Introduction
Organ transplantation is an effective treatment strategy for end-stage organ failure. Despite improvements in immunosuppressive strategies, allograft loss due to acute rejection remains a significant problem. Clinical detection of graft injury and rejection by repeated tissue biopsy is associated with certain risks (1,2). Therefore, it is imperative to develop non-invasive approaches for in vivo imaging to facilitate the diagnosis and prognosis by evaluating the immune status and function of transplanted organs. Nuclear medical imaging is a reliable and highly sensitive technique allowing deep tissue penetration (3,4). Currently, 18F-fluorodeoxyglucose (18F-FDG) is the predominant radiolabel used to visualize the metabolic activity of immune cells. Previous studies have evaluated 18F-FDG in the assessment of acute allograft rejection and the efficacy of immunosuppressive treatment (5,6). Grabner et al (7) reported that 18F-FDG-labeled T lymphocytes accumulated in rat kidney allografts undergoing acute rejection.
As immunosuppressive treatments inhibit or prevent the proliferation and/or function of immune cells, including T cells, B cells and macrophages (8–10), the present study hypothesizes that the reliability of 18F-FDG is impaired following organ transplantation. The aim of administering immunosuppressants is to impair the metabolic activity of effector cells, which results in decreased 18F-FDG uptake. This was demonstrated in our previous research, which revealed that the immunosuppressant rapamycin significantly reduced 18F-FDG uptake and prevented imaging of the graft (11). As an inhibitor of the intracellular kinase, mammalian target of rapamycin (mTOR), rapamycin is an immunosuppressive agent commonly administered to transplant recipients. Rapamycin exerts various effects on target cells, which include plasmacytoid dendritic cells (12), effector T cells (13), regulatory T cells (14,15), B cells (9) and sensitive tumor cell lines (16). One consequence of mTOR inhibition is glycometabolism reduction (17,18). In addition, mTOR inhibition blocks the proliferation, activation and trafficking of lymphocytes and inhibits the production of cytotoxic antibodies. Finlay et al (19) demonstrated that the phosphoinositide 3-kinase-mTOR signaling pathways determined the expression of key lymph node homing receptors (CD62 L and CCR7) and regulated lymphocyte trafficking. Therefore, whether 18F-FDG and 18F-FDG-labeled immune cells are suitable for use in the imaging of rapamycin-treated allografts remains to be elucidated.
In the present study, 18F-FDG-labeled splenocytes (with/without rapamycin treatment: FR and FC, respectively) were investigated for their potential application in allograft in vivo imaging. 18F-FDG-labeled splenocytes were transferred into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice that had previously received skin allografts. Tracking of transferred cells was conducted using whole-body phosphor-autoradiography. The aim of this study was to evaluate whether 18F-FDG-labeled splenocyte accumulation is affected by rapamycin treatment.
Materials and methods
Ethical approval
The animal protocol was reviewed and approved by the Institutional Animal Care and Use Committee at the School of Medicine, Shandong University (Jinan, China).
Animal model
Female NOD/LtSz-PrKdcscid (NOD/SCID; H-2d; n=10) mice, BALB/c (H-2d) mice and C57BL/6 (H-2b; n=40) mice aged 6–8 weeks (weight, 18±2 g) were bred and maintained under defined flora conditions in individually ventilated (HEPA-filtered air) sterile cages (19 days; humidity, 50–60%). All mice were maintained under a 12-h light/dark cycle with access to standard mice chow and sterilized water ad libitum. Experiments were performed in accordance with national animal protection guidelines. Full-thickness skin grafts from donor C57BL/6 mice were harvested and transplanted onto the prepared graft beds on the right shoulders of recipient NOD/SCID mice (20). Surgeries were performed in a sterile environment under anesthesia with 0.6% pentobarbital sodium (0.1 ml/10 g body weight) by intraperitoneal (i.p.) injection. Buprenorphine (Reckitt Benckiser Pharmaceuticals, Richmond, Virginia, USA) was administered (0.1 mg/kg/BW i.p.) for 3 days after surgery to control wound pain. The wounds were then dressed. Bandages were removed 14 days later and the recipients with well-healed grafts were selected as the experimental subjects for imaging at 30 days post-transplantation (n=20 per group).
Cell isolation and labeling
Splenocytes were isolated from BALB/c mice as previously described (21). The cells were cultured in cell culture plates in the constant temperature (37°C) incubator with 10 U/ml recombinant interleukin 2 (rIL-2) or 10 U/ml rIL-2 and 100 nM rapamycin (cat. no. M1768; AbMole Bioscience, Inc., Houston, TX, USA) for 24 h. Cells were centrifuged and washed twice at 400 × g for 6 min at 4°C with fresh PBS and counted using a conventional Neubauer chamber. Splenocytes (1×107) were then incubated with 15 MBq 18F-FDG (HTA Co., Ltd., Beijing, China) in 37°C potassium-enriched physiological saline containing 100 IU/ml insulin (Sigma-Aldrich, St. Louis, MO, USA) for 30 min (22). Splenocytes were collected by centrifugation at 400 × g for 6 min at 4°C, and radioactivity in the supernatant and pelleted cells was measured with a Wipe Test/Well γ-Counter (Capintec, Inc., Ramsey, NJ, USA). Labeling efficiency was defined as the ratio of radioactivity of cells to overall radioactivity. To analyze labeling stability, 18F-FDG-labeled splenocytes (5×106) were incubated in mouse blood plasma (total volume, 400 µl; Shanghai Fanke Biotechnology Co., Ltd., Shanghai, China) for 5, 10, 30, 60 and 90 min. The ratios of radioactivity of cells to overall radioactivity (the labeling stability) were calculated. Cells were divided into two groups: 18F-FDG-labeled control (FC) cells and 18F-FDG-labeled rapamycin-treated (FR) cells.
Image acquisition: Dynamic whole-body phosphor-autoradiography
Recipient mice were injected with 1×107 FC or FR cells via the tail vein. The imaging was performed under isoflurane (Sigma-Aldrich) inhalation anesthesia (induction, 3%; maintenance, 1.5%). Anesthetized mice were placed on the storage phosphor screen plate with their backs to the plate, in subdued light. The plate was exposed for 10 min. At cessation of exposure, the plate was immediately covered with an opaque plastic sheet, then transferred to the Cyclone Plusscanner (PerkinElmer Life Sciences, Waltham, MA, USA). Dynamic in vivo whole-body phosphor-autoradiography was performed at 30, 60 and 90 min following injection of radio-labeled cells. Semi-quantitative analysis was performed by manually drawing rectangular regions of interest (n=5) within the graft at each time point. Digital light units (DLU)/mm2 were obtained using OptiQuant™ image analysis software 5.0 (PerkinElmer Life Sciences).
Histology
Portions of skin grafts were harvested, and stained with hematoxylin and eosin for histologic evaluation according to a previous study (20). The slides were visualized with an inverted microscope (DMIRB; Leica Microsystems GmbH, Wetzlar, Germany) at ×10 and ×20 magnifications. Infiltrating splenocytes were quantified in five ×20 fields.
Statistical analysis
Data are presented as means ± SD (standard deviation). Statistical analysis was performed using the paired t-test by GraphPad Prism version 5 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference. The non-parametric Spearman's rho test was used to assess the association between the accumulation of labeled splenocytes, as measured by radioactivity, and the number of splenocytes, which was assessed histologically.
Results
Mouse model and radiolabeling of splenocytes
A representative C57BL/6 skin graft on the right shoulder of a NOD/SCID mouse is presented in Fig. 1A. Although rapamycin reduces the glycometabolism function of host immune cells during the rejection response, no significant difference was identified in labeling efficiency between the FC and FR cells (Fig. 1B). In the FC cells, labeling stability was evaluated and the percentage of labeled cells slowly decreased in vitro over time, as retention of 18F-FDG in the cells declined from 92.4±1.7% at 5 min to 73.2±8.1% at 30 min, 62.6±2.0% at 60 min and 57.7±6.2% at 90 min (Fig. 1C).
In vivo imaging
Whole-body phosphor-autoradiography of allografts revealed significantly reduced radioactivity (DLU/mm2) in the transplanted skin area (allograft) of mice that received FR cells compared with those that received FC cells, at 30, 60 and 90 min (Fig. 2A). The opposite native skin in all mice exhibited consistently low radioactivity (Fig. 2B). The peak of allograft to opposite native skin ratio (AOR) was significantly lower in the FR cell group (1.29±0.02 at 60 min) compared with the FC cell group (3.29±0.17 at 30 min; P<0.001) (Fig. 2C). Whole-body phosphor-autoradiography revealed a clearly elevated radioactive signal in the allograft area 30 min following injection, which indicated a significant accumulation of FC cells in skin allografts (Fig. 3A). High-uptake organs included the brain, heart and spleen. This may be due to lymphocytes homing to the spleen and non-specific uptake of free 18F-FDG dissociated from FC cells. However, images of FR cells did not enable graft visualization at any time point (Fig. 3B). These results confirmed that the migration of FR cells to graft areas was notably impaired.
Histology
Representative images of allograft sections were stained with hematoxylin and eosin to estimate lymphocytic infiltration, and are presented in Fig. 4A. As exhibited in Fig. 4B, significant infiltration of lymphocytes was absent in the grafts of mice injected with FR cells (P<0.001). The number of splenocytes per field of view was then correlated with radioactivity of the graft (Fig. 4C), and revealed to be significantly correlated (r2= 0.887; P<0.0001).
Discussion
Currently, the lack of effective parameters to assess allograft function and rejection status limits the success of treatment. Lymphocyte migration, a crucial element during the development of the acute alloimmuneresponse, mediates the immune response during acute allograft rejection; therefore, lymphocytes present as attractive biomarkers and targets for graft imaging. Thus, the tracing of lymphocyte trafficking has been evaluated for allograft monitoring and even prediction of allograft survival (23,24). However, the use of immunosuppressants following clinical organ transplantation severely impairs the proliferation and trafficking of lymphocytes, and it therefore remains to be elucidated whether imaging of lymphocytes has clinical applications. In the present study, C57BL/6 skin was transplanted onto NOD/SCID recipient mice, which then received FC or FR BABL/c splenocytes 30 days later. NOD/SCID recipient mice, used to reduce the severity of the immunologically mediated surgical injury, are deficient in T and B lymphocytes and have impaired natural killer cells and complement function. The fully mismatched allografts do not undergo rejection; therefore, the trafficking of transferred cells could be monitored more accurately (25).
The results of the present study revealed that rapamycin had minimal impact on the uptake of 18F-FDG by naive lymphocytes. No significant difference in labeling efficiency and stability was identified between FC and FR cells. FC cell accumulation was observed in the allograft area with high radioactivity and AORs at all time points; however, FR cell accumulation in allografts was never clearly imaged. Histological analysis was performed and confirmed reduced FR cell accumulation in allografts. The present study employs molecular imaging to extend the results of a previous study in which 18F-FDG was determined to be unsuitable as a rapamycin-treated graft biotracer (11). In the present study, the accumulation of FR cells in the allograft was barely visible when compared with FC cells. Furthermore, a significant correlation was observed between allograft radioactivity and splenocyte infiltration, which was determined by histology.
There are several limitations of the present study. The rapamycin dose was selected according to a previous study, which revealed it to be effective at controlling lymphocyte trafficking (26). This was confirmed by the results of the present study; however, no alternative doses were evaluated. In addition, to avoid interference by the recipient's immune response, labeled BALB/c lymphocytes were transferred into NOD/SCID, rather than BALB/c, recipients. This may not be reflective of the physiological situation.
Despite increasing knowledge of the rejection process and tolerance induction, elements of these processes have not been extensively investigated as imaging targets. In acute rejection, the non-specific accumulation of 18F-FDG-labeled immune cells at sites of inflammation following surgery impedes the accurate assessment of the immune response (27,28). Clinically, immunosuppressive treatments have been shown to inhibit immune cell migration, which rendered postoperative detection of grafts impossible (29). Thus, the use of labeled lymphocytes in in vivo imaging remains controversial. The results of the present study suggest that splenocyte trafficking was impaired by the mTOR inhibitor, rapamycin and is therefore not a suitable method for monitoring patients receiving this particular immunosuppressant. Alternative imaging strategies are required, which will enable determination of graft function and immune status in the presence of immunosuppressive therapy.