Alpha-1 antitrypsin inhibits fractalkine-mediated monocyte-lung endothelial cell interactions

Abstract

Chronic obstructive pulmonary disease (COPD) is characterized by nonresolving inflammation fueled by breach in the endothelial barrier and leukocyte recruitment into the airspaces. Among the ligand-receptor axes that control leukocyte recruitment, the full-length fractalkine ligand (CX3CL1)-receptor (CX3CR1) ensures homeostatic endothelial-leukocyte interactions. Cigarette smoke (CS) exposure and respiratory pathogens increase expression of endothelial sheddases, such as a-disintegrin-and-metalloproteinase-domain 17 (ADAM17, TACE), inhibited by the anti-protease α-1 antitrypsin (AAT). In the systemic endothelium, TACE cleaves CX3CL1 to release soluble CX3CL1 (sCX3CL1). During CS exposure, it is not known whether AAT inhibits sCX3CL1 shedding and CX3CR1⁺ leukocyte transendothelial migration across lung microvasculature. We investigated the mechanism of sCX3CL1 shedding, its role in endothelial-monocyte interactions, and AAT effect on these interactions during acute inflammation. We used two, CS and lipopolysaccharide (LPS) models of acute inflammation in transgenic Cx3cr1^gfp/gfp mice and primary human endothelial cells and monocytes to study sCX3CL1-mediated CX3CR1⁺ monocyte adhesion and migration. We measured sCX3CL1 levels in plasma and bronchoalveolar lavage (BALF) of individuals with COPD. Both sCX3CL1 shedding and CX3CR1⁺ monocytes transendothelial migration were triggered by LPS and CS exposure in mice, and were significantly attenuated by AAT. The inhibition of monocyte-endothelial adhesion and migration by AAT was TACE-dependent. Compared with healthy controls, sCX3CL1 levels were increased in plasma and BALF of individuals with COPD, and were associated with clinical parameters of emphysema. Our results indicate that inhibition of sCX3CL1 as well as AAT augmentation may be effective approaches to decrease excessive monocyte lung recruitment during acute and chronic inflammatory states.NEW & NOTEWORTHY Our novel findings that AAT and other inhibitors of TACE, the sheddase that controls full-length fractalkine (CX3CL1) endothelial expression, may provide fine-tuning of the CX3CL1-CX3CR1 axis specifically involved in endothelial-monocyte cross talk and leukocyte recruitment to the alveolar space, suggests that AAT and inhibitors of sCX3CL1 signaling may be harnessed to reduce lung inflammation.

Keywords: TACE; cell-cell interaction; cigarette smoking; soluble fractalkine; α-1 antitrypsin.

Graphical abstract

Figure 1.

Endothelial transmigration of CX3CR1⁺ monocytes during acute inflammation in mice is dependent on AAT effect on the CX3CL1-CX3CR1 axis. A: schematic of two acute inflammation models using LPS (50 μg/mouse) administered via oropharyngeal aspiration on day 1 and CS exposure, 5 days/wk for 4 wk. Intraperitoneal PBS or AAT (20 mg/kg body wt) was administered prophylactically, one individual dose the day prior to LPS administration or as a treatment, weekly dose for 3 wk during 1-mo CS exposure, respectively. B and C: plasma and BALf sCX3CL1 levels measured by ELISA in CX3CR1^gfp/gfp (n = 8–10 mice/group) and WT littermates (n = 4–7 mice/group) at day 3 post LPS administration (B) or at 1-mo post CS exposure (C) treated or not with intraperitoneal AAT. D–I: flow cytometry analysis of CX3CR1⁺ (CD45⁺CD11b⁺Ly6G^-F4/80⁺Ly6C^-CX3CR1⁺) monocytes; neutrophils (CD45⁺CD11b⁺Ly6G⁺); and recruited monocytes (CD45⁺CD11b⁺Ly6G^-F4/80⁺). D: CX3CR1⁺ monocytes, E: neutrophils, and F: recruited monocytes absolute counts in the BAL fluid at day 3 post LPS administration and AAT pretreatment. G: CX3CR1⁺ monocytes, H: neutrophils and I: recruited monocytes counts in the BAL fluid after 1-mo CS exposure and AAT treatment. Data pooled from three independent LPS- or CS-exposure experiments are presented as means ± SD, one-way ANOVA, followed by Tukey’s multiple-comparisons, #P < 0.05 vs. PBS-treated mice, *P < 0.05 vs. LPS-treated or CS-exposed mice; ^P < 0.05 vs. LPS-treated or CS-exposed WT mice, ‡P < 0.05 vs. PBS-treated WT mice. AAT, α-1 antitrypsin; CS, cigarette smoke; LPS, lipopolysaccharide; WT, wild type.

Figure 2.

AAT decreases sCX3CL1 shedding by the endothelium in a TACE-dependent manner. sCX3CL1 shedding measured by ELISA (A and B) and full-length CX3CL1 membrane expression measured by FACS (C and D) in HLMVEC (A and C) and HPAEC (B and D) stimulated by acute serum deprivation (0% serum, 1 h) and treated with AAT (500 μg/mL, 1 h) vs. full media, control conditions. E and F: sCX3CL1 shedding by HLMVECs stimulated by serum deprivation and CS (5%, 16 h, E) and PMA (100 ng/mL, 1 h, F) and treated with AAT (500 μg/mL, 1 h) or the TACE inhibitor, GM6001 (2 μM, 1 h). G: TACE activity in the membrane fraction of HLMVECs stimulated by serum deprivation and PMA (100 ng/mL, 1 h) and treated with AAT (500 μg/mL, 1 h) or GM6001 (2 μM, 1 h). H: Cx3cl1 gene transcription in HLMVECs stimulated by acute and prolonged serum deprivation (0% serum, 1 h or 16 h) and treated with AAT (500 μg/mL, 1 h or 16 h) vs. full media, control conditions. All panels show data points from ≥ 3 independent experiments and means ± SE, one-way ANOVA followed by Tukey’s multiple comparisons, #P < 0.05 vs. serum, *P < 0.05 vs. serum deficiency or vs. PMA. AAT, α-1 antitrypsin; HLMVECs, human lung microvascular endothelial cells; HPAEC, human pulmonary artery endothelial cell; PMA, phorbol 12-myristate 13-acetate; TACE, TNF-α-converting enzyme.

Figure 3.

Monocyte adhesion to endothelium and migration is dependent on AAT effect on CX3CL1–CX3CR1 axis. A–C: monocyte adhesion to HLMVECs monolayer assay measured using time-lapse microscopy and Zeiss 200 M-inverted microscope. A unidirectional and low shear stress flow of THP-1 monocytes inside μ-Slides I^0.4 Luer ibiTreat chamber with a HLMVEC monolayer was achieved and maintained with a KDS pump set at 1.3 mL/h. A: representative static images of THP-1 monocytes (round and clear, top) adhesion to HLMVECs (cobblestone-like, bottom) after 16 h in full media (5% serum, i), serum deprived (0%, 16 h, ii), serum-deprived media supplemented with AAT (500 μg/mL, 16 h, iii), or with TACE inhibitor, TAPI-1 (50 μM, 16 h, iv). Scale bar: 50 μm. B: quantification of THP-1 monocyte adhesion to HLMVECs from time-lapse videos of conditions indicated in (A) using TrackMate nearest neighbor tracker algorithm. Individual dots represent “adhesion events”, aka number of THP-1 cell stopping onto the HLMVEC monolayer for ≥ 2 frames. C: quantification of THP-1 monocytes adhesion to HLMVECs stimulated by serum deprivation (0%, 16 h) or serum-deprived media supplemented with anti-sCX3CL1 inhibitory antibodies (5 μg/mL, 30 min prior to imaging). All quantifications were normalized to full media as control. Red dashed line represent means ± 2SD in full media (control) condition. D and E: migration of THP-1 (0.5 × 10⁵cells/mL) seeded in a chemotaxis tray toward conditioned medium of HLMVECs maintained in full media, stimulated by serum deprivation (0% serum, 16 h), or serum-deprived media supplemented with AAT (500 μg/mL, 16 h) vs. CM (D) or toward sCX3CL1 (50 ng/mL, 4 h) and/or LPS (100 ng/mL, 16 h) vs. THP-1 control media (CM) expressed as relative fluoresce units (RFUs) (E). All panels show data points from ≥3 independent experiments. Data are presented as means ± SE, one-way ANOVA followed by Tukey’s multiple-comparisons or t test, respectively, #P < 0.05 vs. CM THP-1, *P < 0.05 vs. serum-deficiency. AAT, α-1 antitrypsin; HLMVECs, human lung microvascular endothelial cells; TACE, TNF-α-converting enzyme.

Figure 4.

AAT decreases cleaved/mb-bound CX3CL1 ratio in endothelium in a TACE-dependent manner. A: schematic of wild-type and mutant full length CX3CL1 cleaved by TACE into soluble (sCX3CL1) and cleaved CX3CL1 isoforms. HA-tag was introduced in the C-domain to allow tracking via Western blotting of the C-domain containing CX3CL1 isoforms: full length membrane-bound, full length intracellular, and cleaved CX3CL1. CX3CL1 mutants were obtained by amino acid deletion (ΔLeu₃₂₄, ΔVal₃₂₆, ΔThr₃₂₉, ΔVal₃₃₁, and ΔAla₃₃₄) within the putative TACE cleavage site in the extracellular N-domain. B–E: representative immunoblots (B, D) of cleaved (∼27 kDa) and mb-bound full-length (∼90 kDa) CX3CL1 and densitometry (C, E) of the 27 kDa/90 kDa ratio in HPAEC overexpressing wild-type (B, C) or mutant (D, E) HA-tagged CX3CL1 plasmid (5 μg/1 × 10⁶ cells, 48 h). Cells were exposed to proinflammatory stimuli PMA (100 ng/mL, 1 h) or LPS (100 ng/mL, 16 h) in the presence or absence of AAT (500 μg/mL, 1 h and 16 h) or GM6001 (2 μM, 1 h) for the indicated time. All panels show data points from ≥3 independent experiments. Means ± SE, one-way ANOVA with Tukey’s multiple-comparisons, #P < 0.05 vs. full media, *P < 0.05 vs. serum-free PMA or LPS. AAT, α-1 antitrypsin; HPAEC, human pulmonary artery endothelial cell; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; TACE, TNF-α-converting enzyme.

Figure 5.

CX3CL1-CX3CR1 axis in COPD. A–C: plasma and BALf sCx3CL1 levels in COPD subjects enrolled in COPDGene cohort (A) and Pi*Z AATD individuals (B and C) measured by ELISA. BALf sCX3CL1 levels are normalized by the volume of epithelial lining fluid (ELF) imputed from BALf/plasma urea ratio and BAL fluid volume. Median ± IQR, Mann–Whitney test, P < 0.05 vs. smokers (current and former) or Pi*Z individuals without COPD. Pi*Z AATD individuals on augmentation therapy are depicted by dark circles (B and C). AATD, α-1 antitrypsin deficiency; COPD, chronic obstructive pulmonary disease; IQR, interquartile range.