Recombinant Human Neutrophil Elastase Protein [GST]

Aims/hypothesis Levels of neutrophil elastase, a serine protease secreted by neutrophils, are elevated in diabetes. The purpose of this study was to determine whether neutrophil elastase (NE) contributes to the diabetes-induced increase in retinal vascular permeability in mice with streptozotocin-induced diabetes, and, if so, to investigate the potential role of IL-17 in this process. Methods In vivo, diabetes was induced in neutrophil elastase-deficient (Elane(-/-)), Il-17a(-/-) and wild-type mice. After 8 months of diabetes, Elane(-/-) mice and wild-type age-matched control mice were injected with FITC-BSA. Fluorescence microscopy was used to assess leakage of FITC-BSA from the retinal vasculature into the neural retina. The level of NE in Il-17a(-/-) diabetic retina and sera were determined by ELISA. In vitro, the effect of NE on the permeability and viability of human retinal endothelial cells and the expression of junction proteins and adhesion molecules were studied. Results Eight months of diabetes resulted in increased retinal vascular permeability and levels of NE in retina and plasma of wild-type animals. All of these abnormalities were significantly inhibited in mice lacking the elastase. The diabetes-induced increase in NE was inhibited in mice lacking IL-17. In vitro, NE increased retinal endothelial cell permeability, which was partially inhibited by a myeloid differentiation primary response 88 (MyD88) inhibitor, NF-kappa B inhibitor, and protease-activated receptor (PAR)2 inhibitor. NE degraded vascular endothelial-cadherin (VE-cadherin) in a concentration-dependent manner. Conclusions/interpretation IL-17 regulates NE expression in diabetes. NE contributes to vascular leakage in diabetic retinopathy, partially through activation of MyD88, NF-kappa B and PAR2 and degradation of VE-cadherin.

PURPOSE. Neutrophil-secreted effector molecules are one of the primary causes of tissue damage during corneal inflammation. In the present study, we have investigated the effect of stromal cells in regulating neutrophil expression of tissue-damaging enzymes, myeloperoxidase (MPO), and N-elastase (ELANE). METHODS. Bone marrow-purified nonhematopoietic mesenchymal stromal cells and formylmethionyl- leucyl-phenylalanine-activated neutrophils were cocultured in the presence or absence of Transwell inserts for 1 hour. Neutrophil effector molecules, MPO and ELANE, were quantified using ELISA. In mice, corneal injury was created by mechanical removal of the corneal epithelium and anterior stroma approximating one third of total corneal thickness, and mesenchymal stromal cells were then intravenously injected 1 hour post injury. Corneas were harvested to evaluate MPO expression and infiltration of CD11b(+)Ly6G(+) neutrophils. RESULTS. Activated neutrophils cocultured with mesenchymal stromal cells showed a significant 2-fold decrease in secretion of MPO and ELANE compared to neutrophils activated alone (P < 0.05). This suppressive effect was cell-cell contact dependent, as stromal cells cocultured with neutrophils in the presence of Transwell failed to suppress the secretion of neutrophil effector molecules. Following corneal injury, stromal cell-treated mice showed a significant 40% decrease in MPO expression by neutrophils and lower neutrophil frequencies compared to untreated injured controls (P < 0.05). Reduced MPO expression by neutrophils was also accompanied by normalization of corneal tissue structure following stromal cell treatment. CONCLUSIONS. Mesenchymal stromal cells inhibit neutrophil effector functions via direct cell-cell contact interaction during inflammation. The current findings could have implications for the treatment of inflammatory ocular disorders caused by excessive neutrophil activation.