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Alpha-1 antitrypsin (AAT) is an acute phase protein that possesses immune-regulatory and anti-inflammatory functions independent of antiprotease activity. AAT deficiency (AATD) is associated with early-onset emphysema and chronic obstructive pulmonary disease. Of interest are the AATD nonsense mutations (termed null or Q0), the majority of which arise from premature termination codons in the mRNA coding region. We have recently demonstrated that plasma from an AATD patient homozygous for the Null Bolton allele ( Q0 bolton ) contains AAT protein of truncated size. Although the potential to alleviate the phenotypic consequences of AATD by increasing levels of truncated protein holds therapeutic promise, protein functionality is key. The goal of this study was to evaluate the structural features and anti-inflammatory capacity of Q0 bolton -AAT. A low-abundance, truncated AAT protein was confirmed in plasma of a Q0 bolton -AATD patient and was secreted by patient-derived induced pluripotent stem cell-hepatic cells. Functional assays confirmed the ability of purified Q0 bolton -AAT protein to bind neutrophil elastase and to inhibit protease activity. Q0 bolton -AAT bound IL-8 and leukotriene B 4 , comparable to healthy control M-AAT, and significantly decreased leukotriene B 4 -induced neutrophil adhesion ( p = 0.04). Through a mechanism involving increased mRNA stability ( p = 0.007), ataluren treatment of HEK-293 significantly increased Q0 bolton -AAT mRNA expression ( p = 0.03) and Q0 bolton -AAT truncated protein secretion ( p = 0.04). Results support the rationale for treatment with pharmacological agents that augment levels of functional Q0 bolton -AAT protein, thus offering a potential therapeutic option for AATD patients with rare mutations of similar theratype., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Leukotriene B4 (LTB4) contributes to many inflammatory diseases, including genetic and nongenetic forms of chronic obstructive pulmonary disease. α-1 Antitrypsin (AAT) deficiency (AATD) is characterized by destruction of lung parenchyma and development of emphysema, caused by low AAT levels and a high neutrophil burden in the airways of affected individuals. In this study we assessed whether AATD is an LTB4-related disease and investigated the ability of serum AAT to control LTB4 signaling in neutrophils. In vitro studies demonstrate that neutrophil elastase is a key player in the LTB4 inflammatory cycle in AATD, causing increased LTB4 production, and associated BLT1 membrane receptor expression. AATD patients homozygous for the Z allele were characterized by increased neutrophil adhesion and degranulation responses to LTB4. We demonstrate that AAT can bind LTB4 and that AAT/LTB4 complex formation modulates BLT1 engagement and downstream signaling events, including 1,4,5-triphosphate production and Ca(2+) flux. Additionally, treatment of ZZ-AATD individuals with AAT augmentation therapy decreased plasma LTB4 concentrations and reduced levels of membrane-bound neutrophil elastase. Collectively, these results provide a mechanism by which AAT augmentation therapy impacts on LTB4 signaling in vivo, and not only reinforces the utility of this therapy for resolving inflammation in AATD, but supports useful future clinical applications in treatment of other LTB4-related diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) β/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The angiopoietin-like protein 4 (angptl4, also known as peroxisome proliferator-activated receptor [PPAR]γ-induced angiopoietin-related protein) is a multifunctional protein associated with acute-phase response. The mechanisms accounting for the increase in angptl4 expression are largely unknown. This study shows that human α1-antitrypsin (A1AT) upregulates expression and release of angplt4 in human blood adherent mononuclear cells and in primary human lung microvascular endothelial cells in a concentration- and time-dependent manner. Mononuclear cells treated for 1 h with A1AT (from 0.1 to 4 mg/ml) increased mRNA of angptl4 from 2- to 174-fold, respectively, relative to controls. In endothelial cells, the maximal effect on angptl4 expression was achieved at 8 h with 2 mg/ml A1AT (11-fold induction versus controls). In 10 emphysema patients receiving A1AT therapy (Prolastin), plasma angptl4 levels were higher relative to patients without therapy (nanograms per milliliter, mean [95% confidence interval] 127.1 [99.5-154.6] versus 76.8 [54.8-98.8], respectively, p = 0.045) and correlated with A1AT levels. The effect of A1AT on angptl4 expression was significantly diminished in cells pretreated with a specific inhibitor of ERK1/2 activation (UO126), irreversible and selective PPARγ antagonist (GW9662), or genistein, a ligand for PPARγ. GW9662 did not alter the ability of A1AT to induce ERK1/2 phosphorylation, suggesting that PPARγ is a critical mediator in the A1AT-driven angptl4 expression. In contrast, the forced accumulation of HIF-1α, an upregulator of angptl4 expression, enhanced the effect of A1AT. Thus, acute-phase protein A1AT is a physiological regulator of angptl4, another acute-phase protein., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The recognition of peptide Ags by T cells through the TCR has exquisite specificity. Cross-reactive T cell responses have been described; however, the structural basis for these responses is not known. We show that two peptides derived from the same protein can exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive T cell responses. In addition, we explore the structural basis for cross-reactivity. T cell hybridomas recognizing PiM and PiZ allelic forms of human alpha 1-antitrypsin (hAAT) each recognized both PiM 205-220 and PiM 335-350. These two peptides possessed primary sequence identity at only two of 16 amino acid residues. Cross-reactive peptides also exhibited homology at the bulk T cell level because lymph node T cells primed with one peptide proliferated to the other peptide in vitro. Critical amino acids for the responding T cells were determined, and the core was transferred into the less reactive peptide in an attempt to increase homology by increasing sequence identity. Interestingly, as identity increased, homology decreased: peptides with the least primary sequence identity appeared most homologous to the T cells. These results have important implications for understanding the development of autoimmune diseases, and imply that minimal obvious primary sequence identity may be sufficient to initiate cross-reactive T cell responses. The ability of structurally dissimilar peptides to mimic each other when bound to a class II MHC molecule may also be important to the understanding of T development and autoimmunity.

The sequence motif of peptide ligands naturally associated with DR17 has indicated conserved residues at the relative positions P1-P4-P6-P 8.9 or 10. Eight naturally processed DR17 ligands were synthesized to study the role of conserved residues in DR17 binding. In their majority, they showed an excellent ability to bind to purified DR17 molecules. Binding experiments with variant peptides confirmed aspartate as the DR17-specific contact site at P4. In addition, hydrophobic or aromatic residues at P1 and P9, probably interacting with the NH2- and COOH-terminal pockets, and lysine or chemically related amino acids at P6 were important for binding. A core peptide of 10 amino acids, bordered by the terminal contact sites, is sufficient, although the ability to bind is reduced approximately 10-fold compared with the binding capacity of the natural ligand. Ala substitution of flanking stretches at either end completely restores the binding capacity to that of the natural ligand. This suggests that regions flanking the peptide core contribute to the binding strength nonspecifically, i.e., by forming H-bonds to MHC molecules. Natural DR1 and DR12 ligands like HLA-A2 (103-117) and transferrin receptor (140-156) failed to bind to DR17 molecules. However, substituting leucine for aspartate at P4 transformed DR1 and DR12 ligands into excellent DR17 binders. This conversion, enabled by a single amino acid substitution, emphasizes the importance of aspartate as the DR17-specific contact site and suggests that terminal contact residues are shared among DR1, DR12, and DR17 ligands. In contrast, additional aspartates introduced next to the contact site at P4 impaired the binding capacity. Regarding this specific role of asparate we expect that DR17-specific ligands will be rarely found among "promiscuous" peptides binding to several different DR molecules.

Human lymphocytes that have undergone concanavalin A-induced blastogenetic transformation demonstrate surface alpha-1-antitrypsin by immunofluorescence when examined after 72 hr of culture, whereas unstimulated cells do not. These results may indicate a role for alpha-1-antitrypsin in lymphocyte blastogenesis.