Objectives: It has been reported that CXCR3 is related to inflammatory cell infiltration. The purpose of this study was to investigate iodine-125-labeled CXCL10, a ligand of CXCR3, as a tracer targeting CXCR3 to detect acute rejection in a mouse skin transplant model.

Materials and Methods: The isograft and allograft skin models were established with BALB/c and C57BL/6 mouse skin, respectively, as donors and BALB/c mice as recipients. We used reverse transcriptase-polymerase chain reaction and immunochemistry staining to test CXCR3 expression. 125I-labeled CXCL10 was produced with the iodogenic method. Allograft/isograft mice were examined with whole body autoradiography and ex vivo biodistribution after tail vein injection of 125I-labeled CXCL10 on day 8 posttransplant.

Results: CXCR3 expression was higher in allograft tissue than in isograft control. 125I-labeled CXCL10 was prepared with high specificity and affinity. Biodistribution results showed higher 125I-labeled CXCL10 uptake in allograft tissue. The target-to-nontarget ratio was 3.01 ± 0.25 at 24 hours, a result higher than that shown in the isograft group. Pharmacokinetic analyses of 125I-labeled CXCL10 showed that distribution half-life was 0.34 hour and the elimination half-life was 9.83 hours. Dynamic whole body autoradiography images of 125I-labeled CXCL10 showed excellent graft visualization in the allograft compared with the isograft group at all checking points, with visualization much more obvious at 12 and 24 hours.

Conclusions: These data suggest that CXCR3 is a promising imaging target for immune cell infiltration in early-stage acute rejection and 125I-labeled CXCL10 can successfully image acute rejection with good pharmacokinetics.

Key words : Acute allograft rejection, Molecular imaging, Radioiodine, Skin transplant

Introduction

With new advances in transplant medicine, more and more people with end-organ dysfunction are able to survive allograft organ transplant procedures. However, rejection after transplant can reduce graft survival. Different studies have found that the incidence of acute rejection (AR) of different organs varies between 10% and 80% in the first year.^1-4 Although many methods are available to monitor the allograft and to forecast outcomes, a specific noninvasive method for examining early-stage rejection is still urgently needed.

Compelling evidence has demonstrated that the chemokine receptor 3 (CXCR3), along with its ligands (CXCL9, CXCL10, CXCL11), plays an important role in many immune diseases, including rheumatoid arthritis, systemic lupus erythematosus, bacterial infection, and transplant rejection.5-9 Recently, much attention has been paid to investigate the role of these receptors in transplant rejection. Peripheral CXCL9 and CXCL10 have been shown to be closely related to the occurrence of transplant rejection in different organ models; however, this relation could be easily affected by immune incidences outside of the graft.^10-14

There are 3 splice variants of CXCR3 (CXCR3-A, CXCR3-B, CXCR3-alt). CXCR3-A is the major variant expressed on immune cells (natural killer cells, plasmacytoid and myeloid dendritic cells, B cells, and activated T cells) and is associated with the recruitment of immune cells.^15,16 CXCR3-B is mainly associated with its angiogenic function, whereas the function of CXCR3-alt, which coexpresses with CXCR3-A at a low level, is not clear.¹⁷

Among the 3 ligands of CXCR3, CXCL10 can be secreted from a variety of cells, such as leukocytes, activated neutrophils, eosinophils,¹⁸ monocytes, endothelial cells, epithelial cells, stromal cells (fibroblasts), and keratinocytes in response to interferon-gamma (IFN-γ).^19,20 CXCL10 is always the first to be detected when inflammation occurs. After this, immune cells are recruited and IFN-γ is secreted, which in turn causes more secretion of CXCL9, CXCL10, and CXCL11. This suggests that there is a positive feedback loop between the recruitment of immune cells and the secretion of CXCL9, CXCL10, and CXCL11 at the focus. These observations demonstrate that CXCR3 and its ligands play an important role in inflammation onset and amplification.

Acute rejection after transplant is a special kind of inflammation in which the graft is targeted. Graft injury is mainly the result of T-cell infiltration and IFN-γ production, damaging the donor graft directly or indirectly.21,22 As a positive feedback, these results provide a target closely related to AR, which makes it possible to detect AR at the stage of T-cell infiltration before the injury happens. The detection and treatment of AR at this stage are of great importance to protect the graft and to prolong survival.

To image the CXCR3 and its ligands, 131I-labeled anti-CXCL10 monoclonal antibody targeted at CXCL10 has been investigated in our earlier published study.²³ Although graft imaging was successful, because of the large molecular weight of the antibody, the pharmacokinetic results of 131I-labeled anti-CXCL10 monoclonal antibody showed that it was not a good enough option. In this study, we choose a small molecule (CXCL10; 8.5 kDa) to evaluate whether 125I-labeled CXCL10 could be a better radiotracer target to diagnose early-stage AR with allograft-infiltrated CXCR3-positive inflammatory cells.

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.

Establishment of skin graft model
Female C57BL/6 (H-2d) and BALB/c (H-2b) mice (age, 6-8 wk; weight, 18-20 g) were purchased from the Center for New Drug Evaluation of Shandong University. Full-thickness trunk skin grafts were placed using standard techniques.²⁴ After death, skin samples from C57BL/6 (allograft group) and BALB/c (isograft group) mice were obtained, cut into 0.5-cm² pieces, and stored in sterile phosphate-buffered saline (PBS). After anesthetization with intraperitoneal injection of 0.6% pentobarbital sodium 0.1 mL/10 g body weight, recipient mice (BALB/c) were shaved around the chest and abdomen. After a slightly larger graft bed over the right back of the recipient mouse was prepared, the graft was immediately placed and secured with Vaseline gauze and bandage. Bandages were removed 7 days after transplant.

Reverse transcriptase-polymerase chain reaction
Mice from both the allograft and isograft groups were killed on day 9 posttransplant, and grafts underwent reverse transcriptase-polymerase chain reaction (RT-PCR). Normal skin areas of isograft mice were used as controls. The grafts were submitted to lysis using the Trizol reagent (TransGen Biotechnology, Beijing, China), and total RNA was extracted according to the manufacturer’s instructions. Total RNA (5 μg) was reverse transcribed by the EasyScript 2-step RT-PCR SuperMix Kit (TransGen Biotechnology). To quantify CXCR3 mRNA expression, the prepared cDNA was subjected to PCR by using primer for CXCR3 and glyceraldehyde 3-phosphate dehydrogenase as an internal control. The proven primers were designed and provided by Sangon Biotech Co., Ltd. (Shanghai, China). Forward and reverse primers for CXCR3 (316 bp) were 5’-CCTCCTACCTGGGCTTGTAA-3’ and 5’-GCCTCTCCCTCTTCTCACAC-3’, respectively. Cycling conditions for amplification were as follows: 4-minute denaturation step at 94℃, followed by 33 cycles of 30 seconds at 94℃, 30 seconds at 57℃, and 30 seconds at 72℃. The PCR products were analyzed on 1.5% agarose gels and stained with ethidium bromide.

Immunohistochemistry staining
Skin sections from mice at day 9 (early stage of AR) after allograft and isograft transplant underwent histologic examinations. Skin sections were stained with hematoxylin and eosin, and immunohis­tochemistry was performed using rabbit polyclonal antibody against CXCR3. Sections were examined by light microscopy at magnifications of either ×100 or ×200 and photographed.

Radiopharmaceutical preparation
Radioiodination of CXCL10 (PepProTech, Rocky Hill, NJ, USA) with 125-iodine was performed in accordance with the iodogenic method, as previously described.²⁵ Briefly, 10 μg of CXCL10 were added to 9.25 MBq (250 μCi) of 125I-labeled NaI and reacted with the help of iodogen. Radioiodinated CXCL10 was separated from free iodine using size-exclusion columns (PD-10 Sephadex G-25; GE Healthcare, Diegem, Belgium), with flow through collected in sequential fractions. The radioactivity and concen­tration were measured by a gamma counter. An aliquot of 2 μL of radiotracer was mixed in 200 μL of normal saline or 200 μL of serum for in vitro stability analysis. We used paper chromatography for experiments, with radioactivity counted by a gamma counter.

Radioligand-based binding assay
For saturation studies, 1 × 10⁶ splenocytes from BALB/c mice were incubated with increasing concentrations of 125I-labeled CXCL10 (0.022-48 nM) for 4 hours at 4℃. For competition binding, 1 × 10⁶ splenocytes were incubated with a fixed concentration of 125I-labeled CXCL10 (10 nM) for 4 hours at 4℃ in the presence of unlabeled CXCL10 (0.1-250 nM). Bound and free radioactivities were separated by centrifugation (3000× for 1 min), followed by 3 × 2-mL washes in PBS (4℃) and centrifugation. The radioactivity of bound radioligand was assayed by gamma counter.

Pharmacokinetic analyses
Microscale blood samples were taken from tail veins of 5 mice at 0.5, 1, 2, 3, 4, 7, 12, 26, 32, and 48 hours after injection of 0.19 MBq of 125I-labeled CXCL10 (5 μCi). After weights and radioactivities were measured, distribution half-life (T1/2α) and elim­ination half-life (T1/2β) were calculated.

Biodistribution of 125I-labeled CXCL10
Mice from both the allograft and isograft groups were injected intravenously with 0.19 MBq (5 μCi) of 125I-labeled CXCL10 in 200 μL of PBS. Two days before injection, 3% potassium iodide was added to the drinking water to block the thyroid gland. Animals were euthanized 24 hours after injection. Blood, grafts, normal skin samples from the opposite side, and selected tissues were retrieved and weighed, and tissue radioactivity was counted in a gamma counter. After decay correction, the percent injected dose per gram (%ID/g) was calculated.

Whole body autoradiography
Mice from both graft groups received injection of PBS solution (200 μL) with 0.19 MBq of 125I-labeled CXCL10 (5 μCi) into tail vein at day 8 after transplant. Two days before injection, 3% (wt/vol) potassium iodide was added to the drinking water to block the thyroid gland. At each time point (3, 12, and 24 h after injection), mice were imaged by whole body autoradiography. In brief, anesthetized mice were placed on the storage phosphor screen plate with their backs facing the plate in subdued light. The plate was exposed to each mouse for 20 minutes. At cessation of exposure, the plate was immediately scanned by Typhoon Trio+ (GE Healthcare-Amersham, Little Chalfont, UK; laser red 633 nm, pixel size 200 μM, phosphor mode: best sensitivity).

Statistical analyses
Results were analyzed using GraphPad Prism Software version 7.00 (GraphPad Software, Inc., La Jolla, CA, USA). Statistical analyses were performed using t tests. Differences were considered significant at P < .05 (2-sided). Results are presented as mean ± standard deviation.

Results

CXCR3 mRNA expression
Expression of CXCR3 within grafts was evaluated by RT-PCR (Figure 1). There was rare CXCR3 expression in normal skin samples. The marked increase of CXCR3 expression was detected in allografted skin on day 9, whereas there was no increase in the isografted skin.

Immunohistochemistry staining
Consistent with the RT-PCR results, immuno­histochemistry staining showed that expression of CXCR3 was much higher in the allograft than in the isograft skin samples. Hematoxylin and eosin staining of allografts showed obvious necrosis in epithelial and subcutaneous tissues. Infiltrated lymphocytes and neutrophils in the epithelium and dermis were significantly greater than those infiltrated in the isograft (Figure 2).

Radiolabeling of CXCL10
CXCL10 was successfully radiolabeled with higher labeling efficiency. Specific activity of 125I-labeled CXCL10 was 185 MBq/mg. Radiochemical purity by paper chromatography was above 98%. 125-Iodine was bound to protein after size-exclusion column purification, with purity still above 95% in both normal saline and serum at room temperature at 72 hours (Figure 3).

Radioligand-based binding assay
Specific binding of 125I-labeled CXCL10 to CXCR3 was assessed with BALB/c mouse splenocytes. The apparent dissociation constant of 125I-labeled CXCL10 was 3.48 ± 0.31 nM, and the maximum 125I-labeled CXCL10 bound by 1 × 10⁶ splenocytes was 5463 ± 130 counts/min (Figure 4A). Competitive binding assays demonstrated that 125I-labeled CXCL10 was displaced with increasing amounts of unlabeled CXCL10, with inhibition constant of unlabeled CXCL10 shown to be 3.13 ± 0.29 nM (Figure 4B). 125I-labeled CXCL10 binding to splenocytes can be blocked with excess unlabeled CXCL10, proving binding specificity.

Pharmacokinetic analyses
Pharmacokinetic analyses showed that the pharma­cokinetics of 125I-labeled CXCL10 was in accordance with the 2-compartment model. The fast α-phase of the biphasic blood clearance (T1/2α) was 0.34 hours, and the slower β-phase of the biphasic blood clearance (T1/2β) was 9.83 hours (Figure 5).

Biodistribution
To further quantify the uptake of 125I-labeled CXCL10 in different tissues, biodistribution studies were done at 24 hours in the allograft and isograft models (Figure 6). There was a prominent uptake in the allograft group, with targeted-to-nontargeted ratio of 3.01 ± 0.25 at 24 hours. In contrast, in the isograft group, the ratio was 1.14 ± 0.10 at 24 hours (P < .05). Other tissues with noticeable %ID/g were the blood and lung. All other tissues had low %ID/g results.

Whole body autoradiography
Whole body autoradiography images were obtained in allograft and isograft groups at different time points (Figure 7). A higher signal intensity was observed in the allograft group at all time points. Starting at 3 hours after injection, we observed that the allograft imaged more clearly, with contrast much more evident at 12 and 24 hours as the radiotracer eliminated quickly. There was no noticeable uptake in any of the other organs. In the isograft group, there were no apparent radioactivities located in the isograft versus that shown in normal skin on the opposite side.

Discussion

The available techniques for AR detection include graft biopsy, conventional imaging techniques, and measurement of peripheral biomarkers. Although biopsy is still the criterion standard, it is cumbersome and invasive and is not appropriate as a routine examination to monitor graft status. Traditional imaging techniques (such as magnetic resonance imaging, computed tomography, and ultrasonography) can only be used to evaluate the degree of AR through morphologic changes in subsequent stages. Peripheral biomarkers, such as CXCR3 and its ligands, can be easily detected but could be affected by whole body immune incidents.^5-9 However, specific noninvasive detection methods for AR are at early stages of development and still require great improvements.

Molecular imaging is a new interdiscipline, with images able to show the distribution of a specific radiotracer. This method makes it possible to detect a molecule where it is; however, the concept of molecular imaging involves finding an ideal target. CXCR3 is a molecule expressed mainly on activated immune cells, such as Th1 cells. Infiltration of Th1 cells is closely associated with AR initiation.^21,26 Therefore, we can use a tracer targeted at CXCR3 to detect the distribution of CXCR3-positive immune cells. In our early study, we investigated 131I-labeled CXCL10 monoclonal antibody as a target tracer of CXCL10; this method allowed us to clearly image AR. However, the allograft still imaged at 72 hours after injection; that is, it eliminated too slowly.

To improve the pharmacokinetics, here, we explored an 8.5-kDa small molecular 125I-labeled CXCL10 targeted at CXCR3. Our present results showed that 125I-labeled CXCL10 imaged earlier and eliminated more quickly. Because there are 3 ligands of CXCR3 (CXCL9, CXCL10, CXCL11), we suggest that CXCR3 would be a target more sensitive than CXCL10.

Our imaging results suggested that CXCR3 was an ideal target, showing good contrast in AR imaging. We evaluated the biodistribution of 125I-labeled CXCL10 in skin allografted and iso­grafted mouse models. The results showed specific accumulation of radioactivity in the allograft group. No other organ showed high accumulation of radioactivity except for the lung. Thus, we suggest that this technique has the potential to image AR in different organ models (kidney, heart, liver, etc.), which showed low background accumulation of radioactivity in our model. The RT-PCR and immunohistochemistry results confirmed high expression levels of CXCR3 in the rejected allograft. To further determine the specificity, we performed an in vitro radioligand-based binding assay. These results showed good specificity and high immunore­activity. The specificity, target selectivity, and graft-to-skin ratios observed suggested that 125I-labeled CXCL10 imaging could represent invasion of CXCR3-positive cells, which could be associated with AR in the allograft.

There was an interesting phenomenon: we thought that, as the biggest immune organ, there should be many CXCR3-positive immune cells in the spleen. However, in our biodistribution study, we observed no high radiotracer concentration in the spleen. We speculated that there were greater numbers of other cells that were CXCR3 negative in the spleen, which would weaken accumulation of radiotracer. Furthermore, in our study and other studies of different tracers, a high radiotracer concentration is usually shown in the lung. We suggest that this is due to the abundant blood vessels with more nonspecific radiotracer exudation.

Because 125-iodine is not a good isotope for clinical imaging, we would try to label CXCL10 with other isotopes used in the clinic and examine other allograft organ models of AR to further evaluate the potential use of CXCR3.

Conclusions

CXCR3 was a promising target of CXCR3-positive immune cell infiltration for examination of early-stage AR imaging, and 125I-labeled CXCL10 was shown to successfully image the allograft in skin AR models.

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