In vivo migration of PGE2-treated DCs is MMP-9 dependent. (A) DCs generated from B10.A mice were treated with IFN-α + TNF-α with or without PGE2 (10−6 M), in the presence or absence of the MMP-9 inhibitor I (10−5 M) for 48 hours. DCs were collected and labeled with the PKH 26 red fluorescence dye. Two groups of B10.A mice (n = 5) were preinjected with 40 ng TNF-α subcutaneously in the footpads of each of the hind legs. Twenty-four hours later the mice were inoculated with DCs (1 × 106 cells in 60 μL, subcutaneously in the footpads) as follows: mice in group I received untreated DCs in the right leg, and TNF-α + IFN-α–treated DCs in the left leg; mice in group II received TNF-α + IFN-α + PGE2–treated DCs in the right leg and TNF-α + IFN-α + PGE2 + MMP-9 inhibitor I in the left leg. The draining popliteal lymph nodes were harvested 48 hours later, and the numbers of labeled cells were determined by FACS. Results are expressed as numbers of fluorescent cells per 100 000 lymph node cells. (B) DCs generated from WT (FBV/NJ) or MMP-9–deficient mice were treated with IFN-α + TNF-α + PGE2 for 48 hours. Following labeling with the PKH 26 red fluorescence dye, 1 × 106 DCs were injected subcutaneously in the footpads of WT mice (n = 10). The mice were preinjected with TNF-α as described. Each mouse received WT DC in the right leg and MMP-9–deficient DCs in the left leg. Five mice were killed 24 hours later, and the rest were killed 72 hours after the DC inoculation. The number of labeled cells in the draining popliteal lymph nodes was determined by FACS. (C) DCs generated from WT (FBV/NJ) or MMP-9–deficient mice were treated with IFN-α + TNF-α with or without PGE2 and injected into 2 groups (n = 5) of MMP-9–deficient mice. Each mouse in group I received WT DC treated with IFN-α + TNF-α in the right leg and WT DC treated with IFN-α + TNF-α + PGE2 in the left leg. Mice in group 2 received MMP-9–deficient DCs treated with IFN-α + TNF-α in the right leg and MMP-9–deficient DCs treated with IFN-α + TNF-α + PGE2 in the left leg. All mice were killed 48 hours later; draining lymph node cells were collected and stained with Annexin V and the numbers of labeled cells in the Annexin V–negative population were determined by FACS. Results are expressed as numbers of PHK26+ cells per 100 000 cells.
