Cells from joints of tumor necrosis factor-transgenic (TNF-Tg) mice express high levels of vascular endothelial growth factor-C (VEGF-C). Ankle and wrist joints of TNF-Tg mice and wild-type (WT) littermates were subjected to collagenase digestion to isolate cells, which then were stained with fluorescein isothiocyanate (FITC)-anti-CD11b and phycoerythrin-anti-Gr-1 and subjected to fluorescence-activated cell sorting analysis. (a) The percentage of CD11b+/Gr-1-/lo cells is shown in a representative histogram from one pair of TNF-Tg and WT mice. Cells (3 × 105) were cytospun onto glass slides and double-stained with FITC-anti-CD11b and rabbit anti-VEGF-C followed by anti-rabbit Alexa 546. TO-PRO-3 iodide was used as a DNA dye for nuclear staining. (b) Co-localization of CD11b and VEGF-C proteins was observed, and pictures were taken under a confocal microscope at a power of × 20. Cells were cultured with M-CSF for 3 days, and adherent cells were harvested. (c) Expressions of VEGF-A, -C, and -D mRNA were measured by real-time reverse transcription-polymerase chain reaction (RT-PCR). The fold increases in expression in TNF-Tg over WT cells were calculated. Values are the means of triplicate loadings plus standard deviation (SD). (d) The expression levels of TNF and IL-1 mRNA in joint extracts from TNF-Tg mice and WT littermates were determined by real-time RT-PCR. Values are the means of three mice plus SD. Experiments were repeated using four additional pairs of TNF-Tg and WT mice with similar results.