MRI is an excellent imaging modality for both clinical applications as well as basic biomedical research. Recently, it has been shown that in vivo MRI can monitor cell migration and observe cellular biological processes when appropriate contrast agents are used.17-23
We have used a novel rat model and cellular MRI of labeled macrophages to study CR, which continues to be the major cause of graft loss and contributes to the morbidity and mortality in organ transplant patients. The ability to noninvasively track cells in vivo can lead to a better understanding of the complex immune mechanisms involved in chronic cardiac allograft rejection.
Iron oxide— based contrast agents, such as USPIO, SPIO, and MPIO, have been used to label and track cells in vivo by MRI. The in vivo cell labeling efficiency, by direct intravenous injection, is low because of particle dilution and accessibility. In the case of smaller particles, such as USPIO and SPIO, cells must ingest thousands of particles to create a local magnetic field gradient that is detectable by T2
*-weighted MRI, especially at the level of a single cell. Each MPIO, however, can contain picogram quantities of iron, and therefore 1 or only a few MPIO particles loaded into a cell are sufficient for MRI detection.20-23
Electron micrograph studies have shown that macrophages concentrate MPIO within membrane-bound vesicles.22,23
The macrophage then becomes the basic unit of contrast, and because the superparamagnetic iron center can propagate a magnetic field gradient as much as 50 times its radius, individual cells can be easily detected with the imaging parameters used here.22,23
As well, it has been reported that single iron oxide—labeled cells can be detected at 1.5 T, and thus clinical translation of cellular tracking studies with appropriate conditions should be possible.26
In this study, cells were tracked in a rat model of CR by in vivo MRI at 4.7 T for >100 days after a single intravenous injection of MPIO to the recipient 24 hours before transplantation. We have found that the blood half-life of the 0.9-μ
m MPIO is short (<2 minutes) in rats, suggesting that the MPIO particles are immediately taken up by the reticuloendothelial system, including macrophages. Thus, no free particles should be circulating at the time of transplantation or at later time points. Ex vivo cellular labeling studies have also shown that macrophages phagocytose or endocytose the MPIO particles more readily than other cells types, such as B cells and T cells, and thus our in vivo labeling strategy is likely selective for macrophages.22,23
Although we cannot rule out nonmacrophage labeling, the histological correlation of iron staining and ED1-positive cells as well as our previous double fluorescence studies using an acute rejection model in rats22
supports our findings of recipient macrophage trafficking and accumulation in the graft experiencing CR. Consistent with previous results that the 0.9-μ
m MPIO particles do not affect cell function and proliferation, they do not appear to have any long-term toxic effects on the organism.20-23
The present study clearly demonstrates the effectiveness of in vivo macrophage labeling with MPIO for long-term longitudinal cell tracking studies by MRI.
Immunosuppressive therapy used in organ transplantation curtails acute rejection quite effectively but does not prevent CR in either humans or animals. Mononuclear cell infiltration at later stages of CR is primarily presented by macrophages.9-13
This suggests that macrophages are not only involved with acute rejection but can also participate in the promotion of CR. In our rat model of CR, MPIO-labeled macrophages are observed accumulating in the grafted heart as early as 7 days after transplantation. Serial in vivo MRI shows more accumulation as CR develops. We see some labeled-macrophage accumulation in the isograft controls early on; however, the number of labeled cells found in the isografts does not increase over time and is sparse at later time points. The early accumulation in the isograft may be in response to the transient ischemia/reperfusion injury. A few hypointensity contrast spots are seen in high-resolution MR microscopy images of the native hearts after MPIO injection. This may reflect MPIO taken up by resident-tissue macrophages.27
This mechanism is not likely responsible for MPIO accumulation in our transplant animals because the MPIO injection is 24 hours before surgery and the blood clearance of the MPIO is rapid.
Allograft hearts had fewer cells that were positive for both RT1.Aa and ED1 markers than ED1-positive only cells, suggesting that some of the ED1-positive cells may be of donor origin. Alternatively, anti-rat RT1.Aa,b,l-FITC mAb used to identify recipient cells may be less sensitive for immunohistochemical staining. Further investigation of the distribution of these cell populations will help us to understand the role they play in allograft CR. Studies of this nature could identify new targets for immunotherapy in organ transplantation.
The hallmark of CR in heart allograft is arteriosclerosis characterized by diffuse, concentric intimal thickening, resulting in narrowing and ultimate luminal occlusion of the arteries of the graft.3-5
Studies have indicated that vascular narrowing is primarily caused by the proliferation of smooth muscle cells, and recipient monocyte/macrophages seen in the vessel wall at the initial stage of chronic rejection may contribute to the luminal occlusion process.8-11
This study shows that recipient macrophages migrate to the transplanted heart at the early stages of CR and accumulate over time. This finding is in agreement with previous reports.10-13
These recipient macrophages may be involved in an initial inflammatory response that promotes the development of CR by releasing platelet-derived growth factor to prompt smooth muscle cell proliferation, producing tumor necrosis factor-α
and interleukin-1 to promote inflammation and presenting transforming growth factor-β
and matrix metalloprotease to induce fibrosis.8-11
Early macrophage infiltration correlates with a poor prognosis, and persistence of macrophage accumulation after resolution of acute rejection episodes is predictive of CR after transplantation.12,13
Thus, monitoring macrophage activities with the use of in vivo MRI provides a novel methodology for studying the mechanisms of CR progression. This technique may potentially lead to a clinical tool for diagnosing CR, managing treatment, and predicting outcomes after organ transplantation.
In summary, our results show the following: (1) the infiltration of MPIO-labeled recipient immune cells, mainly macrophages, can be detected by in vivo MRI for >100 days; (2) it is primarily the macrophages that contained the MPIO particles, as confirmed by histology and fluorescence microscopy; (3) the biventricular working-heart CR model is a good rat model to mimic the clinical situation for the purpose of studying the migration of recipient cells as well as the process of ongoing recipient-donor alloimmune interaction in the cardiac allograft undergoing CR; and (4) recipient macrophage cells are present at the early stages of CR and remain in the graft for an extended period of time.
In conclusion, our approach of tracking immune cells noninvasively by in vivo MRI has great potential to further our understanding of the cellular mechanisms involved in CR. Moreover, this study demonstrates the feasibility of noninvasively observing individual targeted cells over long periods of time with the use of in vivo MRI.
Numerous studies have shown that immune cells including macrophages play crucial roles in the development of organ rejection. The ability to monitor the migration and localization of specific cell types in vivo, noninvasively, and in real time will greatly improve our understanding of the complex roles that different cells play in cardiac allograft rejection. This study presents the feasibility of imaging individual recipient macrophages in vivo by magnetic resonance imaging over long periods of time in a rodent heterotopic working-heart transplantation model with the use of a sensitive contrast agent, micrometer-sized paramagnetic iron oxide particles. In this study, recipient cells, mainly macrophages, have been labeled in vivo by direct intravenous administration of micrometer-sized paramagnetic iron oxide particles before heart transplantation. This cell-labeling procedure is convenient for clinical application. Thus, this approach provides a novel methodology for studying the mechanisms of cardiac allograft rejection in both animals and humans. Moreover, the current gold standard for diagnosing and staging rejection after organ transplantation is biopsy, which is not only invasive but also prone to sampling errors because rejection sites are highly heterogeneous. The activated macrophages have been found to be the primary cells in the cellular infiltrate of rejecting grafts. Thus, this imaging modality using magnetic resonance imaging to monitor cell migration in real time, with whole-heart visualization of cellular infiltration, could potentially lead to a powerful clinical tool, providing information for noninvasive evaluation of graft rejection, managing treatment, and predicting outcomes after heart transplantation.