Exogenous serpin B1 restricts immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis.

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

Expression and purification of recombinant human serpin B1 yields novel molecules with altered protease inhibitory activities: Functional implications.

Serpin B1 regulates the innate immune system by inhibiting serine and cysteine proteases that control programmed cell death and proliferation pathways. To provide recombinant human proteins for in vitro and in vivo studies we expressed and purified wild-type human serpin B1 and a C344A variant in the yeast S. cerevisiae. Both proteins expressed well and inhibited elastase and chymotrypsin. However, purification of wild-type serpin B1 in the absence of a reducing agent resulted in the specific loss of elastase - but not chymotrypsin - inhibition, concomitant with the formation of two higher molecular weight forms of the protein - a modified monomer and a dimer created via an intermolecular disulfide bond formed between C344 in respective serpin B1 monomers. In contrast to fully reduced serpin B1, both modified forms were good elastase substrates and catalytically cleaved at multiple adjacent sites within the reactive site loop. In contrast, purification of the C344A variant in the absence of a reducing agent yielded only one form of the protein which retained elastase and chymotrypsin inhibitory properties when purified. Furthermore, the elastase inhibitory activity of wild-type serpin B1, but not the C344A variant, was sensitive to oxidation. Thus, wild-type human serpin B1 should be formulated with a pharmaceutically acceptable reducing agent to protect C344 against post-translational oxidative modifications. Alternatively, the C344A variant of this protein may prove to be a suitable drug development candidate. These findings also suggest that inactivation of serpin B1 by oxidation may have a physiological role to play during inflammation.

Internalization by multiple endocytic pathways and lysosomal processing impact maspin-based therapeutics.

UNLABELLED: Patients with metastatic disease face high rates of mortality with a paucity of therapeutic options. Protein-based therapeutics provide advantages over traditional chemotherapy through increased specificity, decreased immune impairment, and more direct means of delivery. However, development is often hindered because of insufficient knowledge about protein processing by cells when exogenously applied. This study focuses on recombinant Maspin (rMaspin), a serine protease inhibitor (SERPINB5), which alters invasive properties when directly applied to cancer cells. Previous evidence suggests differences in the effects of rMaspin treatment when compared with endogenous reexpression, with little explanation for these discrepancies. A leading hypothesis is that exogenously applied rMaspin is subject to different regulatory and/or processing mechanisms in cancer cells when compared with endogenous expression. Therefore, a more detailed understanding of the mechanisms of internalization and subcellular trafficking of rMaspin is needed to guide future translational development. We describe the molecular trafficking of rMaspin in cytoplasmic vesicles of the endosomal/lysosomal pathway and characterize its uptake by multiple endocytic mechanisms. Time-lapse laser scanning confocal microscopy shows the uptake, in real time, of dye-labeled rMaspin in cancer cells. This study indicates that cellular processing of rMaspin plays a key role by affecting its biologic activity and highlights the need for new approaches aimed at increasing the availability of rMaspin when used to treat cancer. IMPLICATIONS: Novel characterization of internalization and subcellular trafficking of rMaspin provides new insights for future therapeutic development.

Maspin: molecular mechanisms and therapeutic implications.

Maspin, a non-inhibitory member of the serine protease inhibitor superfamily, has been characterized as a tumor suppressor gene in multiple cancer types. Among the established anti-tumor effects of Maspin are the inhibition of cancer cell invasion, attachment to extracellular matrices, increased sensitivity to apoptosis, and inhibition of angiogenesis. However, while significant experimental data support the role of Maspin as a tumor suppressor, clinical data regarding the prognostic implications of Maspin expression have led to conflicting results. This highlights the need for a better understanding of the context dependencies of Maspin in normal biology and how these are perturbed in the context of cancer. In this review, we outline the regulation and roles of Maspin in normal and developmental biology while discussing novel evidence and emerging theories related to its functions in cancer. We provide insight into the immense therapeutic potential of Maspin and the challenges related to its successful clinical translation.

Inhaled recombinant alpha 1-antitrypsin ameliorates cigarette smoke-induced emphysema in the mouse.

In alpha 1-antitrypsin deficiency in humans, inadequately regulated activity of serine protease activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function seen in those individuals with emphysema. Typically, disease symptoms in this patient population are exacerbated by cigarette smoke. Here we show that inhaled recombinant alpha 1-antitrypsin (rAAT) can provide significant protection against the development of emphysema in cigarette smoke-treated mice. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of neutrophils and macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In smoking animals treated for 6 months with inhaled rAAT, effects on lavage levels of neutrophils and macrophages were only moderate when compared with untreated animals. Furthermore, neutralizing antibodies to rAAT were generated in all rAAT-treated animals. Despite this, however, reductions in airspace enlargement of up to 73% were observed. These findings demonstrate that delivery of rAAT directly to the lungs of smoke-treated mice can inhibit lung tissue damage mediated by proteases, suggesting that rAAT inhalation therapy might represent a practical approach towards treating emphysema in humans, by modifying the course of the disease.

Alpha1-antitrypsin single dose adjuvant therapy for acute otitis media.

OBJECTIVES: Proteases have been shown to play a role in the pathogenesis of otitis media. Inhibition of these proteases can improve treatment outcomes in certain conditions. The goal of this study was to determine if intratympanic administration of a single dose of the protease inhibitor, recombinant alpha 1-antitrypsin (rAAT), can facilitate resolution of acute otitis media (AOM) in the chinchilla. METHODS AND MEASURES: Pneumococcus was injected into both middle ears of 12 chinchillas. After 3 to 4 days, middle ears were cultured, systemic antibiotics were initiated, and rAAT or its vehicle was administered into the middle ears of all animals. Serial tympanic membrane (TM) scoring, tympanometry, and auditory-evoked brain stem response testing were performed. Animals were sacrificed at varying timepoints and temporal bones studied for objective measures of OM. RESULTS: Although not reaching statistical significance, there was a trend to more rapid resolution of AOM in rAAT-treated ears. Tympanometry, auditory thresholds, and quantitative histologic parameters did not differ between rAAT and vehicle treated ears. CONCLUSIONS: A single dose of intratympanic rAAT likely does not facilitate the resolution of antibiotic-treated pneumococcal AOM in the chinchilla model. Serial administration of this protease inhibitor may be necessary to see a significant treatment effect.

Inflammatory proteases in chronic otitis externa.

OBJECTIVES: Proteases are known to contribute to the pathogenesis of chronic inflammatory skin conditions such as atopic dermatitis and psoriasis. Inhibition of these proteases has shown promise in the treatment of these skin conditions. The purpose of this study was to measure the matrix metalloproteinases (MMP) and human neutrophil elastase (HNE) activities in chronic otitis externa (COE) and to determine whether administration of protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat might reduce these protease activities. STUDY DESIGN: Prospective and ex vivo. METHODS: Twenty-five ear canals with COE and 34 with no pathology (i.e., controls) were debrided and filled with saline. After a tragal pump and 1 to 2 minutes, the washes were collected and analyzed for MMP and HNE activities and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP and HNE levels were significantly higher (P = .0057 and .0112) in ears with COE than normal ears. MMP activity greater than 3 mAU/minute was observed in 30% of COE and 0% of controls (P = .0270). HNE activity greater than 3 mAU/minute was found in 77% of COE versus 7% of controls (P < .0001). Ilomastat and rAAT inhibited 60% of MMP and 98% of HNE activity, respectively, in COE ears. CONCLUSIONS: Elevated levels of proteases found in COE, MMP, and HNE may be inhibited with ilomastat and rAAT. The therapeutic potential of these protease inhibitors warrants investigation.

An inhaled matrix metalloprotease inhibitor prevents cigarette smoke-induced emphysema in the mouse.

Inadequately regulated proteolytic activity is responsible for the chronic lung tissue degeneration and irreversible loss of pulmonary function that define emphysema. In this study, we show that an inhaled broad-spectrum matrix metalloprotease inhibitor, ilomastat, can provide protection against the development of emphysema in cigarette smoke-treated mice. Control animals were exposed to daily cigarette smoke for 6 months. As has been reported previously, cigarette smoke was seen to increase significantly the recruitment of macrophages into the lungs of these animals, leading to concomitant alveolar airspace enlargement and emphysema. In animals treated daily with nebulized ilomastat for 6 months, lung macrophage levels were greatly reduced, and neutrophil accumulation was also inhibited. Corresponding reductions in airspace enlargement of up to 96% were observed. These striking observations suggest that delivery of ilomastat directly into the lungs of smoke-treated mice can not only inhibit lung tissue damage mediated by metalloproteases, but may also reduce that component of tissue degeneration mediated by excess neutrophil-derived products. Our data also suggest that the matrix metalloprotease inhibitors may represent a class of drugs that, when delivered by inhalation, could be used practically to treat cigarette smoking-related chronic obstructive pulmonary disease by modifying the course of the disease.

Production of serpins using yeast expression systems.

Serpins occupy a unique niche in the field of biology. As more of them are discovered, the need to produce sufficient quantities of each to aid experimental and therapeutic research increases. Yeast expression systems are well suited for the production of recombinant serpins. The genetics of many yeast species is well understood and readily manipulated to induce the targeted over-production of many different serpins. In addition, protease-deficient strains of certain species are available and a few species carry out post-translational modifications resembling those of humans. Yeasts are easy to grow and multiply readily in simple culture media hence the cost of production is low, while the scale of production can be small or large. The disadvantages are the inability of most yeast(s) to perform complex post-translational modifications and a lower product yield of secreted protein compared to intracellular protein production. However, for the intracellular production of serpins, in particular the clade B serpins that do not have complex post-translational modifications, yeast expression systems should be among the first systems considered.

Protease inhibitors alpha1-antitrypsin and ilomastat are not ototoxic in the chinchilla.

OBJECTIVES: Proteases of both the serine and the metalloprotease families have been shown to play a role in the pathogenesis of otitis media. Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions. The purpose of this study was to assess the safety of protease inhibitors when instilled into the middle ear, with a view to their potential use in the treatment of human otitis media. STUDY DESIGN: Prospective, randomized, controlled trial in the chinchilla model. METHODS: After completing baseline auditory testing and bilateral transpalatal obstruction of the Eustachian tube, chinchillas received weekly transbullar injections of protease inhibitor (alpha1-antitrypsin, ilomastat, or both), vehicle, or saline. After 1 month, hearing was tested and the animals were sacrificed. Temporal bone histopathologic examination was performed. RESULTS: All treatment groups demonstrated a statistically insignificant average loss in long-term hearing (0 dB) for all measures using clicks and tones (P >.15 for all conditions). All treatment groups were statistically insignificantly different from one another (P =.5625). Histopathologic examination revealed no significant inner ear changes. CONCLUSIONS: Protease inhibitors that are currently under study in animal models and humans for the treatment of inflammatory diseases that are related to imbalances between protease and protease inhibitor have no significant toxic effect on the inner ear of chinchillas. These findings support the safety of further clinical trials using these inhibitors to treat middle ear inflammation.

Alpha 1-antitrypsin and ilomastat inhibit inflammatory proteases present in human middle ear effusions.

OBJECTIVES: Proteases of both the serine and metalloproteinase families have been shown to play a role in the pathogenesis of otitis media (OM). Inhibitors of proteases from each of these families have been shown to beneficially impact disease progression in a number of related chronic inflammatory conditions, but their use has not been studied in OM. The purpose of this study was to assess the activity of the protease inhibitors recombinant alpha 1-antitrypsin (rAAT) and ilomastat on inflammatory proteases present in human middle ear effusions (MEEs), with a view to their potential utility in the treatment of OM. STUDY DESIGN: Prospective and ex vivo. METHODS: MEEs were collected from 100 patients presenting for middle ear surgery, most commonly tympanostomy tube placement or treatment of acute posttympanostomy otorrhea (APTO). MEEs were analyzed for the presence of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE) and the inhibitory activity of rAAT and ilomastat on these proteases, respectively. RESULTS: MMP levels were highest in APTO, and HNE was highest in chronic suppurative OM and APTO. High levels of MMP and HNE (>3 mAU/min) were found in 52% and 37% of MEEs, respectively. Ilomastat and rAAT demonstrated significant inhibition of MMP and HNE activity (>30% reduction), respectively, in 80% and 82% of MEEs with high levels of activity. CONCLUSIONS: Proteases are commonly found in OM. Ilomastat and rAAT are potent inhibitors of proteases in MEEs across a wide range of OM in humans. Investigation into the potential therapeutic benefits of these protease inhibitors is warranted.