Mechanisms and modulation of microvesicle uptake in a model of alveolar cell communication

Abstract

Extracellular vesicles, including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which extracellular vesicles are internalized is incompletely characterized, it is generally considered to involve endocytosis and an initial surface-binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here, we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage-derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptor-dependent endocytosis and is subject to bidirectional modulation by relevant pathologic perturbations.

Extracellular vesicles released in response to respiratory exposures: implications for chronic disease

“…Importantly, CS-exposed mice and human smokers exhibited decreased SOCS concentrations in BALF compared to nonsmoking controls, suggesting a loss of EV-dependent antiinflammatory state in smokers (Bourdonnay et al 2015). In addition, the anti-inflammatory effect of alveolar macrophage-derived EV depends upon their internalization by target cells, which is inhibited by the presence of CS extract (CSE) (Schneider et al 2017). …”

Resident alveolar macrophage‐derived vesicular SOCS3 dampens allergic airway inflammation

“…In initial studies with MHS cells, EVs were pelleted from MHS CM by 17 000×g ultracentrifugation for 30 minutes, with quantification of EV numbers performed as described previously. 16 During the course of our studies, we observed that the yield of EVs by this isolation method was limited due to their rupture owing to the high shear forces from ultracentrifugation. This prompted us to instead employ the gentler method of centrifugal filtration of AM CM through a 100 kDa exclusion filter (MilliporeSigma) 32 and this technique was employed for EV isolation from the J2immortalized AM cell line.…”

“…A continuous SV40‐transformed line of primary AMs originally obtained from lavage fluid of normal mice (MHS, CRL‐2019), which we have utilized previously as a source of EVs, and a transformed human bronchial EC line (BEAS‐2B) were purchased from American Type Culture Collection. Normal primary mouse AMs were obtained by lung lavage of a male C57BL/6 wild type mouse (The Jackson Laboratory) and subsequently immortalized by infecting with the J2 retrovirus carrying v‐raf and v‐myc oncogenes as previously described .…”

“…15 We have demonstrated that these AM-derived EVs are rapidly internalized by, and inhibit JAK-STAT signaling and inflammatory gene expression within, ECs. 13,16 Pharmacologic inhibition of JAK is used clinically in rheumatoid arthritis and its potential is being widely explored in other inflammatory diseases, 17 including allergic asthma. [18][19][20][21][22] While SOCS3 has been suggested to promote type 2 immune responses by its actions in lymphocytes and eosinophils, 23,24 its upregulation in bronchial ECs has been associated with anti-inflammatory actions.…”

The biology of extracellular microvesicles

“…Low pH has also been shown to promote exosome release and uptake, indicating that this may be a conserved EV characteristic. Recent studies have indicated an importance for actin polymerization and dynamin, a key protein involved in membrane budding and fission, in macropinocytic‐like MV uptake by alveolar epithelial cells . An important regulator in EV internalization is heparan sulfate proteoglycans (HSPGs), and treating EVs with heparin reduces their uptake .…”