Short communicationXenogeneic interaction between human CD40L and porcine CD40 activates porcine endothelial cells through NF-κB signaling
Introduction
During T-cell activation, CD40L (CD154) on T cells interacts with CD40, found mainly on antigen-presenting cells (APCs). Consistent with its pivotal role in T cell activation, treatment with monoclonal anti-CD40L antibody prolongs the survival of heart, kidney, and islet allografts both in rodents and primates (Parker et al., 1995, Larsen et al., 1996, Kenyon et al., 1999, Kirk et al., 1997, Kirk et al., 1999, Cho et al., 2001, Pearson et al., 2002, Xu et al., 2002). CD40 is also detected on endothelial cells (Yellin et al., 1995, Hollenbaugh et al., 1995). Once CD40 on endothelial cells is engaged, the expression of adhesion molecules [endothelial adhesion molecule 1 (E-selectin; CD62E), intercellular adhesion molecule-1 (ICAM-1; CD54), and vascular cell adhesion molecule 1 (VCAM-1; CD106)] and chemokines [monocyte chemoattractant protein 1 (MCP-1; CCL2), interleukin-8 (IL-8; CXCL8), and regulated upon activation, normally T-expressed, and presumably secreted (RANTES; CCL5)] increases (Karmann et al., 1995, Stout and Suttles, 1996, Dechanet et al., 1997, Henn et al., 1998, Thienel et al., 1999).
Acute vascular rejection is the most important obstacle in the clinical application of xenotransplantation (Zhong, 2007, Tai et al., 2007), and its key step is the activation of PECs. If xenogeneic interactions between pCD40 on PECs and hCD40L on T cells contribute to the activation of PECs like in syngeneic or allogeneic systems (Pluvinet et al., 2004), it could play an important role in acute vascular rejection. Indeed, human CD40L+ Jurkat cells were reported to interact with CD40 on PECs and thereby stimulate adhesion molecule expression (Rushworth et al., 2000, Rushworth et al., 2001). Here, we explored this xenogeneic interaction further, to elucidate whether xenogeneic interactions between hCD40L and pCD40 can induce chemokine expression and which intracellular signal transduction pathways are involved in these processes.
In this study, we identified that xenogeneic interaction between pCD40 on miniature PECs and hCD40L on human T cells can induce the expression of various chemokines, as well as MHC and adhesion molecules through NF-κB activation.
Section snippets
Cell lines and cell culture
A miniature porcine aortic endothelial cell line (MPN3; Kim et al., 2005) and a conventional domestic porcine endothelial cell line (MYP30; Miyagawa et al., 1994) were used. The Jurkat D1.1 (ATCC no. CRL-10915) clone and the E6.1 clone (ATCC no. TIB-152) were used as human T cells. The reporting cell lines, expressing firefly (Photinus pyralis) luciferase in response to the activation of NF-κB, were constructed by delivering pHTS-NF-κB vector (Biomyx, CA) into HEK 293 (ATCC no. CRL-1573) and
Characterization of porcine CD40 and human CD40L
The sequence alignment of deduced CD40 proteins from pigs and other mammalian species was performed by using the Clustal X program (Jeanmougin et al., 1998). The sequence alignment showed that the pCD40 protein (Sus scrofa, GenBank accession no. NM214194) has over 58% identity to others, especially over 70% to hCD40 (Homo sapiens, GenBank accession no. NM001250) (Fig. 1A). The constructed CD40 gene was transfected into COS-7 cells and the CD40 expression was confirmed by immunofluorescence
Acknowledgement
This work was supported by National R&D Program Grant of The Ministry of Science and Technology in Republic of Korea (M10417060002-05N1706-00210).
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