Elsevier

Experimental Cell Research

Volume 314, Issue 6, 1 April 2008, Pages 1337-1350
Experimental Cell Research

Research Article
Immunosuppression in cardiac graft rejection: A human in vitro model to study the potential use of new immunomodulatory drugs

https://doi.org/10.1016/j.yexcr.2007.12.016Get rights and content

Abstract

CXCL10–CXCR3 axis plays a pivotal role in cardiac allograft rejection, so that targeting CXCL10 without inducing generalized immunosuppression may be of therapeutic significance in allotransplantation. Since the role of resident cells in cardiac rejection is still unclear, we aimed to establish reliable human cardiomyocyte cultures to investigate Th1 cytokine-mediated response in allograft rejection. We used human fetal cardiomyocytes (Hfcm) isolated from fetal hearts, obtained after legal abortions.

Hfcm expressed specific cardiac lineage markers, specific cardiac structural proteins, typical cardiac currents and generated ventricular action potentials.

Thus, Hfcm represent a reliable in vitro tool for allograft rejection research, since they resemble the features of mature cells. Hfcm secreted CXCL10 in response to IFNγ and TNFαα; this effect was magnified by cytokine combination. Cytokine synergy was associated to a significant TNFα-induced up-regulation of IFNγR.

The response of Hfcm to some currently used immunosuppressive drugs compared to rosiglitazone, a peroxisome proliferator-activated receptor γ agonist and Th1-mediated response inhibitor, was also evaluated. Only micophenolic acid and rosiglitazone halved CXCL10 secretion by Hfcm.

Given the pivotal role of IFNγ-induced chemokines in Th1-mediated allograft rejection, these preliminary results suggest that the combined effects of immunosuppressive agents and rosiglitazone could be potentially beneficial to patients receiving heart transplants.

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.

References (58)

  • P.F. Halloran

    Immunosuppressive drugs for kidney transplantation

    N. Engl J. Med.

    (2004)
  • E. Lazzeri et al.

    CXCR3-binding chemokines: novel multifunctional therapeutic targets

    Curr. Drug Targets Immune Endocr. Metabol. Disord.

    (2005)
  • G. Tellides

    Th1 adaptive immune responses in cardiac graft arteriosclerosis: deleterious or beneficial?

    Circulation.

    (2006)
  • A. Saiura et al.

    Detection of an up-regulation of a group of chemokine genes in murine cardiac allograft in the absence of interferon-gamma by means of DNA microarray

    Transplantation

    (2002)
  • M. Melter et al.

    Expression of the chemokine receptor CXCR3 and its ligand IP-10 during human cardiac allograft rejection

    Circulation

    (2001)
  • D.X. Zhao et al.

    Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T cell alpha chemoattractant in human cardiac allografts: association with cardiac allograft vasculopathy and acute rejection

    J. Immunol.

    (2002)
  • W.W. Hancock et al.

    Donor-derived IP-10 initiates development of acute allograft rejection

    J. Exp. Med.

    (2001)
  • W.W. Hancock et al.

    Requirement of the chemokine receptor CXCR3 for acute allograft rejection

    J. Exp. Med.

    (2000)
  • R. Klingenberg et al.

    Endothelial inducible costimulator ligand expression is increased during human cardiac allograft rejection and regulates endothelial cell-dependent allo-activation of CD8+ T cells in vitro

    Eur. J. Immunol.

    (2005)
  • J.A. Balfour et al.

    Rosiglitazone

    Drugs

    (1999)
  • G. Villanueva et al.

    Rosiglitazone therapy of posttransplant diabetes mellitus

    Transplantation

    (2005)
  • N. Marx et al.

    Peroxisome proliferator-activated receptor- activators inhibit IFNγ-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells

    J. Immunol.

    (2000)
  • C. Crescioli et al.

    Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes

    J. Endocrinol.

    (2007)
  • L. Formigli et al.

    Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell–cell contacts and relaxin treatment

    Am. J. Physiol. Cell Physiol.

    (2005)
  • G.B. Vannelli et al.

    Neuroblast long-term cell cultures from human fetal olfactory epithelium respond to odors

    J. Neurosci

    (1995)
  • L. Cosmi et al.

    CRTH2 is the most reliable marker for detection of human circulating Th2 and Tc2 cells in health and disease

    Eur. J. Immunol.

    (2000)
  • P. Romagnani et al.

    CD14+CD34low cells with stem cell phenotypic and functional features are the major source of circulating endothelial progenitors

    Circ. Res.

    (2005)
  • A. De Lean et al.

    Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose–response curves

    Am. J. Physiol.

    (1978)
  • L. Sartiani et al.

    Developmental changes in cardiomyocytes differentiated from human embryonic stem cells: a molecular and electrophysiological approach

    Stem Cells

    (2007)
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