Sabine Wrenger

α1-Antitrypsin Modulates Lung Endothelial Cell Inflammatory Responses to TNF-α

α₁-Antitrypsin (A1AT) is an acute-phase reactant, but also a major protective factor against the development of chronic obstructive pulmonary disease, a complex disease with sustained chronic inflammation. The lung-protective effects of A1AT have been attributed to the inhibition of proteases involved in lung matrix fragmentation, macrophage activation, and endothelial-cell apoptosis. More recently, A1AT has been shown to directly interact with or modulate the actions of cytokines such as TNF-α or IL-1 in inflammatory cells, but its effect on the lung endothelium, an active participant in the amplification and resolution of inflammation, has received little attention. An important role of A1AT in modulating lung endothelial inflammatory responses is expected, given the high concentrations of circulating A1AT during inflammation and its active uptake by endothelial cells. We investigated the role of A1AT in primary lung microvascular endothelial cell activation by relevant cytokines such as TNF-α or IL-1β. Despite an initial marked augmentation of TNF-α self-induced transcription, A1AT inhibited TNF-α receptor 1 up-regulation and significantly reduced TNF-α secretion, effects that were associated with inhibition of TNF-α-converting enzyme activity. Furthermore, A1AT inhibited calpain activity, whose activation by TNF-α contributed to decreased intracellular A1AT concentrations. These data indicate that A1AT initially facilitates acute responses of the endothelium to TNF-α, followed by selective inhibition of TNF-α-induced-self amplification, which may assist the vasculature in the resolution of chronic inflammation.

α1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression

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.

Acute-Phase Protein α1-Antitrypsin—A Novel Regulator of Angiopoietin-like Protein 4 Transcription and Secretion

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.

Krüppel-like zinc finger proteins in end-stage COPD lungs with and without severe alpha1-antitrypsin deficiency

BackgroundChronic obstructive pulmonary disease (COPD) is influenced by environmental and genetic factors. An important fraction of COPD cases harbor a major genetic determinant, inherited ZZ (Glu342Lys) α1-antitrypsin deficiency (AATD). A study was undertaken to investigate gene expression patterns in end-stage COPD lungs from patients with and without AATD.MethodsExplanted lungs of end-stage ZZ AATD-related (treated and non-treated with AAT augmentation therapy) and “normal” MM COPD, and liver biopsies from patients suffering from liver cirrhosis with and without ZZ AATD were used for gene expression analysis by Affymetrix microarrays or RT-PCR.ResultsA total of 162 genes were found to be differentially expressed (p-value ≤ 0.05 and |FC| ≥ 2) between MM and ZZ COPD patients. Of those, 134 gene sets were up-regulated and 28 were down-regulated in ZZ relative to MM lung tissue. A subgroup of genes, zinc finger protein 165, snail homolog 1 (Drosophila) (SNAI1), and Krüppel-like transcription factors (KLFs) 4 (gut), 9 and 10, perfectly segregated ZZ and MM COPD patients. The higher expression of KLF 9 and KLF10 has been verified in the replication cohort with AATD-related end-stage lung emphysema and liver cirrhosis. Furthermore, higher expression of KLF9, SNAI1 and DEFA1 was found in ZZ COPD lungs without augmentation therapy relative to MM COPD or ZZ COPD with augmentation therapy.ConclusionsThese results reveal the involvement of transcriptional regulators of the zinc-finger family in COPD pathogenesis and provide deeper insight into the pathophysiological mechanisms of COPD with and without AATD.

The Multifaceted Effects of Alpha1-Antitrypsin on Neutrophil Functions

Neutrophils are the predominant immune cells in human blood possessing heterogeneity, plasticity and functional diversity. The activation and recruitment of neutrophils into inflamed tissue in response to stimuli are tightly regulated processes. Alpha1-Antitrypsin (AAT), an acute phase protein, is one of the potent regulators of neutrophil activation via both -protease inhibitory and non-inhibitory functions. This review summarizes our current understanding of the effects of AAT on neutrophils, illustrating the interplay between AAT and the key effector functions of neutrophils.

Alpha1‐antitrypsin binds hemin and prevents oxidative activation of human neutrophils: putative pathophysiological significance

Heme is a ubiquitous compound of human tissues, and it is involved in cellular physiology and metabolism. Once released from the cell, free heme oxidizes to the ferric state (hemin). High levels of hemin can cause oxidative stress and inflammation if not neutralized immediately by specialized scavenger proteins. Human alpha1‐antitrypsin (A1AT), an acute‐phase glycoprotein and important inhibitor of neutrophil proteases, is also a hemin‐binding protein. A short‐term exposure of freshly isolated human blood neutrophils to 4 µM hemin results in cell spreading, surface expression of filament protein, vimentin, free radical production, expression of heme oxygenase‐1 (HO‐1), release of IL‐8, and enhanced neutrophil adhesion to human endothelial cells. Consequently, the phosphorylation of protein kinase C (PKC) occurs after 25 min. Under the same experimental conditions, addition of 1 mg/ml A1AT markedly reduces or abolishes neutrophil‐activating effects of hemin and prevents PKC phosphorylation. In a mouse model of acute kidney injury (AKI) plus injection of hemin, monotherapy with 4 mg/mouse A1AT significantly lowered serum levels of free hemin at 2 h after surgery. Moreover, a tendency toward lower AKI scores, reduced infiltration of neutrophils, and lower levels of serum chemokine [CXCL1/keratinocyte‐derived chemokine (KC)] was observed. Our findings highlight A1AT as a potential serum scavenger of hemin and suggest that the commercial preparations of human plasma A1AT might prove to be useful therapeutics in conditions associated with hemolysis.

Elastase inhibition is not a requirement for the anti-inflammatory effects of Alpha-1 Antitrypsin: studies in neutrophil models

As an acute phase protein, alpha-antitrypsin (AAT) is thought to play an important role in limiting host tissue injury by proteases released from activated myeloid cells, specifically neutrophil elastase (NE). The clinical importance of AAT is highlighted in individuals with inherited deficiency in circulating AAT who have an increased susceptibility to early onset pulmonary emphysema, chronic bronchitis, bronchiectasis, and liver diseases. Administration of exogenous human plasma-derived AAT is used in various animal models to test the value of AAT augmentation therapy. Unrelated to emphysema models, administration of exogenous AAT inhibits HIV 1 expression, prevents murine islet cell allografts from rejection, blocks cell apoptosis and increases survival in an allogeneic marrow transplantation models. The mechanism of the above mentioned anti-inflammatory activities of AAT has been assumed to be due to its anti-proteolytic activity; however this has not been studied. We hypothesized that the anti-elastase activity of AAT was not required for its anti-inflammatory properties. To investigate this prediction, effects of native human plasma derived (nAAT, inhibitory form) and recombinant (rAAT, non-inhibitory form) on lipopolysaccharide (LPS)-induced IL-8 and TNFα release from neutrophils isolated from human blood or the bone marrow from wild type (WT) and elastase deficient mice (NE-/-) were examined. When WT or NE deficient neutrophils were treated with LPS plus rAAT (50 nM), release of TNFα (42%, p=0.002 and 24%, p=0.011, respectively) as well as of IL-8 was decreased (57%, p=0.004 and 40%, p=0.01, respectively) relative to LPS challenged cells. By comparison, to achieve the same reduction in TNFα from either WT or NE deficient neutrophils, a concentration of nAAT of 2000 nM was required. Next, human blood neutrophils were incubated with LPS (10ng/ml) alone or LPS plus increasing concentrations of nAAT or rAAT. After 8 and 18 hours, cytokines were measured in the supernatants. At 20.000 nM, nAAT reduced TNFα release by 46% (p=0.002) and IL-8 by 29%, (p=0.024). However, markedly lower concentrations of rAAT (5nM) reduced TNFα release by 41% (p=0.004) and IL-8 by 40% (p=0.05). We conclude that anti-proteolytic activity of AAT may be required but less important for its anti-inflammatory function. Acknowledgments: This study was supported by Soohyun Kim who provided the rAAT and our collaborator Charles A. Dinarello.