Research ArticleImmunosuppression in cardiac graft rejection: A human in vitro model to study the potential use of new immunomodulatory drugs
Introduction
Long-term success after cardiac allograft transplantation is limited by chronic inflammatory processes, despite prolonged immunosuppression [1]. Current immunosuppressive regimens have little or no impact on the long-term rate of graft loss and, indeed, may themselves contribute to chronic graft rejection [2]. The goal to attenuate inflammation has focused the attention on chemokines, a family of chemotactic peptides that provide signals (acting via specific G protein-coupled receptors) for activation and recruitment of effector cells at inflammation sites [3].
Allograft rejection, a T helper 1 (Th1)-type-mediated immune response, is characterized by the presence of CXCR3-bearing T cells and by the expression of CXC chemokines inducible by interferon (IFN) γ, such as CXCL9, CXCL10, CXCL11 [4]. Among chemokines, CXCL10 plays a pivotal role during cardiac rejection both in vitro and in vivo [5], [6], [7], [8]; it is the first CXCR3 ligand detected after allografting and the only chemokine induced by isografting [9]. In particular, donor-derived CXCL10 seems to play a critical role in initiating alloresponses [9]. Since CXCL10–CXCR3 axis emerged as the most important one in cardiac allograft rejection [9], [10], interfering with CXCR3 or CXCL10 production might considerably reduce the inflammatory process [3]. The immune response mechanisms involved in allograft injury are still to be fully understood. Whereas the participation of endothelial cells or alloreactive T cells, is well documented [11], [12], the role of graft resident cells still remains to be elucidated. Since CXCL10 appears the main mediator of allogen-specific graft infiltration [11], our study was performed to further investigate the association between this chemokine and cardiac cells. Indeed, clarifying CXCL10 source(s) and pathway(s) would be very advantageous to design therapeutic strategies, targeted to abrogate inflammatory processes and to reduce harmful side effects. Actually, readily available cultures of cardiomyocytes would be a valuable tool for allograft rejection research. Studies at cellular level have been often hampered by a lack of technique for isolating and culturing cardiac cells suitable for electrical, biochemical and signalling assays. Thus, we first aimed to establish and characterize reliable cultures of functional human cardiomyocytes; next, we assessed the response of isolated cardiomyocytes to IFNγ and TNFα, in terms of CXCL10 protein secretion; last, we screened the effect, if any, of different immunomodulatory agents, such as micophenolate mofetil (MMF), tacrolimus (FK-506), sirolimus (Sir), methilprednisolone (MeP), and cyclosporin A (CsA) [1], onto cardiomyocytes. In addition, we utilized a peroxisome proliferator-activated receptor γ (PPARγ) agonist, rosiglitazone (RGZ), a drug currently used for type 2 diabetes treatment [13] and recently used also for the treatment of post-transplant diabetes mellitus [14], since it has been demonstrated its ability to modulate Th1-mediated inflammatory responses [15].
Section snippets
Chemicals
Dulbecco Modified Eagle Medium (DMEM)/Ham's F-12 medium (1:1) with and without phenol red, phosphate buffered saline Ca2+/Mg2+-free (PBS), bovine serum albumin (BSA) fraction V, glutamine, antibiotics, NaOH, absolute ethanol, EDTA-trypsin solution, Bradford reagent, rabbit anti-myosin Iβ antibody (Ab), mouse anti-α-smooth muscle actin monoclonal Ab (mAb) (α-SMA, clone 1A4), mouse anti-connexin 43 (Cx43) mAb (clone CXN-6) and all reagents for western blot and for electrophysiological experiments
Cell morphology and lifespan
Apparent morphological changes in time were observed in culture. Isolated round-shaped cells began to attach and grow as monolayer within 24–48 h from seeding. First adherent cells exhibited a round-shaped morphology within 2nd–3rd passage (p), as examined by phase contrast microscopy (Fig. 1A, left panel, p1/2). At the later passages the cells acquired a spindle-shape fibroblast like morphology (Fig. 1A, middle panel, p5/6). A more rodshaped, adult-like, phenotype was observed along with time (
Discussion
Human cardiac cells secreted large amount of CXCL10 protein in response to proinflammatory Th1 cytokines. In Hfcm, IFNγ and TNFα, in addition to the independent activation of Stat1 and NF-kB pathways, exerted a synergistic effect on CXCL10 protein secretion, associated to a significant up-regulation of IFNγR driven by TNFα. MPA and RGZ reduced more than 50% Th1 cytokine-induced CXCL10 secretion by Hfcm, differently from the other current immunosuppressive agents.
Human cardiomyocytes in vitro
Acknowledgments
This research was supported by TRESOR (Tuscany REgional Study On Rosiglitazone).
The authors wish to thank Prof. Paola Romagnani, Center for Research Transfer and High Education “DENOthe”, University of Florence, Italy, for her suggestions; Dr. Linda Vignozzi, Dept. Clinical Pathophysiology, Unit of Andrology, University of Florence, Italy, for her help with sigmoid curves analysis.
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