Inflammation imaging of atherosclerosis in Apo-E-deficient mice using a (99m)Tc-labeled dual-domain cytokine ligand.

UNLABELLED: Interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) play a critical role in initiating and accelerating atherosclerosis. This study evaluated the imaging properties of (99m)Tc-TNFR2-Fc-IL-1RA ((99m)Tc-TFI), a dual-domain cytokine radioligand that targets TNF-α and IL-1ß pathways, in assessing atherosclerosis development in apolipoprotein-E-deficient (ApoE(-)(/)(-)) mice. METHODS: The feasibility and specificity of detecting atherosclerosis with (99m)Tc-TFI SPECT imaging were investigated in ApoE(-)(/)(-) and ApoE(+)(/)(+) mice. Fifty-four ApoE(-)(/)(-) mice were fed either an atherogenic diet (AGD) or a normal diet (ND) beginning at 5 weeks of age. Eighteen Apo-E wild-type (ApoE(+)(/)(+)) mice were fed an ND. Two groups of ApoE(-)(/)(-) mice (n=12 each group) on AGD and ND were imaged three times with (99m)Tc-TFI and a high-resolution SPECT system at 20-25, 30-40, and 48-52 weeks to study the evolution of atherosclerotic plaque. RESULTS: Focal radioactive accumulations in the aortic arch region were observed in the ApoE(-)(/)(-) mice (n=12) on AGD but not in the ApoE(+)(/)(+) mice on ND (n=10). Apo-E(-)(/)(-) mice on ND (n=11) exhibited lower radioactive uptake than ApoE(-)(/)(-) mice on AGD (P<0.05). Co-injection of an excess of cold ligand with (99m)Tc-TFI resulted in significant reduction of (99m)Tc-TFI uptake in the ApoE(-)(/)(-) mice on AGD. Longitudinal studies showed that (99m)Tc-TFI uptake in the aortas of ApoE(-)(/)(-) mice progressively increased from 20 to 48 weeks. Real-time PCR assays demonstrated that atherosclerotic aortas expressed significantly higher IL-1ß and TNF-α than the aortas from wild-type controls. CONCLUSIONS: Atherosclerotic plaques were detected by (99m)Tc-TFI imaging in ApoE(-)(/)(-) mice. (99m)Tc-TFI is promising for specific detection of inflammatory response in atherosclerotic plaques.

SPECT imaging of inflammatory response in ischemic-reperfused rat hearts using a 99mTc-labeled dual-domain cytokine ligand.

UNLABELLED: Soluble tumor necrosis factor (TNF) receptor-2 (TNFR2) and interleukin-1 receptor antagonist (IL-1ra) were fused to the Fc portion of IgG1 using recombinant DNA technology. The resulting dual-domain cytokine ligand, TNFR2-Fc-IL-1ra, specifically binds to TNF and to the type I IL-1 receptor (IL-1RI). This study was designed to characterize the kinetic profile of (99m)Tc-labeled TNFR2-Fc-IL-1ra (TFI) for imaging inflammatory response in an ischemic-reperfused (IR) rat heart model. METHODS: The IR model was created by ligating the left coronary artery for 45 min, followed by 2-h reperfusion. Cardiac SPECT images of TFI in the IR model (n = 6) were dynamically acquired for 3 h. Correlative data of myocardial TFI distribution versus microsphere-determined tissue blood flow were acquired in 3 extra IR hearts. Inflammation targeting affinity of TFI was compared with 2 individual cytokine radioligands, (99m)Tc-IL-1ra-Fc (IF) and (99m)Tc-TNFR2-Fc (TF) (n = 6 each group). Myocardial cytokine expression was evaluated by immunochemical assay. RESULTS: Increased TFI uptake was found in the ischemic area and correlated with the severity of ischemia. At 3 h after injection, the ratio of hot-spot accumulation in the ischemic area to a remote viable zone was 5.39 ± 1.11 for TFI, which was greater than that for IF (3.28 ± 0.81) and TF (3.29 ± 0.75) (P < 0.05). The in vivo uptake profiles of TFI, TF, and IF were consistent with ex vivo radioactive measurements and correlated with upregulated IL-1 and TNF expression. CONCLUSION: The dual-domain TFI is promising for noninvasive detection of inflammatory reactions in IR myocardium because of its more potent affinity to the inflammatory sites compared with TF and IF.

Characterization of 99mTc-labeled cytokine ligands for inflammation imaging via TNF and IL-1 pathways.

INTRODUCTION: TNFR2-Fc and IL-1ra-Fc are recombinant cytokine ligands that target TNF and IL-1. TNFR2-Fc-IL-1ra, a dual-domain agent that incorporates both ligands, allows bifunctional binding of IL-1 receptors and TNF. This study was designed to characterize (99m)Tc-labeled forms of these ligands, (99m)Tc-IL-1ra-Fc (IF), (99m)Tc-TNFR2-Fc (TF), and (99m)Tc-TNFR2-Fc-IL-1ra (TFI), for inflammation imaging. METHODS: The cytokine ligands were labeled with (99m)Tc by a direct approach via 2-iminothiolane (2-IT) reduction at various 2-IT/protein molar ratios. In vivo inflammation targeting studies were carried out in a mouse ear edema model created by topical application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on the right ear of ICR mice. RESULTS: Radiolabeling yields increased with increasing amounts of 2-IT. When the 2-IT/protein ratio reached 1000, the radiolabeling yield was greater than 90% without significant colloid production. TPA-treated ears showed high radioligand uptake, which was clearly detected by SPECT and autoradiographic imaging. The activities (%ID/g) in the inflamed and control ears at 3h after injection were 2.76 ± 0.20 vs. 0.69 ± 0.12 for IF, 5.86 ± 0.40 vs. 2.86 ± 0.61 for TF, and 7.61 ± 0.86 vs. 1.99 ± 0.31 for TFI (P<0.05 vs. controls). TFI showed significantly higher uptake in the inflamed ears compared to TF and IF (P<0.05). Blocking study results indicated specificity of radioligand binding with decreased radioactive uptake in the inflamed ears. Western blotting and ELISA analysis further confirmed a high expression of IL-1ß and TNF-α in the inflamed ears. CONCLUSIONS: (99m)Tc-labeled cytokine ligands are a promising approach for detecting and understanding the inflammatory process. TFI may be more useful than the single-domain ligands for noninvasive detection of inflammatory sites.

A (99m)Tc-labeled dual-domain cytokine ligand for imaging of inflammation.

INTRODUCTION: Interleukin (IL)-1 and IL-18 are potent proinflammatory cytokines in inflammation-related diseases. Their actions are regulated by IL-1 receptor antagonist (IL-1ra) and IL-18 binding protein (IL-18bp). This study was designed to (99m)Tc-radiolabel an IL-1ra and IL-18bp dual-domain cytokine ligand, IL-18bp-Fc-IL-1ra, for specific inflammation targeting. METHODS: The (99m)Tc-IL-18bp-Fc-IL-1ra was obtained by direct labeling via 2-iminothiolane reduction. Competitive binding of (99m)Tc-labeled and unlabeled IL-18bp-Fc-IL-1ra to rat polymorphonuclear leukocytes was assessed in vitro. A mouse ear edema model was used to evaluate specific targeting properties of (99m)Tc-IL-18bp-Fc-IL1ra in vivo. The correlation between (99m)Tc-IL-18bp-Fc-IL-1ra uptake and (111)In-labeled polymorphonuclear neutrophil infiltration was studied using ischemic-reperfused rat hearts. RESULTS: Direct (99m)Tc-labeling yielded a stable dual-domain cytokine radioligand with radiochemical purity greater than 95% after gel filtration. Competitive binding studies showed specific targeting of (99m)Tc-IL-18bp-Fc-IL-1ra to inflammatory cells. The (99m)Tc-IL-18bp-Fc-IL-1ra uptake was 1.80±0.17 % injected dose per gram (%ID/g) in the inflamed ear without blocking, whereas uptake in the presence of IL-18bp-Fc-IL-1ra was 1.09±0.08 %ID/g (P<.05). The amounts of IL-1ß and IL-18 were significantly increased in the inflamed ears compared to the vehicle controls. A significant correlation of (99m)Tc-IL-18bp-Fc-IL-1ra with (111)In-labeled neutrophil distribution was observed in the ischemic-reperfused hearts (P<.001). CONCLUSION: Targeting proinflammatory cytokines with (99m)Tc-IL-18bp-Fc-IL-1ra may provide a suitable approach for specific detection of inflammatory sites.