Tracking the migration of dendritic cells by in vivo optical imaging

Abstract

We report herein a method to track the migration of dendritic cells (DCs) using optical imaging. With the assistance of the delivery module, fluorescein isothiocyanate (FITC) could internalize inside DCs within 15 minutes of incubation. The fluorescent signal was mostly cytoplasmic and could be detected using in vivo imaging. Furthermore, we observed that the probe did not interfere with the DCs maturation as we assessed the expression of several surface markers. The labeled DCs secreted interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) and stimulated the proliferation of CD4+ T lymphocytes responding to lipopolysaccharide (LPS) stimulation. We have systematically compared the probe uptake between mature and immature DCs. The study showed that the latter phagocytosed the probe slightly better than the former. Intravital imaging of treated mice showed the migration of DCs to lymph nodes (LNs), which is confirmed by immunohistochemistry. Taken together, we demonstrated the potential use of optical imaging for tracking the migration of DCs and homing in vivo. The delivery molecules could also be used on other imaging modalities or for delivery of antigens.

Keywords: Dendritic cells; antigen-presenting cells.; delivery; lymph node; optical imaging.

Figure 1

Mature versus immature DCs on the ability to uptake the probe. Half of the isolated DCs was incubated with FITC or the MAP₁₁P-FITC probe at two different concentrations and times. Cell-associated materials were quantified by FACS analysis. The other half of the population was allowed to mature for 2 days, followed by a similar study.^a The fluorescent signals were 2- and 4-fold compared to the control for mature and immature DCs, respectively.^b The signal was 3-fold compared to the control for both mature and immature DCs. The results shown represent the mean of two independent experiments.

Figure 2

Comparison of the uptake of DCs at different stages of cellular development. (A) Confocal microscopy on live cells plated on the eight-well Nalge Nunc chambers on incubation with either 0.2 µM of a fluorescent dye of FITC or with probe MPA₁₁P-FITC for 15 minutes at 37°C. The morphologies of mature and immature cells are distinguished on the DIC channel. Images were color-coded green for FITC. Fluorescence images indicate that the delivery module shuttles the dye inside DCs with remarkable efficiency. The signal is perinuclear and mostly cytosolic. To generate a better localization, the fluorescence image was merged with DIC. (B) Immature DCs uptake better than mature counterparts. Bone marrow-derived DCs were isolated; on day 6, half of the cells were treated with the probe at the indicated time and the uptake was quantified by FACS analysis. LPS was added to the rest of the cells and they were allowed to grow over a period of 2 days. The mature cells were then treated in a similar manner as described for the immature counterparts. The uptake of immature DCs was, to some extent, better at every tested concentration. The results shown represent the mean of two independent experiments.

Figure 3

Phenotypical and functional study of labeled DCs. (A) FACS profiles of surface markers expression of CD11c, CD80, CD86, and CCR7. The shaded histogram represents the indicated specific antibody; the open histogram is the isotype control antibody. (B) Isolated bone marrow-derived DCs were divided into three groups. The first group was treated with buffer; the second and third groups were activated with LPS, and one of them was treated with buffer as control and the other with the MPA₁₁P-FITC probe. Secretions of IL-12 and TNF-α were measured in the supernatants using ELISA. Results are shown as means ± SD of triplicate values and statistical analysis was performed using Student's t test, P < .05. (C) The abilities of the labeled and mature DCs to induce ${\text{CD}}_4^{\text{+}}$ T-cell proliferation were compared with the ability of the unlabeled mature DCs. In this assay, the incorporation of BrdU into the newly synthesized T-cells was stained with specific anti-BrdU fluorescent antibodies. The degree of cell-associated BrdU was then measured by FACS analysis. Labeling DCs when they were immature (Pre) or mature (Post) without activation by LPS did not induce T-cell proliferation; consequently, we observed the levels of T-cell counts to be as low as the control. In contrast, activation of DCs with LPS in either mature (LPS + Post) or immature status (Pre + LPS) stimulated ${\text{CD}}_4^{\text{+}}$ T-cell proliferation.

Figure 4

Mature DCs were incubated with the MPA₁₁P-FITC probe for 15 minutes and the fluorescent signal was readily detected in phantom imaging.

Figure 5

Images of a mouse injected s.c. with DCs labeled with 6 µM MPA₁₁P-FITC probe. (A) Twenty-four-hour postinjection, the digital image showed the exposed inguinal LN as indicated by an arrowhead. (B) Intravital optical imaging of labeled DCs migration to the LN. (C) False-color merged image of the LN. (D) The LN was dissected for ex vivo imaging compared with the muscle; an arrow indicates the muscle. (E) Color-coded image. (F) Immunohistological analysis of a frozen section of a LN. Partial view of a LN at the follicle area: the labeled DCs (green) are associated with Rhodamine-labeled CD3 (red) indicating that the DCs are in the T-cell area. The image was taken using a confocal microscope; original magnification, x40.