Human serum albumin nanoparticles as nanovector carriers for proteins: Application to the antibacterial proteins "neutrophil elastase" and "secretory leukocyte protease inhibitor".

The use of proteins and defined amino acid sequences as therapeutic drugs have gained a certain interest in the past decade. However, protein encapsulation within protein nanoparticles was never endeavored. For this reason, human serum albumin (HSA) nanoparticles were prepared by nanoprecipitation method. The process was optimized, and particles were obtained with a size of 120 nm and zeta potential of -25 mV. Neutrophil elastase (NE) and secretory leukocyte protease inhibitor (SLPI) were encapsulated separately within HSA nanoparticles. Gel electrophoresis and western blot studies demonstrate the successful encapsulation and the stability of the particles. On the other hand, enzymatic assays show that encapsulated NE lost its proteolytic activity, whereas encapsulated SLPI maintained its inhibitory property. In addition, the antibacterial studies showed that both formulations were able to drastically reduce bacterial growth of Pseudomonas aeruginosa. This work showed the possibility of using both NE and SLPI as anti-bacterial agents through encapsulation within HSA nanoparticles.

Tiotropium inhibits mucin production stimulated by neutrophil elastase but not by IL-13.

Tiotropium, a muscarinic antagonist, is approved for the treatment of chronic obstructive pulmonary disease and poorly controlled asthma. Because mucus hypersecretion is characteristic of both of these diseases, and muscarinic agonists stimulate mucus secretion, we hypothesized that tiotropium would attenuate airway MUC5AC expression. We grew normal human bronchial epithelial (NHBE) cells to a goblet cell phenotype with 1 or 5 ng/mL of IL-13 and exposed these cells to 10 nM tiotropium or excipient for the full 14 days. Normally differentiated NHBE cells (without IL-13) were exposed to neutrophil elastase (NE) 1 × 10-7 or 5 × 10-7 M for 1 h. MUC5AC was measured by quantitative PCR and ELISA. Acetylcholine production by the epithelium was evaluated by quantitative PCR and by choline/acetylcholine quantification. Tiotropium had no effect on IL-13-stimulated MUC5AC, but attenuated MUC5AC stimulated by NE (p = 0.007 at 5 × 10-7 M). IL-13 increased CarAT mRNA (p < 0.001 at 5 ng/mL) and acetylcholine concentration in the medium (p = 0.018 at 5 ng/mL), while NE had no effect. Tiotropium had no direct effect on IL-13 or NE-induced CarAT or acetylcholine concentration. Tiotropium decreased MUC5AC stimulated by NE, but had no effect on MUC5AC stimulated by IL-13. These results may be due to IL-13, but not NE, increasing acetylcholine production.

Autoprocessing of neutrophil elastase near its active site reduces the efficiency of natural and synthetic elastase inhibitors.

An imbalance between neutrophil-derived proteases and extracellular inhibitors is widely regarded as an important pathogenic mechanism for lung injury. Despite intense efforts over the last three decades, attempts to develop small-molecule inhibitors for neutrophil elastase have failed in the clinic. Here we discover an intrinsic self-cleaving property of mouse neutrophil elastase that interferes with the action of elastase inhibitors. We show that conversion of the single-chain (sc) into a two-chain (tc) neutrophil elastase by self-cleavage near its S1 pocket altered substrate activity and impaired both inhibition by endogenous α-1-antitrypsin and synthetic small molecules. Our data indicate that autoconversion of neutrophil elastase decreases the inhibitory efficacy of natural α-1-antitrypsin and small-molecule inhibitors, while retaining its pathological potential in an experimental mouse model. The so-far overlooked occurrence and properties of a naturally occurring tc-form of neutrophil elastase necessitates the redesign of small-molecule inhibitors that target the sc-form as well as the tc-form of neutrophil elastase.

Degradation of noncollagenous components by neutrophil elastase reduces the mechanical strength of rat periodontal ligament.

BACKGROUND AND OBJECTIVE: We have previously shown that increases in neutrophil elastase in periodontal ligament with chronic periodontitis results in degradation of the noncollagenous components. The purpose of this study was to investigate whether the destruction of noncollagenous components by treatment with elastase in vitro causes changes in the mechanical properties of the periodontal ligament. MATERIAL AND METHODS: The transverse sections of mandibular first molars, prepared from male Wistar rats at 6 wk of age, were digested with 0-50 microg/mL of neutrophil elastase at 37 degrees C for 4 h. Then, their mechanical properties and morphological features were examined. RESULTS: Digestion with elastase dose-dependently decreased the maximum shear stress and failure strain energy density of the periodontal ligament (p < 0.05-0.01). The histological observations after digestion revealed marked degradation of oxytalan fibers, but no marked changes of the collagen fibers, which was confirmed by the detection of very low quantities of hydroxyproline in the digest. The light and scanning electron micrographs showed that the elastase degraded the interfibrillar substances in the periodontal ligament and exposed individual collagen fibrils. CONCLUSION: These results suggest that the increased neutrophil elastase observed in periodontal disease degrades the oxytalan fibers and interfibrillar substances in the periodontal ligament to decrease its mechanical strength.

Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase.

Human neutrophil elastase (HNE) and proteinase 3 (PRO3) are myeloid tissue-restricted serine proteases, aberrantly expressed by myeloid leukemia cells. PRO3 and HNE share the PR1 peptide sequence that induces HLA-A*0201-restricted cytotoxic T cells (CTLs) with antileukemia reactivity. We studied the entire HNE protein for its ability to induce CTLs. In an 18-hour culture, HNE-loaded monocytes stimulated significant intracellular interferon gamma (IFN-gamma) production by CD4+ and CD8+ T cells in 12 of 20 and 8 of 20 healthy individuals, respectively. Lymphocytes from 2 HNE responders were pulsed weekly for 4 weeks to generate HNE-specific CTLs. One of 2 HLA-A*0201-negative individuals inhibited the colony formation of HLA-identical chronic myelogenous leukemia progenitor cells (73% inhibition at 50:1 effector-target [E/T] ratio), indicating that peptides other than PR1 can induce leukemia-reactive CTLs. Repetitive stimulations with HNE in 2 of 5 HLA-A*0201+ individuals increased PR1 tetramer-positive CD8+ T-cell frequencies from 0.1% to 0.29% and 0.02% to 0.55%, respectively. These CTLs recognized PR1 peptide or killed HNE-loaded targets. These results indicate that exogenously processed HNE is a source of PR1 peptide as well as other peptide sequences capable of inducing leukemia-specific CD8+ and CD4+ T cells. HNE could, therefore, be used in an HLA-unrestricted manner to induce leukemia-reactive CTLs for adoptive immunotherapy.

A novel orally active inhibitor of HLE.

Human leukocyte elastase (HLE) is a serine proteinase, capable of degrading a variety of structural matrix proteins. SSR69071 2-[(4-isopropyl-6-methoxy-1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methoxy]-9-(2-piperidin-1-ylethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one was selected as a novel orally active HLE inhibitor for treatment of chronic obstructive pulmonary diseases, asthma, emphysema, cystic fibrosis and several inflammatory diseases (WO 01/44245 A1) (J. Pharm. Exp. Ther., submitted for publication).

Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Systemic injection of products of activated neutrophils and H2O2 in myeloperoxidase-immunized rats leads to necrotizing vasculitis in the lungs and gut.

The strong association of anti-neutrophil cytoplasmic antibodies with various forms of systemic vasculitis suggests a role for these autoantibodies in the pathophysiology of systemic vasculitis. In the present study, we tested the hypothesis that release of neutrophil lysosomal enzymes in the presence of an anti-myeloperoxidase (anti-MPO) immune response may underlie the development of systemic vasculitis. Brown Norway rats were immunized with MPO in complete Freund's adjuvant or complete Freund's adjuvant alone. Two weeks after immunization, rats bad developed antibodies to human and rat MPO as measured by enzyme-linked immunosorbent assay. Next, rats were intravenously infused with 400 micrograms of a human neutrophil lysosomal extract containing 200 micrograms of MPO followed by 0.5 ml of a 1 mmol/L solution of H2O2 through a cannula inserted into the right jugular vein. Rats were sacrificed at 4 hours, 24 hours, 7 days, or 14 days, and several organs (lungs, heart, liver, spleen, gut, and kidneys) were examined for vasculitic lesions and inflammatory cell infiltrates. Macroscopically, patchy hemorrhagic spots were observed in the lungs and gut of MPO-immunized rats at days 7 and 14 after systemic infection of the neutrophil lysosomal extract and H2O2. Such changes were not observed at earlier time points or in control immunized rats. Histologically, the lungs of MPO-immunized rats sacrificed at days 7 and 14 showed patchy inflammatory cell infiltrates associated with vasculitis, granuloma formation, giant cells, and foci of hemorrhage. At 14 days, early signs of fibrosis were found with deposition of collagen and proliferation of fibroblasts. Furthermore, a prominent leukocytoclastic vasculitis was found in the small intestine of these rats characterized by fibrinoid necrosis and an extensive neutrophilic infiltrate. No inflammatory changes were found in the other organs studied (heart, liver, spleen, and kidneys). Control immunized rats, sacrificed at days 7 and 14 showed only some small foci of inflammatory infiltrates in the lungs whereas no inflammatory changes were found in the gastrointestinal tract. These studies show that release of products from activated neutrophils in the presence of anti-MPO autoantibodies may be relevant to the pathogenesis of anti-MPO-associated vasculitides.

Abnormal mucous cell phenotype induced by neutrophil elastase in hamster bronchi.

Bronchial mucous cell metaplasia (MCM) is a histologic component of chronic mucus hypersecretion. The hamster model of elastase-induced MCM appears to involve an irreversible conversion of Clara cells to mucous cells. The present study questioned whether the mucous cells seen in hamster bronchi exposed to neutrophil elastase produce and maintain a form of glycoconjugate secretory product different from that normally found in mucous cells or Clara cells. Ultrastructural cytochemistry using the gold-labeled lectin HPA revealed a difference in the cell surface and stored secretory granules of elastase-derived mucous cells compared to normal mucous cells and Clara cells at 3 weeks and 4 months following exposure. The results suggest that elastase irreversibly alters the glycoconjugate character of the Clara cells normally present so that they produce an abnormal form of mucus. Because secreted glycoconjugates can affect the rate of mucociliary clearance and receptor-mediated binding of microorganisms, this change in phenotype may be involved in the pathogenesis of diseases associated with chronic mucus hypersecretion in humans.