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WIREs Nanomed Nanobiotechnol
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19F magnetic resonance imaging of endogenous macrophages in inflammation

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Abstract In this article, we review the use of 19F MRI (magnetic resonance imaging) for in vivo tracking of monocytes and macrophages in the course of tissue inflammation. Emulsified perfluorocarbons (PFCs) are preferentially phagocytized by monocytes/macrophages and are readily detected by 19F MRI. Because of the lack of any 19F background in the body, observed signals are robust and exhibit an excellent degree of specificity. As a consequence of progressive infiltration of the labeled immunocompetent cells into inflamed areas, foci of inflammation can be localized as hot spots by simultaneous acquisition of morphologically matched proton (1H) and fluorine (19F) MRI. The identification of inflammation by 19F MRI—at a time when the inflammatory cascade is initiated—opens the possibility for an early detection and more timely therapeutic intervention. Since signal intensity in the 19F images reflects the severity of inflammation, this approach is also suitable to monitor the efficacy of pharmaceutical treatment. Because PFCs are biochemically inert and the fluorine nucleus exhibits high magnetic resonance (MR) sensitivity, 19F MRI may be applicable for clinical inflammation imaging. WIREs Nanomed Nanobiotechnol 2012, 4:329–343. doi: 10.1002/wnan.1163 This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging

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Uptake of PFCs by mononuclear cells. (a) Matching 1H and 19F MRIs of a 15‐mL Falcon tube after centrifugation of the collected mouse blood over histopaque density gradient show a time‐dependent accumulation of 19F signal in mononuclear cells after tail vein injection of 500 µL PFC emulsion. Blood samples were taken 2 h and 1, 2, and 3 days after PFC injection. (b and c) Flow cytometry of murine mononuclear cells 2 h after tail vein injection of rhodamine‐labeled PFCs. (b) Peripheral blood mononuclear cells (PBMCs) from a control mouse (top) and a mouse treated with rhodamine‐labeled PFCs (bottom) were analyzed for rhodamine fluorescence by flow cytometry. Dot blots show rhodamine versus FITC fluorescence; numbers in the top left quadrants indicate the percentage of rhodamine‐positive PBMCs. (c) PBMCs from both mice were stained with FITC‐labeled anti‐CD11b, anti‐B220, and anti‐CD3 monoclonal antibodies. Gated on rhodamine‐positive cells, histograms display staining of specific (open) and isotype‐matched (gray) control monoclonal antibodies. Numbers indicate the percentage of rhodamine‐positive cells expressing the specific cell marker. (Reprinted with permission from Ref 63. Copyright 2008 Lippincott Williams & Wilkins: Circulation)

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Imaging of PFCs with magnetically different 19F nuclei. 1H and 19F MRI phantom experiments at 9.4 T on five microfuge tubes immersed in a snap cap vial filled with water. (a) 1H MR reference image: White arrows indicate mircofuge tubes filled with two different PFC emulsions [either 10% PFCE or 20% perfluorodecalin (PFD)]. The three other tubes contained only water. (b) Non‐volume selective 19F MR spectrum recorded over the entire field of view showing one signal for the 20 magnetically equivalent fluorine nuclei of PFCE and five signal groups for PFD (for an exact assignment of the individual PFD signals please refer to Refs 97 and 98). Blue arrows indicate the frequencies used for acquisition of the two 19F MR images shown in (c). (c) Corresponding 19F MR images to (a): Because of the large chemical shift range of 19F MR resonances, almost artifact free MR images can be obtained even in conventional manner (in this case turbo spin echo without any frequency‐selective suppression pulses), when signals are well separated as shown on the left (acquired with a basic frequency of 376.465 MHz (on‐resonance with PFCE, indicated as ν = 0 Hz) and an excitation pulse bandwidth of 6210 Hz). However, when the same imaging sequence is used at a basic frequency moved by ∼14,400 Hz into the bulk of PFD resonances, the reconstructed image (right) is compromised by severe chemical shift artifacts due to signal splitting of the magnetically different 19F nuclei.

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19F labeling of inflamed tissues. Inflammation results in the release of cytokines and chemokines attracting macrophages from neighboring tissue as well as activating endothelial cells (1) which leads to the immigration of monocytes and neutrophil granulocytes from the circulating blood into the inflamed area.90,91 After intravenous injection (2), PFC emulsion particles are taken up within the blood stream predominantly by monocytes (3) and to a minor extent by neutrophil granulocytes.62,63 Owing to the small size (100 nm) emulsion particles can also passively diffuse into the inflamed area via the inflamed endothelium (4).63,66 After migration into the inflamed tissue, monocytes differentiate into macrophages or DCs6,25,26 because of the effect of the inflammatory cytokine milieu (5). Because of the chemical inertness of the PFCs, it can be assumed that the PFC label is not lost during this differentiation process. Monocyte‐derived DCs exit the inflamed area via the afferent lymphatics into the draining lymph nodes (6) to orchestrate adaptive immune responses. By this way, the 19F label within the DCs is also transported into the lymph nodes.63,71 In contrast to neutrophils in the blood, neutrophil granulocytes located within the inflamed area contain a significant amount of PFC emulsion particles (7),62,63 possibly acquired by phagocytosis of tissue resident emulsion particles. Neutrophil granulocytes have only a relatively short life span and die by apoptosis after immigration into the inflamed area (8).92 Apoptotic neutrophils are removed by local macrophages67 which themselves could acquire 19F label by uptake of apoptotic PFC‐labeled neutrophils (9). Moreover, it is conceivable that tissue macrophages can phagocytose PFC emulsion particles that leaked into the tissue through the inflamed endothelial barrier (10).

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Infiltration of PFCs after myocardial infarction. (a) Anatomically corresponding 1H and 19F images from the mouse thorax recorded 4 days after ligation of the left anterior descending (LAD) coronary artery showing accumulation of 19F signal near the infarcted region (I) and at the location of surgery where the thorax was opened (T). (b and c) Colocalization of rhodamine‐labeled PFCs and monocytes/macrophages in the heart 4 days after myocardial infarction. (b) Overview images of the heart from frozen sections (8 µm) obtained from the same mouse shown in (a). (c) Anatomically matching sections before (PFC) and after processing for immunofluorescence of CD11b as marker for monocytes/macrophages (DAPI, CD11b). The black rectangle in the bright‐field image (scale bar, 500 µm) represents the section displayed in the adjoining fluorescence images. Because of water solubility of the rhodamine‐labeled PFCs and the impossibility of adequate histological fixing of the particles, rhodamine fluorescence images had to be recorded before immunohistochemistry. Therefore, the sections selected for photographs were carefully related to anatomic landmarks to ensure retrieval of the same area after immunohistochemistry. Although the PFC image is slightly shifted to the left compared with the CD11b and DAPI images, colocalization of red and green fluorescence can be unequivocally recognized. PFCs were injected at day 3 after LAD ligation via the tail vein. Scale bar = 50 µm. (Reprinted with permission from Ref 63. Copyright 2008 Lippincott Williams & Wilkins: Circulation)

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Location of incorporated PFCs within the cell. (a) Confocal fluorescent (left panel) and differential interference contrast (right panel) images of labeled DCs. Fluorescein‐isothiocyanate‐dextran (green) is contained in macropinosomes, as are the DiI‐labeled PFC particles, which appear as punctate yellow spots (left panel). The scale bar represents 5 µm (right panel). (b) Electron micrograph of a labeled DC at low magnification (left panel) and at higher magnification (right panel). Numerous light spots (that is PFC particles)77 are observed inside the cell, but are absent in unlabeled cells (data not shown). Particles appear as smooth spheroids (right panel). The particle diameters are 100–200 nm. Arrows show endosomal compartments. In (b), the scale bars represent 1 µm (left panel) and 200 nm (right panel). (Reprinted with permission from Ref 71. Copyright 2005 Macmillan Publishers Ltd: Nature Biotechnology)

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FACS analysis of inflamed lungs—monocytes and neutrophils transport 19F. Forty‐eight hours after intratracheal lipopolysaccharide challenge lungs were processed for flow cytometric analysis of leukocytes. (a) Monocytes were identified as F4/80high and clone 7/4low; neutrophils as clone 7/4high and F4/80low. T‐cells were defined as CD3+ and B‐cells as CD19+. Cell purity was determined after cell sorting by flow cytometry, and morphology was confirmed after azur and eosin staining using optical microscopy. (b) Sorted cells were analyzed using 1H and 19F MRI. Superimposing of the images revealed detectable amounts of PFCs in monocytes and to a lesser extent in neutrophils. The water‐filled capillary shown in the figure served as geometric landmark for accurate sample identification (small white dot in 1H MR reference image). B, B cells; M, monocytes; N, neutrophils; T, T cells. (Reprinted with permission from Ref 62. Copyright 2010 Lippincott Williams & Wilkins: Circ Cardiovasc Imaging)

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