VIEWPOINT
The Dying Stem Cell Hypothesis
Immune Modulation as a Novel Mechanism for Progenitor Cell Therapy in Cardiac Muscle
Thomas Thum, MD*, ,*,
Johann Bauersachs, MD,
Philip A. Poole-Wilson, MD, FRCP*,
Hans-Dieter Volk, MD, PhD and
Stefan D. Anker, MD, PhD*, ,*
* Department of Clinical Cardiology, National Heart and Lung Institute, Imperial College of Medicine, London, United Kingdom
Medizinische Klinik I, Universitätsklinikum, Bayerische Julius-Maximilians-Universität, Würzburg, Germany
Institute of Medical Immunology, Charité Campus Mitte, Berlin, Germany
Applied Cachexia Research, Department of Cardiology, Charité Campus Virchow-Klinikum, Berlin, Germany
Manuscript received June 3, 2005;
revised manuscript received July 1, 2005,
accepted July 12, 2005.
* Reprint requests and correspondence: Dr. Thomas Thum, Julius-Maximilians University, Cardiology, Josef-Schneider Str. 2, Würzburg, Bavaria 97080, Germany. (Email: thum_t{at}klinik.uni-wuerzburg.de). * Prof. Stefan Anker, National Heart and Lung Institute, Department of Clinical Cardiology, Dovehouse Street, London SW3 6LY, United Kingdom. (Email: s.anker{at}imperial.ac.uk).
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Abstract
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Stem cell transplantation after myocardial infarction has been claimed to restore cardiac function, but the underlying mechanism remains unclear. A minority of transplanted cells become adherent in heart tissue and contribute to neovascularization, whereas many donor cells die from apoptosis. We propose that apoptosis of transplanted cells modulates local tissue reactions. Apoptotic cells impact on immune reactivity by down-regulating innate and adaptive immunity, deactivating macrophages and dendritic cells, and stimulating regulatory T cells. This leads to reduced scar formation, repressed myocardial apoptosis, and improved cardiac outcome.
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Abbreviations and Acronyms
| | AMI = acute myocardial infarction | | IL = interleukin | | PS = phosphatidylserine | | TGF = transforming growth factor | | TLR = Toll-like receptor |
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Acute myocardial infarction (AMI) often leads to congestive heart failure and is one of the most frequent causes of hospital stays in developed countries (1). Despite pharmacological treatment and mechanical revascularization techniques, there is no effective therapy to replace infarcted myocardium. Transplantation of autologous stem cells isolated from peripheral blood or bone marrow into the site of cardiac ischemia has been claimed to improve cardiac function (reviewed in von Harsdorf et al. [2]). It is not known whether such benefit is mediated by the transplanted cells themselves, recruitment of resident cardiac stem cells (3), or by activation of, as yet, unidentified paracrine or immunologic mechanisms. The low number of transplanted cells detectable in heart tissue makes a direct mechanism unlikely.
Acute immune activation plays a role in most acute clinical manifestations of coronary atherosclerosis, and AMI is associated with local and systemic inflammation (4,5). After AMI, inflammatory cytokines contribute to myocardial apoptosis, necrosis, and scar formation, leading to the development of heart failure (6). In humans, apoptosis after myocardial infarction occurs mainly in the border zones or even in the remote areas of ischemia (7,8); however, necrosis is the predominant mode of cell death in the infarcted myocardium and might be primarily responsible for triggering the inflammatory response (9).
In previous clinical trials, in the context of AMI, stem cells from peripheral blood or whole bone marrow were isolated from patients and were either injected into the coronary vessel of the infarct area (2,10,11) or directly into the ischemic heart muscle (2,12). The viability of cells before injection was in the range of 75% to 95% (2,10–12), which means that a proportion of injected cells were already apoptotic or necrotic at the time of injection (or transplantation). A further proportion of healthy injected living cells likely become apoptotic within heart tissue because of exposure to various proapoptotic or cytotoxic factors in an ischemic environment (13).
Apoptotic cells are able to regulate local immune reactions, and the effect is amplified after AMI (4,14). The effects of exogenous transplanted apoptotic cells into areas of AMI on immunity, neovascularization, and cardiac outcome are unknown.
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The dying stem cell hypothesis
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We propose that improved cardiac function after application of stem cells to patients with AMI can be explained by the modulation of local immune reactions in response to apoptosis of transplanted cells (Fig. 1).
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Figure 1 (A) Immune pathophysiology of myocardial injury. Ischemic injury damages parenchymal cells, resulting in exposition of heat-shock proteins (HSPs) that trigger tissue-resident macrophages and immature dendritic cells (DCs) via Toll-like receptor 4 (TLR-4) activation. Activated macrophages release several inflammatory cytokines, further amplifying tissue injury, whereas immature DCs undergo maturation and migration to secondary lymphoid tissues. After DC–T-cell interaction, HSP-specific T cells undergo clonal expansion and effector–T-cell differentiation. Homing of these cells to the injured tissue further amplifies local inflammation. (B) Apoptotic cells interact with the immune pathophysiology of myocardial injury. Transplanted, ex vivo, induced apoptotic cells or cells undergoing in vivo massive apoptosis inhibit macrophages and DCs via interaction of their surface phosphatidylserine with the respective receptors (PS-R) on the immune cells. As a result, local release of anti-inflammatory cytokines like transforming growth factor (TGF)-beta and interleukin (IL)-10 rises (14,20), maturation and migration of DCs and T helper type 1 (Th1) activation is inhibited (16,17), and activation of regulatory T cells (Treg) is enhanced (18). Consequently, less myocardial inflammation is observed, resulting in less myocardial apoptosis and scar formation as well as enhanced angiogenesis.
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Delivery of "danger" signals via Toll-like receptor (TLR) activation plays a key role in activating both innate (activation of phagocytes) and adaptive (activation of antigen-presenting cells, particularly dendritic cells) immunity (15). It is well established that microbial products, like endotoxin, bacterial CpG-rich deoxyribonucleic acid, viral ribonucleic acid, among others, trigger a broad set of TLRs. Endogenous ligands (e.g., heat-shock proteins) are released by stressed and necrotic cells after ischemia/reperfusion injury and stimulate, particularly, TLR-4, resulting in local inflammation. In addition, tissue-resident immature antigen-presenting cells are triggered, via TLR-4, to mature and migrate to secondary lymphoid organs, where they stimulate the clonal expansion and differentiation of effector T cells (16). Homing of activated T cells into the injured tissue further amplifies local inflammation (Fig. 1A). In contrast to stressed and necrotic cells, apoptotic cells inhibit inflammation, as detailed in Figure 1B (17,18). Recognition of apoptotic cells by professional phagocytes (macrophages and dendritic cells) requires exposure of phosphatidylserine (PS), a phospholipid normally limited to the inner leaflet of the plasma membrane bilayer, on the surface of the apoptotic cell and ligation by the PS receptor of the phagocyte (Fig. 1). Apoptotic cell ingestion by macrophages induces expression of the anti-inflammatory cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-beta. The increased expression of anti-inflammatory cytokines by macrophages phagocytosing apoptotic cells might suppress synthesis of pro-inflammatory mediators (e.g., tumor necrosis factor [TNF]-alpha, IL-1-beta, and IL-6) in the infarcted heart, resulting in accelerated resolution of the inflammatory response. Apoptotic cells are also recognized by immature dendritic cells, which are present in all tissues, including heart (16,19). This might additionally lead to enhanced tissue-specific production of anti-inflammatory cytokines and down-regulation of inflammatory mediators, as detailed in Figure 1.
A variety of inflammatory cytokines, including IL-6, enhance scar formation after AMI (6). Apoptotic transplanted cells could change, at least in part, the immune status of the infarcted myocardium from inflammation to an anti-inflammatory state (20). Recruited inflammatory cells also undergo apoptotic cell death, and their phagocytic elimination might play a role in resolution of inflammation by promoting secretion of anti-inflammatory cytokines such as TGF-beta or IL-10 (21). Apoptosis occurs mainly at the border zones of ischemia after myocardial infarction (7,8); injection of cells directly to the site of ischemia would significantly increase the amount of cells that, in turn, could become apoptotic. Such modulation of the local inflammatory response might suppress inflammatory injury, prevent expansion of the inflammatory infiltrate, and might, in part, explain the beneficial effects of cell transplantation in patients with AMI. In contrast, transplantation of a significant amount of necrotic cells would lead to an acceleration of inflammation in the infarcted heart. This problem could further be exaggerated when dead or necrotic cells enter or are transplanted to adjacent normal myocardium. This must be avoided in future clinical trials, and testing cells for necrosis should be a prerequisite before transplantation.
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Support of the hypothesis
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Autologous blood exposed ex vivo to oxidative stress to induce cell apoptosis and administered intramuscularly decreases the production of inflammatory mediators, increases anti-inflammatory cytokines, and decreases cellular injury (22–24). Immune modulation therapy has been shown to be safe and led to significantly improved vascular endothelial function in the first human trials in peripheral arterial disease (23). In a pilot study in patients with advanced chronic heart failure, immune modulation therapy significantly reduced risk of death and hospitalization (25). Pre-treatment of spontaneously hypertensive rats with apoptotic cells reduced severe renal ischemia reperfusion injury (26). Direct instillation of apoptotic cells enhanced the resolution of acute inflammation in the lung (27). In this study, apoptotic cells with externally exposed PS induced TGF-beta1 secretion, resulting in an accelerated resolution of inflammation. This in vivo study nicely demonstrates the proposed concept of anti-inflammatory actions of apoptotic cells.
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Implications of the hypothesis
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If our hypothesis was true, the cell type of stem cells transplanted to the infarcted myocardium would play only a minor role for improved cardiac function. The local immune responses to implanted cells might be of greater importance. Anti-inflammatory and cytoprotective signaling mechanisms, in response to transplanted apoptotic cells or cells becoming apoptotic soon after transplantation, might explain the advantageous cardiac outcome after stem cell or bone marrow transplantation after AMI. Transplantation to areas of infarction of any cells (e.g., blood leukocytes) triggered ex vivo to undergo apoptosis could lead to a better cardiac outcome.
If the beneficial effects of progenitor cell therapy are due to PS-mediated deactivation of macrophages and dendritic cells, drugs that engage the PS receptors might accelerate resolution of inflammation, decreasing injury in the infarcted myocardium. That would negate the need to transplant cells at all.
The hypothesis might be applicable to any cell transplantation therapy that applies cells to immunologically active tissue.
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Testing the hypothesis
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We suggest transplanting apoptotic progenitor cells after modification by ex vivo exposure to specific physicochemical stressors, including oxygen, ultraviolet light, and elevated temperature into areas of ischemia in an animal model of AMI. For comparison, healthy endothelial progenitor cells and ex vivo stressed autologous whole blood should be used to investigate whether the beneficial effects can also be detected with cells other than progenitor cells. The cardiac function and outcome should be measured in the different treatment groups over time. Mechanistically, the use of inhibitory antibodies against potentially involved anti-inflammatory mediators, such as the cytokines TGF-beta or IL-10, could help to gain in-depth molecular insight into the suppression of inflammation by apoptosis.
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Footnotes
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Dr. Thum is supported by the Ernst und Berta Grimmke-Stiftung. Dr. Bauersachs is supported by the IZKF Würzburg (D22). Dr. Anker is supported by a Vanderville fellowship and a donation from Dr. Bailey. The Department of Clinical Cardiology (Imperial College London) is supported by the British Heart Foundation.
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Abstract
The dying stem cell...