Inhibition of Neutrophil Exocytosis Ameliorates Acute Lung Injury in Rats

Abstract

Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALf). Administration of TAT-SNAP-23, but not TAT-Control, significantly reduced albumin leakage, total protein levels in the BALf, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALf neutrophils and a decrease in neutrophil granule proteins in BALf. Similar degree of neutrophil accumulation in the lungs and/or BALf suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALf revealed that components of the complement and coagulation pathways were significantly reduced in BALf from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI.

TAT-SNAP-23 treatment inhibits the priming of neutrophil functions contributing to shock and/or sepsis-induced extra-pulmonary acute lung injury

“…It has been shown in vitro that blocking human neutrophil granule exocytosis by TAT-SNAP-23 inhibited TNF-α-induced priming of the respiratory burst [34] . In the current study, we wanted to test the effect of TAT-SNAP-23 in a mouse model of in vivo ‘priming’ by hemorrhage followed by septic challenge.…”

“…They demonstrated that TAT-SNAP-23’s ability to inhibit tumor necrosis factor-α (TNF-α) and platelet-activating factor-induced priming of the normal human neutrophil respiratory burst by up to 80% in vitro by blocking exocytosis selectively. They further confirmed that neutrophil granule exocytosis contributing to phagocytosis-induced respiratory burst played a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase [34] . They recently showed that intravenous administration of TAT-SNAP-23 resulted in amelioration of injury in a rat model of direct ALI by inhibiting neutrophil exocytosis [35] .…”

“…This, in turn, affects processes related to neutrophil function such as granule exocyctosis, migration and/or their respiratory capacity. Interestingly, it has been reported that inhibition of SNARE protein function with TAT-SNAP-23 was able to selectively block neutrophil degranulation and reduced TNF-mediated respiratory burst priming in human neutrophils [34] . However, until now, no studies have focused on these proteins or the effect of TAT-SNAP-23 inhibition of these proteins on the sepsis-induced neutrophil respiratory burst response.…”

“…Mouse interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1 (MCP-1) and TNF-α ELISA kits were purchased from BD Bioscience, San Diego, CA. The TAT-SNAP-23 fusion protein was provided by Uriarte et al [34,35] . All other chemicals were of analytical reagent grade and purchased from Sigma Chemical, St Louis, MO.…”

Functionally and morphologically distinct populations of extracellular vesicles produced by human neutrophilic granulocytes

“…EV samples (45 mg) were lysed using 2% SDS at 65°C for 30 min, reduced, alkylated, and digested using trypsin (Promega, Madison, WI, USA) as described previously [29], with modifications for the filter-assisted sample preparation protocol [30]. Before trypsinization, the samples were pipetted into a Microcon-10 Ultracel YM-10 10,000 NMWL centrifugal filter (EMD Millipore, Billerica, MA, USA) and rinsed (3 times) using 250 ml of 8 M urea/ 0.1 M Tris-HCl (pH 8.5) to remove SDS.…”