Log₁₀ of telomere length in the lung vs. log₁₀ of telomere length in the blood.

<p>Log₁₀ of telomere length in the lung vs. log₁₀ of telomere length in the blood.</p

Log₁₀ of telomere length in α₁-antitrypsin deficient COPD patients vs. log₁₀ of telomere length in COPD controls.

<p>Log₁₀ of telomere length in α₁-antitrypsin deficient COPD patients vs. log₁₀ of telomere length in COPD controls.</p

Mean log₁₀ of telomere length in the blood and lung tissue.

<p>Mean log₁₀ of telomere length in the blood and lung tissue.</p

Cytosolic, Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

<div><h3>Rationale</h3><p>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.</p> <h3>Methods</h3><p>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.</p> <h3>Results</h3><p>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.</p> <h3>Conclusions</h3><p>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.</p> </div

Demographic and genotypic characteristics of subjects from the Alpha-1 Foundation DNA and Tissue Bank and the Lung Health Study.

<p>*Mean ± standard error of the mean.</p

A1PI Impacts ASC Dependent IL-1β Release in Transfected Monocytes.

<p>A-B) THP-1 cells were nucleofected with plasmids encoding wild type A1PI (A1PI-M) or Z isoform A1PI (A1PI-Z). Cells were stimulated with PMA or PBS control for 5 days, and A1PI (A) and IL-1β (B) were detected in supernatant by ELISA. C) Secreted IL-6, IL-8, TNF-α and IL-1 β were detected in supernatants of THP-1 cells or THP-1 cells lacking ASC, incubated with PMA or PBS control for 5 days. D-E) THP-1 cells or THP-1 cells lacking ASC were nucleofected with vectors expressing A1PI-M or A1PI-Z, then challenged with PMA. Secreted IL-1β (D) and TNF- α (E) were detected in supernatants collected on day 5. F) THP-1 cells stably expressing A1PI-Z were pre-treated with caspase-1 inhibitor (40 µM) or plasma-derived A1PI (1 mg/ml), then PMA stimulated for 18 hours followed by 20 µM nigericin challenge. Secreted IL-1β was measured in supernatants collected up to 120 minutes post nigericin. Asterisks denote P≤0.05.</p

A1PI and Cytokine Secretion in Cultured Monocytes.

<p>A) IL-1β and TLR4 transcripts were evaluated by quantitative PCR in alveolar macrophages acquired by bronchoalveolar lavage from PIZZ (squares) or PIMM (circles) outpatient volunteers (n = 3–4). Bars indicate the mean values for PIZZ (filled bar) and PIMM (dashed bar) samples. B) Elutriated peripheral blood monocytes of healthy volunteers (n = 3) were cultured 5 days with or without PMA and LPS stimuli as indicated. Secreted A1PI and IL-1β were detected in collected supernatants by ELISA. C-E) Monocytic cell lines were cultured 5 days with or without PMA and LPS stimuli as indicated. Secreted A1PI (C), IL-1β (D), and TNF-α (E) were detected in collected supernatants by ELISA. E) THP-1 and U937 cultured 4 days with PBS control or with PMA were washed, and LPS (100 ng/ml) was added to selected wells. Released nucleosome:DNA complexes were assessed in supernatants collected at 24 hours. Asterisks denote P≤0.05 compared with unstimulated control cells. For (A) through (E), representative results of 3 experiments are shown.</p

A1PI Interacts with Caspase-1 in Monocyte Cytosol.

<p>A) Cytosolic preparations of THP-1 cells activated 18 hours with PMA were applied to a Superdex S-200 column. Fractions 6 through 24 were analyzed by SDS-PAGE and immunoblot with antibodies directed to A1PI (top panel), caspase-1 (middle panel), and ASC (bottom panel). B) THP-1 cells activated overnight with PMA were pulsed for 1 hour with 20 µM nigericin before hypotonic lysis. Immunoprecipitation was performed using an irrelevant control IgG or A1PI-directed antibodies. Precipitated proteins were analyzed by immunoblot with anti-A1PI (top panel) or anti-caspase-1 (lower panel). C) Graded amounts of plasma derived A1PI, BSA control, or small molecule caspase-1 inhibitor, were coincubated with recombinant active caspase-1 prior to addition of a fluorescent peptide substrate. Reactions were incubated at 37 °C, and fluorescence over time in duplicate wells (mean and STD) were measured by platereader. D) Gelatin control protein or plasma derived A1PI were conjugated to agarose beads, then incubated with recombinant caspase-1. After extensive washing, proteins were eluted with SDS-PAGE buffer and analyzed by immunoblot for bands reactive with a caspase-1 directed polyclonal antibody.</p

Cytosolic Monocyte-Expressed A1PI is Glycosylated.

<p>A) Immunoblot detection of A1PI in whole cell extracts of U937 and THP-1 monocytes with and without PMA stimulation. B) U937 cells or primary elutriated monocytes of two healthy donors (Mono-1, Mono-2) were lysed with hypotonic buffer. Cytosol (C) and membrane (M) fractions were analyzed by immunoblot for A1PI and the membrane associated protein CD44. C) THP-1 cytosolic samples prepared by hypotonic lysis and plasma-derived A1PI were incubated at 37°C with or without N-glycanase prior to immunoblot detection of A1PI. D) THP-1 cells were cultured with PMA with or without nocodazole stimulation for 18 hours. Cytosolic samples prepared by hypotonic lysis, and A1PI (upper panel) and β-actin loading control (lower panel) were analyzed by immunoblot. A1PI bands were quantified by densitometry (right).</p

Cell Associated A1PI Detection in Human Lung Tissue.

<p>Lung tissue acquired at autopsy from patients with advanced lung disease associated with cystic fibrosis (n = 8, top row) and acute fatal respiratory syncytial virus infection (n = 9, bottom row). Representative detection of macrophage antigen CD68 (left panels), A1PI (middle panels), or irrelevant species-matched control antibody (right panels) are shown. Bar = 30 µm top row, 15 µm bottom row.</p