Monitoring cellular movement in vivo with photoconvertible fluorescence protein "Kaede" transgenic mice

Abstract

Kaede is a photoconvertible fluorescence protein that changes from green to red upon exposure to violet light. The photoconversion of intracellular Kaede has no effect on cellular function. Using transgenic mice expressing the Kaede protein, we demonstrated that movement of cells with the photoconverted Kaede protein could be monitored from lymphoid organs to other tissues as well as from skin to the draining lymph node. Analysis of the kinetics of cellular movement revealed that each subset of cells in the lymph node, such as CD4(+) T, CD8(+) T, B, and dendritic cells, has a distinct migration pattern in vivo. Thus, the Kaede transgenic mouse system would be an ideal tool to monitor precise cellular movement in vivo at different stages of immune response to pathogens as well as in autoimmune diseases.

Fig. 1.

Photoconversion of Kaede in cells and in transgenic mice. (A) Kaede-expressing HeLa cells were photoconverted with UV light as described in Materials and Methods. The photoconverted Kaede signal was monitored for the indicated times after photoconversion. (Scale bar: 30 μm.) (B) A neonatal Kaede transgenic mouse (Upper) was exposed to violet light as described in Materials and Methods for indicated times (Lower) and observed with a fluorescence stereoscopic microscope. (C) Spleen cells from Kaede transgenic mice were exposed to violet light for 0, 5, or 10 min and subjected to flow cytometry. (D) The indicated numbers of spleen cells were stimulated with either Con A (5 μg/ml) or LPS (10 μg/ml), and cell proliferation was measured by [³H]thymidine incorporation as described in Materials and Methods. Mean ± SD of triplicate wells is shown. Results are representative of two experiments.

Fig. 2.

Movement of photoconverted Kaede-positive cells in the inguinal lymph node. (A) Cells from nonphotoconverted and photoconverted (exposure to violet light for 5 min as described in Materials and Methods) inguinal lymph nodes were subjected to flow cytometry. (B) The inguinal lymph node was exposed in the same manner as above, and the wound was surgically closed on day 0. One day after photoconversion, cells from peripheral blood (PBL) and different lymphoid organs as indicated were subjected to flow cytometry. Three animals were independently analyzed, and the results of one representative analysis are shown. (C) FTY-720 (1 mg/kg) was administered i.p. to Kaede transgenic mice 4 h before photoconversion of the inguinal lymph node. Twenty-four hours after photoconversion, the photoconverted inguinal lymph node and the pool of nonphotoconverted other peripheral lymph nodes were analyzed. Control mice received vehicle only. (D) The inguinal lymph node was exposed to violet light, and the wound was closed as indicated in B. On days 1, 3, and 7 after photoconversion, the presence of photoconverted cells in the original photoconverted inguinal lymph node (LN) and nonphotoconverted other peripheral lymph nodes (PLNs) (pool of contralateral inguinal and axillary lymph nodes and bilateral popliteal, brachial, and cervical lymph nodes) was analyzed. Three animals were independently analyzed for each group with similar results, and the results of one representative analysis are shown.

Fig. 3.

Anatomical connection between inguinal and axillary lymph nodes via lymph. The inguinal lymph node was visualized in the same manner as that shown in Fig. 2. Patent blue was injected into the inguinal lymph node to visualize lymph. Five minutes after injection lymph from inguinal to axillary lymph node was observed as a blue line (A). (Scale bar: 5 mm.) (B) After removing connective tissue to visualize the ipsilateral axillary lymph node and lymph, lymph from the inguinal lymph node (blue) connected to the ipsilateral axillary lymph node was observed. (Scale bar: 0.5 mm.) (C–F) Immunofluorescence microscopy of frozen sections of the axillary lymph node 6 h after photoconversion of the inguinal lymph node. (C and E) Frozen sections (merging green and red signals). (D and F) Serial frozen sections stained with APC-conjugated anti-B220 mAbs (blue). (Scale bars: 30 μm.)

Fig. 4.

Movement of lymphocyte subsets in the inguinal lymph node. (A) The inguinal lymph node was exposed to violet light in the same manner as that shown in Fig. 2A. One day after photoconversion, cells from photoconverted inguinal lymph node (photoconverted inguinal LN) and the pool of nonphotoconverted other peripheral lymph nodes (nonphotoconverted other PLNs) were stained with indicated Abs (APC-conjugated anti-CD4, anti-CD8, or anti-B220 mAb) and subjected to flow cytometry. (B) The inguinal lymph node was exposed to violet light in the same manner as that shown in Fig. 2A. At indicated time points after photoconversion, cells from photoconverted inguinal lymph node, ipsilateral axillary lymph node, and the pool of nonphotoconverted other peripheral lymph nodes were stained according to the method described in A. More than four mice were independently analyzed for each group (mean ± SE).

Fig. 5.

Migration of dendritic cells in the steady state. (A) CD11c⁺ cells from photoconverted inguinal lymph node (immediately after exposure to violet light for 5 min) and nonphotoconverted inguinal lymph node were gated and analyzed for Kaede signals. (B) The inguinal lymph node was photoconverted with the same methods as those in Fig. 2A. Twenty-four hours later, CD11c⁺ cells from photoconverted inguinal lymph node, ipsilateral axillary lymph node, and the pool of nonphotoconverted other peripheral lymph nodes were subjected to flow cytometry. Four animals were independently analyzed with similar results, and the results of one representative analysis are shown. (C) Dorsal skin of mice was exposed to violet light. Immediately (0 h) or 24 h after photoconversion, dendritic cells in draining brachial lymph node were gated by means of anti-CD11c mAb staining and analyzed for the presence of photoconverted Kaede-positive skin-derived cells. Four animals were independently analyzed for each group with similar results, and the results of one representative analysis are shown.