![Figure 1. Inhibition of MMPs associated with endothelial cells but not lymphocytes blocks lymphocyte migration across endothelial cells. (A) Confluent monolayers of mHEVc cells in 12-μm pore Transwells were treated for 30 minutes with GM6001 or BB3103, general MMP inhibitors, or the solvent control, 0.1% DMSO. (B) mHEVc cells were treated for 30 minutes with 10 μM GM6001, and then the inhibitor was left in or GM6001-treated mHEVc cells were washed 5 times to remove excess inhibitor. (C) Spleen cells were treated with 10 μM GM6001 for 30 minutes and then washed 5 times to remove the inhibitor. For pretreated lymphocytes that were not washed, the final concentration of GM6001 in the coculture was 5 μM. In addition, medium from the last wash was added to nontreated spleen cells to ensure that the inhibitor was sufficiently removed. (A-C) Spleen cells were added on top of the mHEVc monolayer. At 24 hours, cells were collected from the bottom chamber and counted. (D) Confluent monolayers of mHEVc cells on glass slides were treated for 30 minutes with 10 μM GM6001, 50 μM MMP-2/MMP-9 inhibitor II set ((2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide; Calbiochem), or the solvent control, 0.1% DMSO (data not shown), and then washed 5 times. In addition, medium from the last wash was added to nontreated spleen cells to ensure that the inhibitor was sufficiently removed (data not shown). Spleen lymphocytes were added to the monolayer, and the coculture was exposed to 2 dynes/cm2 laminar flow for 30 minutes. Medium was removed, and cells were fixed in 3% paraformaldehyde for 1 hour. Lymphocyte migration was examined by phase contrast microscopy (micrograph). The image was acquired using an Olympus BH-2 microscope (SPlan 40 × objective lens with 0.7 aperture and a 3.3 × internal lens) equipped with an Olympus C-35AD-2 camera. The micrograph was digitized using a Nikon SLC D1X digital camera and Adobe Photoshop 7.0. Nonmigrated lymphocytes are phase light (open arrow). In contrast, migrated lymphocytes appear as phase dark (closed arrow). Bar, 15 μm. GM6001 and BB3103 had no effect on cell viability, as determined by trypan blue exclusion (data not shown). Data are presented as mean ± SD from a representative experiment of 2 experiments with duplicate samples. *P < .05 compared to nontreated and DMSO controls.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/104/8/10.1182_blood-2004-02-0665/6/zh80200468050001.jpeg?Expires=1724268971&Signature=D5BzIjZoktoXkm7sTEQ2coFBP9tNipcCj62foOfpumvaFClYeps8tR85pOYeWEQTgMs3da-j6Hfse8I5au42f8ov1MSS4F1CWmcEW3hxoqEFu3qQF2gye0r28oVmfLd5RPHDV0YPKch72BYOBRQifo0dC758tdw39~qKUtBCO2gO75bt15pvVNXPA2FcZyNpzZt3Eyqztn9qqO-52m55FjkCOV8qq1LI8C98mOXACrVirpKKB7HQF~~TiMAuypIeA2lfmIi1gSOCxDdspjvyeVjLBxXIZDkWTbL6NOpIxwhv8soA6~JgRtV57Ji7Ax7g9RvC2k21ZzZRNj~hrQJJEw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Inhibition of MMPs associated with endothelial cells but not lymphocytes blocks lymphocyte migration across endothelial cells. (A) Confluent monolayers of mHEVc cells in 12-μm pore Transwells were treated for 30 minutes with GM6001 or BB3103, general MMP inhibitors, or the solvent control, 0.1% DMSO. (B) mHEVc cells were treated for 30 minutes with 10 μM GM6001, and then the inhibitor was left in or GM6001-treated mHEVc cells were washed 5 times to remove excess inhibitor. (C) Spleen cells were treated with 10 μM GM6001 for 30 minutes and then washed 5 times to remove the inhibitor. For pretreated lymphocytes that were not washed, the final concentration of GM6001 in the coculture was 5 μM. In addition, medium from the last wash was added to nontreated spleen cells to ensure that the inhibitor was sufficiently removed. (A-C) Spleen cells were added on top of the mHEVc monolayer. At 24 hours, cells were collected from the bottom chamber and counted. (D) Confluent monolayers of mHEVc cells on glass slides were treated for 30 minutes with 10 μM GM6001, 50 μM MMP-2/MMP-9 inhibitor II set ((2R)-[(4-Biphenylylsulfonyl)amino]-N-hydroxy-3-phenylpropionamide; Calbiochem), or the solvent control, 0.1% DMSO (data not shown), and then washed 5 times. In addition, medium from the last wash was added to nontreated spleen cells to ensure that the inhibitor was sufficiently removed (data not shown). Spleen lymphocytes were added to the monolayer, and the coculture was exposed to 2 dynes/cm2 laminar flow for 30 minutes. Medium was removed, and cells were fixed in 3% paraformaldehyde for 1 hour. Lymphocyte migration was examined by phase contrast microscopy (micrograph). The image was acquired using an Olympus BH-2 microscope (SPlan 40 × objective lens with 0.7 aperture and a 3.3 × internal lens) equipped with an Olympus C-35AD-2 camera. The micrograph was digitized using a Nikon SLC D1X digital camera and Adobe Photoshop 7.0. Nonmigrated lymphocytes are phase light (open arrow). In contrast, migrated lymphocytes appear as phase dark (closed arrow). Bar, 15 μm. GM6001 and BB3103 had no effect on cell viability, as determined by trypan blue exclusion (data not shown). Data are presented as mean ± SD from a representative experiment of 2 experiments with duplicate samples. *P < .05 compared to nontreated and DMSO controls.
