All the stress stimuli tested were proinflammatory because they all induced secretion of IL-8 from HCE cells. Therefore, we were interested in determining what kind of messages these vesicles could convey to the corneal epithelium. Consequently, IL-8 secretion from naïve HCE cells (i.e., cells not exposed to any environmental stress) incubated with isolated stress-induced vesicles was measured (
Fig. 6A). For this purpose HO-, UV-B–, and LPS-induced vesicles were isolated, resuspended in PBS, and added to the cell culture media of the naïve HCEs. After 18 hours of incubation, we observed that, independent of the amount of HO or UV-B stress to which the producing cells were exposed, the vesicles did not affect the proinflammatory profile of the resting (nonstimulated) HCEs. Conversely, the LPS-induced vesicles stimulated a vigorous response from the naïve HCEs (
Fig. 6A, LPS). Most likely, this finding is due to residual LPS molecules attached to the isolated extracellular vesicles. For our experimental setup, this result was valuable because it showed that the isolated vesicles come in contact with the underlying cells and affect their expression pattern. With this in mind, in the following experiment, naïve HCE cells were incubated with either HO- (100 mM of NaCl) or UV-B– (5 mJ/cm
2) induced vesicles, while control cells were incubated in serum-free medium. After 18 hours, the medium was replaced with hyperosmolar medium (70–100 mM of added salt), and IL-8 secretion was measured after an additional 4 hours (
Fig. 6B). Surprisingly, the cells incubated with extracellular vesicles induced under UV-B or HO stress secreted lower amounts of IL-8 (
Fig. 6B,
P < 0.05). Furthermore, after 18 hours of incubation with the stress-induced vesicles, IL-8 gene expression was reduced by 50% (
Fig. 6C,
P < 0.05) when compared to the cells without added vesicles. Decreased levels of mRNA for IL-8 also were observed after the 4 hours of salt stress, although the differences were not statistically significant. The vesicles did not affect the viability of the cells in any way (data not shown).