2022 Annual Meeting and Alumni Reunion
Category : Basic and Translational Research Candidate : Changrim Lee Poster #: B14
Conjunctival Goblet Cells Stimulated with an Allergic Mediator Histamine Secrete Extracellular Vesicles that Exhibit Paracrine Secretagogue Activity
Changirm Lee , Darlene A . Dartt
Purpose : Goblet cell functions , especially secretion of mucins , are relatively well studied owing to their protective role on the ocular surface ; however , little attention is given to the other secretory products , such as extracellular vesicles ( EVs ). The purpose of this study is to examine the existence and the function of goblet cell secreted EVs and test whether EVs produced by inflammatory stimuli will cause distinct actions compared to those produced by healthy ( baseline ) cells on recipient goblet cells .
Methods : First , serum-starved primary human conjunctival goblet cells ( HCGCs , grown from conjunctiva explants ) were incubated for 4 h with and without the allergic mediator histamine ( His , 10-3 ~ 10-5 M ) to induce inflammation or to indicate normal cells , respectively . EVs isolated from each treatment were denoted as EVs- H ( Histamine ) and EVs-B ( Basal ), respectively . Second , a nanoparticle tracking analyzer and western blotting were used to analyze the size and the molecular identity of the isolated EV particles . Third , to examine the secretagogue activity of EVs , freshly prepared recipient HCGCs were treated for 4 h with either EVs-H or EVs- B diluted to 1 , 10 , 100 , and 1000 ng / mL in Hank ’ s balanced salt solution ( HBSS ). The amount of secreted high molecular weight glycoproteins ( HMWP ) in the cell culture media during this 4 h period was measured using an enzyme-linked lectin assay ( ELLA ). Fourth , to test the paracrine and endocrine actions of HCGC EVs , fluorescent Ca2 + indicator Fura-2 loaded HCGCs and primary human corneal epithelial cells were spiked with EVs-H or EVs-B and observed for changes in intracellular Ca2 + concentration ( delta [ Ca2 +] i ).
Results : HCGCs stimulated with His secreted a 2-fold higher amount of high molecular weight glycoproteins ( HMWP ) as well as a 7-fold higher number of cell-derived nano-scale particles ( EVs-H ) into the media compared to the control group , which confirmed that His stimulation increases both mucin secretion and EV release in primary HCGCs . The mean diameter of isolated EVs-H and EVs-B was 204.4 and 170.9 nm , respectively . Isolated EVs were enriched with CD9 , ALIX , and CD81 but negative for GM130 ( Golgi marker ) and calnexin ( ER marker ), showing comparable biophysical and molecular characteristics to the standard set forth by the international community ( MISEV 2018 ). When 10 ng / mL of EVs-H were incubated with recipient HCGCs , HMWP secretion was increased by 14-fold compared to HBSS treated HCGCs ( negative cntl ). This increase was also 6-fold and 4-fold higher compared to the 10 ng / mL EVs-B treated HCGCs and 10-5 M His treated HCGCs ( positive cntl ), respectively . An increase of 2 ~ 3-fold was observed at other EV concentrations ( 1 , 100 , and 1,000 ng / mL ); however , all were lower than the activity seen in 10 ng / mL EVs-H . EVs-H also induced an increase in intracellular Ca2 + concentration ([ Ca2 +] i ) not only in HCGCs but also in primary human corneal epithelial cells . In HCGCs , 100 ng / mL EVs-H increased [ Ca2 +] i by 7-fold compared to mock treatment ( negative cntl ) and this increase was comparable to 10-5 M His ( positive cntl ) treatment . In corneal epithelial cells , only 10 and 100 ng / mL EVs-H showed positive delta [ Ca2 +] i while all other doses showed negative values for delta [ Ca2 +] i upon addition . EVs-H ’ s ability to mobilize intracellular Ca2 + in both HCGCs and corneal epithelial cells suggests that they are capable of inducing intracellular signaling in paracrine and endocrine manner on the ocular surface .
Conclusions : We conclude that EVs secreted from inflammatory mediator stimulated HCGCs ( EVs-H ) have differential secretagogue activity and intracellular Ca2 + mobilization capacity when compared to EVs secreted