Figure 12.

Co-culture with 21dBLMFbs, MNLFbs grown in the lower chamber with PBMCs (mouse leukocytes), U937 cells (human leukocytes) on confluent EC (C166), or HUVECs to determine the leukocyte migration across the endothelial cell monolayers (see “Experimental procedures.” A, leukocyte migration is expressed as mean percentage ± S.E. (error bars) of added cells migrating across control EC monolayers on inserts over 16 h. Data are from replicate wells in three independent experiments using three different 21dBLMFb cultures or three different MNLFb cultures. Migrated cells underneath the inserts were counted by hemocytometer. 21dBLMFbs significantly increased migration of PBMCs through the EC layer, which was further increased by cytokine activation of 21dBLMFbs (1 μg/ml LPS for 8 h) prior to co-culture with ECs. *, p < 0.01, statistically significantly greater than migration across resting EC layer. B, PBMCs attached to the insert 16 h after migration were measured by colorimetry (see “Experimental procedures”). Activation of the EC cell layer with cytokines reduced the number of PBMCs migrating across the EC but increased the PBMC binding to the EC monolayer. C and D, pretreatment of 21dBLMFbs with 1 μg/ml LPS for 8 h followed by treatment with 1 μg/ml V6-PEP/nanoparticle or 1 μg/ml V6 shRNA/nanoparticle reduced PBMC migration through the control EC monolayer (C) and also reduced U937 (leukocyte) cell migration on the HUVEC monolayer (D). *, p < 0.005. E, promotion of U937 (leukocyte) cell migration by CM derived from IPFFbs, by recombinant monocyte chemoattractant protein-1 (MCP-1), and by IL-8 are shown. ∼250 × 10³ U937 cells were put onto confluent HUVEC monolayers that were treated with human recombinant MCP-1, with CM from IPFFb cultures alone or cultures that were treated with IL-8 (50 ng/ml), with 100 μg of each of the indicated nanoparticles, or with a 500 μg/ml concentration of either anti-MCP-1 or anti-CD44v6 for 12 h. U937 cell numbers in the lower chamber were counted in triplicate wells 16 h after the addition of the reagents and the CMs to the U937 cells and HUVECs on the inserts.