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Study Objectives: To describe asthma features in a cohort with alpha(1)-antitrypsin (AAT) deficiency, and determine the impact of asthma on FEV(1) decline.Background: Asthma may be common in those with AAT deficiency, and may lead to accelerated airflow obstruction.Design: Analysis of data obtained from a 5-year, prospective National Heart, Lung, and Blood Institute registry.Setting: A multicenter registry consisting of 37 clinical centers, a central phenotyping laboratory, and a data analysis center.Participants: A cohort of 1,052 subjects with AAT deficiency.Measurements and Results: Asthma was defined as reversible airflow obstruction, recurrent attacks of wheezing, and a reported diagnosis of asthma or allergy with or without an elevated serum IgE level. FEV(1) decline was calculated by least-square means with adjustments for covariables. Asthma was present in 21% of the cohort and in 12.5% of those with a normal FEV(1). Attacks of wheezing were reported in 66%, the first attack occurring at a mean +/- SD age of 31 +/- 16 years. Allergy and asthma was reported in 29% and 38%, respectively. An elevated IgE level occurred in 17% and was significantly associated with signs and symptoms of asthma and an allergy history. Unadjusted FEV(1) decline was less in the group without asthma and a normal IgE level (- 48.5 mL/yr) vs the groups with asthma features (> or = 64 mL/yr) [p = 0.002]. Multivariable analysis showed that bronchodilator response, age, and smoking were significant predictors for FEV(1) decline but not asthma.Conclusions: Symptoms and signs of asthma are common in AAT deficiency and may start at the age of most rapid FEV(1) loss. Adjusting for other risk factors such as bronchodilator response, asthma as defined does not lead to an accelerated FEV(1) decline. In AAT deficiency, augmentation therapy is not more effective in preventing the loss of lung function in those with asthma compared to those without. [ABSTRACT FROM AUTHOR]

Rationale: Alpha-1 antitrypsin deficiency, caused primarily by homozygosity for the Z allele of the SERPINA1 gene, is a well-established genetic cause of chronic obstructive pulmonary disease (COPD). Whether the heterozygous PiMZ genotype for alpha-1 antitrypsin confers increased risk for COPD has been debated., Objectives: We analyzed 8,271 subjects in the Genetic Epidemiology of COPD (COPDGene) Study, hypothesizing that PiMZ would independently associate with COPD and COPD-related phenotypes., Methods: The COPDGene Study comprises a multiethnic, cross-sectional, observational cohort of non-Hispanic white and African American current and former smokers with at least 10 pack-years of smoking who were enrolled for detailed clinical and genetic studies of COPD and COPD-related traits. We performed multivariate logistic regression analysis for moderate to severe COPD and assessed Pi genotype with other relevant covariates in models stratified by race. We analyzed quantitative characteristics on the basis of volumetric computed tomography with generalized linear models controlling for genotype, scanner type, and similar covariates., Results: White PiMZ COPDGene subjects had significantly lower lung function, FEV 1 percent predicted (68 ± 28 vs. 75 ± 27; P = 0.0005), and FEV 1 /FVC ratio (0.59 ± 0.18 vs. 0.63 ± 0.17; P = 0.0008), as well as more radiographic emphysema (P = 0.001), than subjects without alpha-1 antitrypsin Z risk alleles. Similarly, African American PiMZ subjects had lower lung function, FEV 1 percent predicted (65 ± 33 vs. 84 ± 25; P = 0.009) and FEV 1 /FVC (0.61 ± 0.21 vs. 0.71 ± 0.15; P = 0.03)., Conclusions: In the COPDGene Study, we demonstrate that PiMZ heterozygous individuals who smoke are at increased risk for COPD and obstructive lung function impairment compared with Z-allele noncarriers, regardless of race. Although severe alpha-1 antitrypsin deficiency is uncommon in African Americans, our study adds further support for initial targeted detection of all subjects with COPD for alpha-1 antitrypsin deficiency, including African Americans. Clinical trial registered with www.clinicaltrials.gov (NCT00608784).

Alpha-1 antitrypsin (AAT) deficiency-associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (Mɸ) express AAT, we propose that the expression and intracellular accumulation of mutated Z-AAT (the most common mutation) compromises Mɸ function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, Mɸ from individuals with Z-AAT (Z-Mɸ) have greater proteolytic activity on ECM than do normal Mɸ. This abnormal Z-Mɸ activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z-AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z-Mɸ. Importantly, compared with normal Mɸ, the membrane-bound serine protease, matriptase, is present in Z-Mɸ at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)-14, a membrane-anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP-14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z-Mɸ, both directly and through MMP-14 activation. In summary, the expression of Z-AAT in Mɸ confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency-associated emphysema.

Alpha-1 antitrypsin deficiency is a monogenic disorder resulting in emphysema due principally to the unopposed effects of neutrophil elastase. We previously reported achieving plasma wild-type alpha-1 antitrypsin concentrations at 2.5%-3.8% of the purported therapeutic level at 1 year after a single intramuscular administration of recombinant adeno-associated virus serotype 1 alpha-1 antitrypsin vector in alpha-1 antitrypsin deficient patients. We analyzed blood and muscle for alpha-1 antitrypsin expression and immune cell response. We also assayed previously reported markers of neutrophil function known to be altered in alpha-1 antitrypsin deficient patients. Here, we report sustained expression at 2.0%-2.5% of the target level from years 1-5 in these same patients without any additional recombinant adeno-associated virus serotype-1 alpha-1 antitrypsin vector administration. In addition, we observed partial correction of disease-associated neutrophil defects, including neutrophil elastase inhibition, markers of degranulation, and membrane-bound anti-neutrophil antibodies. There was also evidence of an active T regulatory cell response (similar to the 1 year data) and an exhausted cytotoxic T cell response to adeno-associated virus serotype-1 capsid. These findings suggest that muscle-based alpha-1 antitrypsin gene replacement is tolerogenic and that stable levels of M-AAT may exert beneficial neutrophil effects at lower concentrations than previously anticipated., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

α1-Antitrypsin is primarily synthesised in the liver, circulates to the lung and protects pulmonary tissues from proteolytic damage. The Z mutant (Glu342Lys) undergoes inactivating conformational change and polymerises. Polymers are retained within the hepatocyte endoplasmic reticulum (ER) in homozygous (PiZZ) individuals, predisposing the individuals to hepatic cirrhosis and emphysema. Latency is an analogous process of inactivating, intra-molecular conformational change and may co-occur with polymerisation. However, the relationship between latency and polymerisation remained unexplored in the absence of a suitable probe. We have developed a novel monoclonal antibody specific for latent α1-antitrypsin and used it in combination with a polymer-specific antibody, to assess the association of both conformers in vitro, in disease and during augmentation therapy. In vitro kinetics analysis showed polymerisation dominated the pathway but latency could be promoted by stabilising monomeric α1-antitrypsin. Polymers were extensively produced in hepatocytes and a cell line expressing Z α1-antitrypsin but the latent protein was not detected despite manipulation of the secretory pathway. However, α1-antitrypsin augmentation therapy contains latent α1-antitrypsin, as did the plasma of 63/274 PiZZ individuals treated with augmentation therapy but 0/264 who were not receiving this medication (p<10(-14)). We conclude that latent α1-antitrypsin is a by-product of the polymerisation pathway, that the intracellular folding environment is resistant to formation of the latent conformer but that augmentation therapy introduces latent α1-antitrypsin into the circulation. A suite of monoclonal antibodies and methodologies developed in this study can characterise α1-antitrypsin folding and conformational transitions, and screen methods to improve augmentation therapy., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Oxidative stress is involved in the pathogenesis of airway obstruction in α1-antitrypsin deficient patients. This may result in a shortening of telomere length, resulting in cellular senescence. To test whether telomere length differs in α1-antitrypsin deficient patients compared with controls, we measured telomere length in DNA from peripheral blood cells of 217 α1-antitrypsin deficient patients and 217 control COPD patients. We also tested for differences in telomere length between DNA from blood and DNA from lung tissue in a subset of 51 controls. We found that telomere length in the blood was significantly longer in α1-antitrypsin deficient COPD patients compared with control COPD patients (p = 1×10(-29)). Telomere length was not related to lung function in α1-antitrypsin deficient patients (p = 0.3122) or in COPD controls (p = 0.1430). Although mean telomere length was significantly shorter in the blood when compared with the lungs (p = 0.0078), telomere length was correlated between the two tissue types (p = 0.0122). Our results indicate that telomere length is better preserved in α1-antitrypsin deficient COPD patients than in non-deficient patients. In addition, measurement of telomere length in the blood may be a suitable surrogate for measurement in the lung.

Rationale: Pulmonary emphysema overlaps partially with spirometrically defined chronic obstructive pulmonary disease and is heritable, with moderately high familial clustering., Objectives: To complete a genome-wide association study (GWAS) for the percentage of emphysema-like lung on computed tomography in the Multi-Ethnic Study of Atherosclerosis (MESA) Lung/SNP Health Association Resource (SHARe) Study, a large, population-based cohort in the United States., Methods: We determined percent emphysema and upper-lower lobe ratio in emphysema defined by lung regions less than -950 HU on cardiac scans. Genetic analyses were reported combined across four race/ethnic groups: non-Hispanic white (n = 2,587), African American (n = 2,510), Hispanic (n = 2,113), and Chinese (n = 704) and stratified by race and ethnicity., Measurements and Main Results: Among 7,914 participants, we identified regions at genome-wide significance for percent emphysema in or near SNRPF (rs7957346; P = 2.2 × 10(-8)) and PPT2 (rs10947233; P = 3.2 × 10(-8)), both of which replicated in an additional 6,023 individuals of European ancestry. Both single-nucleotide polymorphisms were previously implicated as genes influencing lung function, and analyses including lung function revealed independent associations for percent emphysema. Among Hispanics, we identified a genetic locus for upper-lower lobe ratio near the α-mannosidase-related gene MAN2B1 (rs10411619; P = 1.1 × 10(-9); minor allele frequency [MAF], 4.4%). Among Chinese, we identified single-nucleotide polymorphisms associated with upper-lower lobe ratio near DHX15 (rs7698250; P = 1.8 × 10(-10); MAF, 2.7%) and MGAT5B (rs7221059; P = 2.7 × 10(-8); MAF, 2.6%), which acts on α-linked mannose. Among African Americans, a locus near a third α-mannosidase-related gene, MAN1C1 (rs12130495; P = 9.9 × 10(-6); MAF, 13.3%) was associated with percent emphysema., Conclusions: Our results suggest that some genes previously identified as influencing lung function are independently associated with emphysema rather than lung function, and that genes related to α-mannosidase may influence risk of emphysema.

Maintaining serum levels of alpha-1-proteinase inhibitor (A1PI) >11 μM by augmentation with plasma-derived human A1PI is currently the only specific therapy available to treat patients with the genetic deficiency of A1PI. In this study, a new, high-purity (≥90% A1PI in monomeric form), ready-to-use, liquid formulation of A1PI-GLASSIA (Kamada, Ness Ziona, Israel) was compared to PROLASTINÆ (Talecris, Research Triangle Park, NC, now Grifols), both commercially available, FDA-approved products. This multicenter, double-blind, randomized controlled trial with partial cross-over was designed to test the non-inferiority and safety of GLASSIA compared to PROLASTIN, assessing both antigenic and functional A1PI trough levels in subject serum. Non-inferiority of GLASSIA to PROLASTIN was demonstrated by remaining within the lower bounds of the confidence intervals (≤3 μM) for both antigenic and functional A1PI. The study concluded that GLASSIA, a new liquid, ready to use, formulation of A1PI, was not inferior to PROLASTIN and it was well tolerated with a safety profile comparable to PROLASTIN.

Recombinant adeno-associated virus (rAAV) vectors have shown promise for the treatment of several diseases; however, immune-mediated elimination of transduced cells has been suggested to limit and account for a loss of efficacy. To determine whether rAAV vector expression can persist long term, we administered rAAV vectors expressing normal, M-type α-1 antitrypsin (M-AAT) to AAT-deficient subjects at various doses by multiple i.m. injections. M-specific AAT expression was observed in all subjects in a dose-dependent manner and was sustained for more than 1 year in the absence of immune suppression. Muscle biopsies at 1 year had sustained AAT expression and a reduction of inflammatory cells compared with 3 month biopsies. Deep sequencing of the TCR Vβ region from muscle biopsies demonstrated a limited number of T cell clones that emerged at 3 months after vector administration and persisted for 1 year. In situ immunophenotyping revealed a substantial Treg population in muscle biopsy samples containing AAT-expressing myofibers. Approximately 10% of all T cells in muscle were natural Tregs, which were activated in response to AAV capsid. These results suggest that i.m. delivery of rAAV type 1-AAT (rAAV1-AAT) induces a T regulatory response that allows ongoing transgene expression and indicates that immunomodulatory treatments may not be necessary for rAAV-mediated gene therapy.

Background: Intravenous alpha-1 antitrypsin protein (AAT) augmentation is a prescribed therapy for severe, genetically determined, alpha-1 antitrypsin deficiency (AATD), a genetic basis for pulmonary emphysema. AAT, a predominant systemic inhibitor of neutrophil elastase thus far has not been shown to decrease elastin degradation in a significant number of patients on this therapy. The objective of this study was to compare levels of biomarkers of elastin degradation in plasma, bronchoalveolar lavage (BALF) fluid and urine before and after beginning AAT augmentation therapy in patients with AATD., Methods: Desmosine and isodesmosine (DI), which occur only in elastin, are amino acid cross-links in mature elastin. Levels of DI in body fluids measure degradation of elastin and can be measured more specifically by mass spectrometry. This method was used to measure DI levels in plasma, bronchoalveolar lavage fluid and urine in cohorts of severe AATD patients on augmentation, not on augmentation and before and after the initiation of augmentation therapy., Results: Statistically significant reductions in plasma DI and in BALF DI were demonstrated in AATD patients receiving intravenous (IV) augmentation therapy as compared with those not receiving it. Administration by aerosol also produced statistically significant reductions in levels of DI in BALF., Conclusions: Results indicate that the currently prescribed doses of AAT augmentation inhibit neutrophil elastase adequately to reduce elastin degradation, both systemically and in the lung per se. The currently prescribed doses did not reduce elastin degradation to control levels, which may be possible with higher doses.

Evaluation of human antibody responses to alpha-1 antitrypsin (AAT) in clinical trials and clinical practice has been limited by the lack of a validated assay. Here we describe the development and validation of an ELISA method for quantification of human and nonhuman primate antibody responses to human AAT. A reference anti-human AAT serum standard was generated using sera from a cynomolgus macaque injected with a recombinant adeno-associated virus vector expressing human AAT. The ELISA was validated for use with human serum dilutions as low as 1:10 and was able to distinguish between specific responses in cynomolgus serum and non-specific increases in apparent antibody titer in serum from subjects in a clinical trial of an AAT gene therapy vector., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Rationale: Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli., Methods: IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro., Results: IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner., Conclusions: Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.

Objective: Deficiency of α(1) -antitrypsin (α(1) AT) may be a determinant of susceptibility to Wegener's granulomatosis (WG). Several previous, mainly small, case-control studies have shown that 5-27% of patients with WG carried the α(1) AT deficiency Z allele. It is not clear whether the S allele, the other major α(1) AT deficiency variant, is associated with WG. This study investigated the relationship of the α(1) AT deficiency Z and S alleles with the risk of developing WG in a large cohort., Methods: We studied the distribution of the α(1) AT deficiency alleles Z and S in 433 unrelated Caucasian patients with WG and 421 ethnically matched controls. Genotyping was performed using an allele discrimination assay. Results were compared between cases and controls using exact statistical methods., Results: Among the patients with WG, the allele carriage frequencies of Z and S were 7.4% and 11.5%, respectively. The frequencies of the 6 possible genotypes differed in a statistically significant manner between cases and controls (P = 0.01). The general genetic 2-parameter codominant model provided the best fit to the data. Compared with the normal MM genotype, the odds ratio (OR) for MZ or MS genotypes was 1.47 (95% confidence interval [95% CI] 0.98-2.22), and the OR for ZZ, SS, or SZ genotypes was 14.58 (95% CI 2.33-∞). ORs of similar direction and magnitude were observed within the restricted cohorts that excluded cases and controls carrying ≥1 Z or ≥1 S allele., Conclusion: Both Z and S alleles display associations with risk of WG in a codominant genetic pattern. These findings strengthen the evidence of a causal link between α(1) AT deficiency and susceptibility to WG., (Copyright © 2010 by the American College of Rheumatology.)

Rationale: Individuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown., Objectives: To investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied., Methods: Forty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed., Measurements and Main Results: Concentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P < 0.001). In vitro, HPS-1 pulmonary fibrosis alveolar macrophages, which did not express HPS1 mRNA, secreted significantly higher concentrations of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and regulated upon activation, normal T cell expressed and secreted (RANTES) protein compared with normal cells (P = 0.001, P = 0.014, and P = 0.011, respectively). Pirfenidone suppressed HPS-1 alveolar macrophage cytokine and chemokine secretion in vitro in a dose-dependent manner., Conclusions: In HPS-1, alveolar inflammation predominantly involves macrophages and is associated with high lung concentrations of cytokines and chemokines. HPS-1 alveolar macrophages provide a model system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released to the extracellular space by two mechanisms, the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains. Both pathways appear to be regulated by an interaction between TNFR1 and ARTS-1 (aminopeptidase regulator of TNFR1 shedding). Here, we sought to identify ARTS-1-interacting proteins that modulate TNFR1 release. Co-immunoprecipitation identified an association between ARTS-1 and RBMX (RNA-binding motif gene, X chromosome), a 43-kDa heterogeneous nuclear ribonucleoprotein. RNA interference attenuated RBMX expression, which reduced both the constitutive release of TNFR1 exosome-like vesicles and the IL-1beta-mediated inducible proteolytic cleavage of soluble TNFR1 ectodomains. Reciprocally, over-expression of RBMX increased TNFR1 exosome-like vesicle release and the IL-1beta-mediated inducible shedding of TNFR1 ectodomains. This identifies RBMX as an ARTS-1-associated protein that regulates both the constitutive release of TNFR1 exosome-like vesicles and the inducible proteolytic cleavage of TNFR1 ectodomains.

Extracellular type I tumor necrosis factor receptors (TNFR1) are generated by two mechanisms, proteolytic cleavage of TNFR1 ectodomains and release of full-length TNFR1 in the membranes of exosome-like vesicles. Here, we assessed whether TNFR1 exosome-like vesicles circulate in human blood. Immunoelectron microscopy of human serum demonstrated TNFR1 exosome-like vesicles, with a diameter of 27-36nm, while Western blots of human plasma showed a 48-kDa TNFR1, consistent with a membrane-associated receptor. Gel filtration chromatography revealed that the 48-kDa TNFR1 in human plasma co-segregated with LDL particles by size, but segregated independently by density, demonstrating that they are distinct from LDL particles. Furthermore, the 48-kDa exosome-associated TNFR1 in human plasma contained a reduced content of N-linked carbohydrates as compared to the 55-kDa membrane-associated TNFR1 from human vascular endothelial cells. Thus, a distinct population of TNFR1 exosome-like vesicles circulate in human plasma and may modulate TNF-mediated inflammation.

Extracellular tumor necrosis factor (TNF) receptors function as TNF-binding proteins that modulate TNF activity. In human vascular endothelial cells (HUVEC), extracellular TNFR1 (type I TNF receptor, TNFRSF1A) is generated by two mechanisms, proteolytic cleavage of soluble TNFR1 ectodomains and the release of full-length 55-kDa TNFR1 in the membranes of exosome-like vesicles. TNFR1 release from HUVEC is known to involve the association between ARTS-1 (aminopeptidase regulator of TNFR1 shedding), an integral membrane aminopeptidase, and TNFR1. The goal of this study was to identify ARTS-1 binding partners that modulate TNFR1 release to the extracellular space. A yeast two-hybrid screen of a human placenta cDNA library showed that NUCB2 (nucleobindin 2), via its helix-loop-helix domains, binds the ARTS-1 extracellular domain. The association between endogenous ARTS-1 and NUCB2 in HUVEC was demonstrated by co-immunoprecipitation experiments, which showed the formation of a calcium-dependent NUCB2.ARTS-1 complex that associated with a subset of total cellular TNFR1. Confocal microscopy experiments demonstrated that this association involved a distinct population of NUCB2-containing intracytoplasmic vesicles. RNA interference was utilized to specifically knock down NUCB2 and ARTS-1 expression, which demonstrated that both are required for the constitutive release of a full-length 55-kDa TNFR1 within exosome-like vesicles as well as the inducible proteolytic cleavage of soluble TNFR1 ectodomains. We propose that calcium-dependent NUCB2.ARTS-1 complexes, which associate with TNFR1 prior to its commitment to pathways that result in either the constitutive release of TNFR1 exosome-like vesicles or the inducible proteolytic cleavage of TNFR1 ectodomains, play an important role in mediating TNFR1 release to the extracellular compartment.

Objective: To assess whether tumor necrosis factor (TNF) antagonism can attenuate eosinophilic airway inflammation in patients with mild-to-moderate allergic asthma., Design: Randomized, double-blind, placebo-controlled trial., Setting: National Institutes of Health (NIH) Clinical Center., Patients: Twenty-six patients with mild-to-moderate allergic asthma, receiving only inhaled beta-2-agonists, who demonstrated both an early and late phase response to inhalational allergen challenge., Intervention: Injection of a soluble TNF receptor (TNFR:Fc, etanercept, Enbrel) or placebo, 25mg subcutaneously, twice weekly for 2 weeks, followed by a bronchoscopic segmental allergen challenge., Measurements: The primary outcome measure was whether TNFR:Fc can access the lung and inhibit TNF bioactivity. Secondary outcome measures included pulmonary eosinophilia, Th2-type cytokines, and airway hyperresponsiveness., Results: Anti-TNF therapy was associated with transient hemiplegia in one patient, which resulted in suspension of the study. Data from the 21 participants who completed the study were analyzed. Following treatment, patients receiving anti-TNF therapy had significantly increased TNFR2 levels in epithelial lining fluid (ELF) (P<0.001), consistent with delivery of TNFR:Fc to the lung. TNF antagonism did not attenuate pulmonary eosinophilia and was associated with an increase in ELF IL-4 levels (P=0.033) at 24h following segmental allergen challenge. TNF antagonism was not associated with a change in airway hyperresponsiveness to methacholine., Conclusions: TNF antagonism may not be effective for preventing allergen-mediated eosinophilic airway inflammation in mild-to-moderate asthmatics. Transient hemiplegia, which may mimic an evolving stroke, may be a potential toxicity of anti-TNF therapy.

The type 1 55-kDa TNF receptor (TNFR1) is an important modulator of lung inflammation. Here, we hypothesized that the proteasome might regulate TNFR1 shedding from human airway epithelial cells. Treatment of NCI-H292 human airway epithelial cells for 2 h with the specific proteasome inhibitor clasto-lactacystin beta-lactone induced the shedding of proteolytically cleaved TNFR1 ectodomains. Clasto-lactacystin beta-lactone also induced soluble TNFR1 (sTNFR1) release from the A549 pulmonary epithelial cell line, as well as from primary cultures of human small airway epithelial cells and human umbilical vein endothelial cells. Furthermore, sTNFR1 release induced by clasto-lactacystin beta-lactone was not a consequence of apoptosis or the extracellular release of TNFR1 exosome-like vesicles. The clasto-lactacystin beta-lactone-induced increase in TNFR1 shedding was associated with reductions in cell surface receptors and intracytoplasmic TNFR1 stores that were primarily localized to vesicular structures. As expected, the broad-spectrum zinc metalloprotease inhibitor TNF-alpha protease inhibitor 2 (TAPI-2) attenuated clasto-lactacystin beta-lactone-mediated TNFR1 shedding, which is consistent with its ability to inhibit the zinc metalloprotease-catalyzed cleavage of TNFR1 ectodomains. TAPI-2 also reduced TNFR1 on the cell surface and attenuated the clasto-lactacystin beta-lactone-induced reduction of intracytoplasmic TNFR1 vesicles. This suggests that TNFR1 shedding induced by clasto-lactacystin beta-lactone involves the zinc metalloprotease-dependent trafficking of intracytoplasmic TNFR1 vesicles to the cell surface. Together, these data are consistent with the conclusion that proteasomal activity negatively regulates TNFR1 shedding from human airway epithelial cells, thus identifying previously unrecognized roles for the proteasome and zinc metalloproteases in modulating the generation of sTNFRs.

TNF-alpha-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18-54 (N-TACE(18-54)) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE(18-54) attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE(18-54) inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30-48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.

Individuals with alpha(1)-antitrypsin (alpha(1)-AT) deficiency are at risk for early-onset destructive lung disease as a result of insufficient lower respiratory tract alpha(1)-AT and an increased burden of neutrophil products such as elastase. Human neutrophil peptides (HNP), the most abundant protein component of neutrophil azurophilic granules, represent another potential inflammatory component in lung disease characterized by increased numbers of activated or deteriorating neutrophils. The purpose of this study was to determine the role of HNP in lower respiratory tract inflammation and destruction occuring in alpha(1)-AT deficiency. alpha(1)-AT-deficient individuals (n = 33) and healthy control subjects (n = 21) were evaluated by bronchoalveolar lavage. HNP concentrations were significantly higher in alpha(1)-AT-deficient individuals (1,976 +/- 692 vs. 29 +/- 12 nM, P < 0.0001), and levels correlated with markers of neutrophil-mediated lung inflammation. In vitro, HNP produced a dose-dependent cytotoxic effect on alveolar macrophages and stimulated production of the potent neutrophil chemoattractants leukotriene B(4) and interleukin-8 by alveolar macrophages, with a 6- to 10-fold increase in chemoattractant production over negative control cultures (P < 0.05). A synergistic effect was noted between HNP and neutrophil elastase with regard to leukotriene B(4) production. Importantly, the proinflammatory effects of HNP were blocked by alpha(1)-AT. HNP likely play an important role in amplifying and maintaining neutrophil-mediated inflammation in the lungs.

Soluble tumor necrosis factor receptors (TNFRs) are important modulators of TNF bioactivity. Proteolytic cleavage of the 28-kDa ectodomain of TNFR1 has been recognized as the mechanism by which soluble TNFR is shed. We now describe the release of exosome-like vesicles as a mechanism for the generation of soluble, full-length 55-kDa TNFR1. We found unexpectedly that the predominant form of soluble TNFR1 in human serum and lung epithelial lining fluid is a full-length 55-kDa protein. Furthermore, supernatants from human vascular endothelial cells contain only full-length 55-kDa TNFR1 that can be sedimented by high-speed centrifugation, floated on sucrose gradients at a density of 1.1 g/ml, and associated with vesicles that range in diameter from 20 nm to 50 nm. We conclude that the release of TNFR1 exosome-like vesicles represents a previously unrecognized mechanism by which constitutive production of soluble cytokine receptors may be regulated, independent of ectodomain cleavage by receptor sheddases.

Proteolytic cleavage of the extracellular domain of the type II IL-1 decoy receptor (IL-1RII) generates soluble IL-1-binding proteins that prevent excessive bioactivity by binding free IL-1. In this study we report that an aminopeptidase, aminopeptidase regulator of TNFR1 shedding (ARTS-1), is required for IL-1RII shedding. Coimmunoprecipitation experiments demonstrate an association between endogenous membrane-associated ARTS-1 and a 47-kDa IL-1RII, consistent with ectodomain cleavage of the membrane-bound receptor. A direct correlation exists between ARTS-1 protein expression and IL-1RII shedding, as cell lines overexpressing ARTS-1 have increased IL-1RII shedding and decreased membrane-associated IL-1RII. Basal IL-1RII shedding is absent from ARTS-1 knockout cell lines, demonstrating that ARTS-1 is required for constitutive IL-1RII shedding. Similarly, PMA-mediated IL-1RII shedding is almost entirely ARTS-1-dependent. ARTS-1 expression also enhances ionomycin-induced IL-1RII shedding. ARTS-1 did not alter levels of membrane-associated IL-1RI or IL-1R antagonist release from ARTS-1 cell lines, which suggests that the ability of ARTS-1 to promote shedding of IL-1R family members may be specific for IL-1RII. Further, increased IL-1RII shedding by ARTS-1-overexpressing cells attenuates the biological activity of IL-1beta. We conclude that the ability of ARTS-1 to enhance IL-1RII shedding represents a new mechanism by which IL-1-induced cellular events can be modulated. As ARTS-1 also promotes the shedding of the structurally unrelated 55-kDa, type I TNF receptor and the IL-6R, we propose that ARTS-1 may play an important role in regulating innate immune and inflammatory responses by increasing cytokine receptor shedding.

Aminopeptidase regulator of TNFR1 shedding (ARTS-1) binds to the type I tumor necrosis factor receptor (TNFR1) and promotes receptor shedding. Because hydroxamic acid-based metalloprotease inhibitors prevent shedding of both TNFR1 and the interleukin-6 receptor (IL-6Ralpha), we hypothesized that ARTS-1 might also regulate shedding of IL-6Ralpha, a member of the type I cytokine receptor superfamily that is structurally different from TNFR1. Reciprocal co-immunoprecipitation experiments identified that membrane-associated ARTS-1 directly binds to a 55-kDa IL-6Ralpha, a size consistent with soluble IL-6Ralpha generated by ectodomain cleavage of the membrane-bound receptor. Furthermore, ARTS-1 promoted IL-6Ralpha shedding, as demonstrated by a direct correlation between increased membrane-associated ARTS-1 protein, increased IL-6Ralpha shedding, and decreased membrane-associated IL-6Ralpha in cell lines overexpressing ARTS-1. The absence of basal IL-6Ralpha shedding from arts-1 knock-out cells identified that ARTS-1 was required for constitutive IL-6Ralpha shedding. Furthermore, the mechanism of constitutive IL-6Ralpha shedding requires ARTS-1 catalytic activity. Thus, ARTS-1 promotes the shedding of two cytokine receptor superfamilies, the type I cytokine receptor superfamily (IL-6Ralpha) and the TNF receptor superfamily (TNFR1). We propose that ARTS-1 is a multifunctional aminopeptidase that may modulate inflammatory events by promoting IL-6Ralpha and TNFR1 shedding.

Proteolytic cleavage of TNF receptor 1 (TNFR1) generates soluble receptors that regulate TNF bioactivity. We hypothesized that the mechanism of TNFR1 shedding might involve interactions with regulatory ectoproteins. Using a yeast two-hybrid approach, we identified ARTS-1 (aminopeptidase regulator of TNFR1 shedding) as a type II integral membrane protein that binds to the TNFR1 extracellular domain. In vivo binding of membrane-associated ARTS-1 to TNFR1 was confirmed by coimmunoprecipitation experiments using human pulmonary epithelial and umbilical vein endothelial cells. A direct relationship exists between membrane-associated ARTS-1 protein levels and concordant changes in TNFR1 shedding. Cells overexpressing ARTS-1 demonstrated increased TNFR1 shedding and decreased membrane-associated TNFR1, while cells expressing antisense ARTS-1 mRNA demonstrated decreased membrane-associated ARTS-1, decreased TNFR1 shedding, and increased membrane-associated TNFR1. ARTS-1 neither bound to TNFR2 nor altered its shedding, suggesting specificity for TNFR1. Although a recombinant ARTS-1 protein demonstrated selective aminopeptidase activity toward nonpolar amino acids, multiple lines of negative evidence suggest that ARTS-1 does not possess TNFR1 sheddase activity. These data indicate that ARTS-1 is a multifunctional ectoprotein capable of binding to and promoting TNFR1 shedding. We propose that formation of a TNFR1-ARTS-1 molecular complex represents a novel mechanism by which TNFR1 shedding is regulated.