y, Sirois MG, Cloutier I, Tanguay JF (2006) Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: an ERalpha-mediated pathway. Cardiovasc Res 71(3):566–573
Graf D, Muller S, Korthauer U, van Kooten C, Weise C, Kroczek RA (1995) A soluble form of TRAP (CD40 ligand) is rapidly released after T cell activation. Eur J Immunol 25(6):1749–1754
Hassan GS, Merhi Y, Mourad M (2012) CD40 Ligand: a neo-inflammatory molecule in vascular diseases. Immunobiology 217:521–532
He C, Hu H, Braren R, Fong SY, Trumpp A, Carlson TR, Wang RA (2008) c-myc in the hematopoietic lineage is crucial for its angiogenic function in the mouse embryo. Development 135(14):2467–2477
Heinold A, Hanebeck B, Daniel V, Heyder J, Tran TH, Dohler B, Greil J, Muller FM (2010) Pitfalls of “hyper”-IgM syndrome: a new CD40 ligand mutation in the presence of low IgM levels. A case report and a critical review of the literature. Infection 38(6):491–496
Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, Kroczek RA (1998) CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells. Nature 391(6667):591–594
Hsu HC, Ema H, Osawa M, Nakamura Y, Suda T, Nakauchi H (2000) Hematopoietic stem cells express Tie-2 receptor in the murine fetal liver. Blood 96(12):3757–3762
Inwald DP, McDowall A, Peters MJ, Callard RE, Klein NJ (2003) CD40 is constitutively expressed on platelets and provides a novel mechanism for platelet activation. Circ Res 92(9):1041–1048
Jelcick AS, Yuan Y, Leehy BD, Cox LC, Silveira AC, Qiu F, Schenk S, Sachs AJ, Morrison MA, Nystuen AM, DeAngelis MM, Haider NB (2011) Genetic variations strongly influence phenotypic outcome in the mouse retina. PLoS ONE 6(7):e21858
Kisanuki YY, Hammer RE, Miyazaki J, Williams SC, Richardson JA, Yanagisawa M (2001) Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev Biol 230(2):230–242
Lee PP, Fitzpatrick DR, Beard C, Jessup HK, Lehar S, Makar KW, Perez-Melgosa M, Sweetser MT, Schlissel MS, Nguyen S, Cherry SR, Tsai JH, Tucker SM, Weaver WM, Kelso A, Jaenisch R, Wilson CB (2001) A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity 15(5):763–774
Levy J, Espanol-Boren T, Thomas C, Fischer A, Tovo P, Bordigoni P, Resnick I, Fasth A, Baer M, Gomez L, Sanders EA, Tabone MD, Plantaz D, Etzioni A, Monafo V, Abinun M, Hammarstrom L, Abrahamsen T, Jones A, Finn A, Klemola T, DeVries E, Sanal O, Peitsch MC, Notarangelo LD (1997) Clinical spectrum of X-linked hyper-IgM syndrome. J Pediatr 131(1 Pt 1):47–54
Li C, Li J, Li Y, Lang S, Yougbare I, Zhu G, Chen P, Ni H (2012) Crosstalk between platelets and the immune system: old systems with new discoveries. Adv Hematol 2012:384685
Lievens D, Zernecke A, Seijkens T, Soehnlein O, Beckers L, Munnix IC, Wijnands E, Goossens P, van Kruchten R, Thevissen L, Boon L, Flavell RA, Noelle RJ, Gerdes N, Biessen EA, Daemen MJ, Heemskerk JW, Weber C, Lutgens E (2010) Platelet CD40L mediates thrombotic and inflammatory processes in atherosclerosis. Blood 116(20):4317–4327
Mach F, Schonbeck U, Sukhova GK, Bourcier T, Bonnefoy JY, Pober JS, Libby P (1997) Functional CD40 ligand is expressed on human vascular endothelial cells, smooth muscle cells, and macrophages: implications for CD40-CD40 ligand signaling in atherosclerosis. Proc Natl Acad Sci USA 94(5):1931–1936
Montagutelli X (2000) Effect of the genetic background on the phenotype of mouse mutations. J Am Soc Nephrol 11(Suppl 16):S101–S105
Nowakowski A, Alonso-Martin S, Arias-Salgado EG, Fernandez D, Vilar M, Ayuso MS, Parrilla R (2011) Megakaryocyte gene targeting mediated by restricted expression of recombinase Cre. Thromb Haemost 105(1):138–144
Nurden AT (2011) Platelets, inflammation and tissue regeneration. Thromb Haemost 105(Suppl 1):S13–S33
Pericacho M, Alonso-Martin S, Larrucea S, Gonzalez-Manchon C, Fernandez D, Sanchez I, Ayuso MS, Parrilla R (2011) Diminished thrombogenic responses by deletion of the Podocalyxin gene in mouse megakaryocytes. PLoS ONE 6(10):e26025
Prasad KS, Andre P, He M, Bao M, Manganello J, Phillips DR (2003) Soluble CD40 ligand induces beta3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling. Proc Natl Acad Sci USA 100(21):12367–12371
Reul RM, Fang JC, Denton MD, Geehan C, Long C, Mitchell RN, Ganz P, Briscoe DM (1997) CD40 and CD40 ligand (CD154) are coexpressed on microvessels in vivo in human cardiac allograft rejection. Transplantation 64(12):1765–1774
Schonbeck U, Mach F, Libby P (2000) CD154 (CD40 ligand). Int J Biochem Cell Biol 32(7):687–693
Seijkens T, Engel D, Tjwa M, Lutgens E (2010) The role of CD154 in haematopoietic development. Thromb Haemost 104(4):693–701
Semple JW, Italiano JE Jr, Freedman J (2011) Platelets and the immune continuum. Nat Rev Immunol 11(4):264–274
Shimadzu M, Nunoi H, Terasaki H, Ninomiya R, Iwata M, Kanegasaka S, Matsuda I (1995) Structural organization of the gene for CD40 ligand: molecular analysis for diagnosis of X-linked hyper-IgM syndrome. Biochim Biophys Acta 1260(1):67–72
Tiedt R, Schomber T, Hao-Shen H, Skoda RC (2007) Pf4-Cre transgenic mice allow the generation of lineage-restricted gene knockouts for studying megakaryocyte and platelet function in vivo. Blood 109(4):1503–1506
Tousoulis D, Androulakis E, Papageorgiou N, Briasoulis A, Siasos G, Antoniades C, Stefanadis C (2010) From atherosclerosis to acute coronary syndromes: the role of soluble CD40 ligand. Trends Cardiovasc Med 20:153–164
Turlo KA, Gallaher SD, Vora R, Laski FA, Iruela-Arispe ML (2010) When Cre-mediated recombination in mice does not result in protein loss. Genetics 186(3):959–967
Wagner AH, Guldenzoph B, Lienenluke B, Hecker M (2004) CD154/CD40-mediated expression of CD154 in endothelial cells: consequences for endothelial cell-monocyte interaction. Arterioscl Thromb Vasc Biol 24(4):715–720
Wolf D, Jehle F, Ortiz Rodriguez A, Dufner B, Hoppe N, Colberg C, Lozhkin A, Bassler N, Rupprecht B, Wiedemann A, Hilgendorf I, Stachon P, Willecke F, Febbraio M, von zur Muhlen C, Binder CJ, Bode C, Zirlik A, Peter K (2012) CD40L deficiency attenuates diet-induced adipose tissue inflammation by impairing immune cell accumulation and production of pathogenic IgG-antibodies. PLoS ONE 7(3):e33026
Xu J, Foy TM, Laman JD, Elliott EA, Dunn JJ, Waldschmidt TJ, Elsemore J, Noelle RJ, Flavell RA (1994) Mice deficient for the CD40 ligand. Immunity 1(5):423–431
Zhu X, Chung I, O’Gorman MR, Scholl PR (2001) Coexpression of normal and mutated CD40 ligand with deletion of a putative RNA lariat branchpoint sequence in X-linked hyper-IgM syndrome. Clin Immunol 99(3):334–339
Acknowledgments
CIBER de Enfermedades Raras (CIBERER) is an initiative of the Spanish Health Institute Carlos III (ISCIII). The work was funded with grants SAF2007-61701 and BFU2010-15237 from the Spanish Plan of Research & Development, and grant PIE 201020E018 from CSIC.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Horrillo, A., Fontela, T., Arias-Salgado, E.G. et al. Generation of mice with conditional ablation of the Cd40lg gene: new insights on the role of CD40L. Transgenic Res 23, 53–66 (2014). https://doi.org/10.1007/s11248-013-9743-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-013-9743-2