α-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8

Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor–mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT–IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

α-1 Antitrypsin (AAT), a 52-kDa glycosylated protein primarily synthesized in the liver, is the major physiological inhibitor of a range of serine proteases, and within the lung maintains a protease-antiprotease balance. Recent studies indicate that AAT also possesses antiinflammatory capabilities that extend beyond its antiprotease role, including regulation of CD14 expression (1), inhibition of TNF-α gene upregulation (2), and inhibition of lipopolysaccharide activation of human monocytes and neutrophil migration in vitro (3, 4). In addition, AAT has been shown to downregulate apoptosis (5) and to inhibit antiproteinase 3 antibody activation of neutrophils (6).

Studying the function of AAT is facilitated by the existence of an in vivo model, namely AAT deficiency (AATD). This hereditary condition provides us with the most definitive evidence for the physiological and clinical importance of AAT. AATD is a syndrome, the unifying features of which are a predisposition to emphysema, liver disease, and skin panniculitis. The liver disease associated with AATD involves a gain-of-function mutation (PiZZ) that results in accumulation of polymers of Z-AAT within rough endoplasmic reticulum, leading to activation of ER stress responses (7–9). Pathogenesis of AATD-associated skin panniculitis is largely undefined, and most active research to date has focused on the pulmonary manifestations of the disease. In the past, the protease-antiprotease imbalance theory was accepted as a reason for the pulmonary emphysema associated with AATD. Studies focused upon the role of proteases as a primary contributor to lung tissue damage (10, 11), and the protease-antiprotease hypothesis was consolidated further, as AAT augmentation therapy reversed the biochemical abnormalities in lung fluid and impacted on proteolytic activity (12).

Evidence exists that the neutrophil is the main source of the proteolytic burden within the AATD lung, and airway neutrophilic inflammation plays a major role in the pathogenesis of AATD-associated emphysema. An increased lung neutrophil burden has been described even in AATD subjects with mild functional lung impairment (13) and also in asymptomatic nonsmoking heterozygotes for the Z allele or intermediate deficiency (PiMZ) without airflow obstruction (14). However, the reason for the observed increased neutrophil burden has never been fully elucidated, and with the aim of clarifying the important role of AAT in chronic neutrophilic infiltration, we investigated whether dysregulated neutrophil chemotaxis is associated with changes in neutrophil properties of AATD individuals.

Our results show an inhibitory effect of AAT on neutrophil chemotaxis and illustrate that a low-AAT environment, such as that occurring in the circulation of ZZ-AATD individuals (3–7 μmol/l compared with normal plasma levels of 20–50 μmol/l), correlates with increased chemotactic responses of both CXCR1 and immune complex receptor (FcγRIIIb) signaling. We demonstrate that neutrophil chemotaxis is dependent on opposing gradient concentrations of both IL-8 and AAT and that AAT–IL-8 complex formation inhibits CXCR1 engagement. We further show that AAT is associated with neutrophil membrane lipid rafts, interacting with the glycosylphosphatidylinositol-linked (GPI-linked) membrane protein FcγRIIIb. We demonstrate that AAT can control immune complex–mediated neutrophil chemotaxis by inhibiting ADAM-17 (TACE) activity and preventing the release of FcγRIIIb from the cell. This AAT-induced modulatory effect was also observed in vivo, in AATD individuals receiving augmentation therapy. After infusion, increased serum levels of AAT bound IL-8– and FcγRIIIb-expressing neutrophils and normalized AATD neutrophil chemotactic responses. This study provides a mechanism for the pathway by which AAT may mediate its antiinflammatory effect upon the circulating neutrophil and expands our understanding of the role of AAT in health and disease.

AAT activity regulates IL-8 and immune complex–induced neutrophil chemotaxis. The chemotactic index of ZZ neutrophils obtained from 6 clinically stable AATD individuals was compared with that of control neutrophils (MM) from 6 healthy donors. Results revealed that in response to IL-8 (10 ng), ZZ neutrophils exhibited a 2- or 8-fold increase in chemotactic activity when experiments were performed in PBS or serum (50% v/v), respectively (Figure 1A). In addition, in response to IL-8 the chemotactic index of MM neutrophils suspended in PBS was superior to that of MM cells suspended in serum (P = 0.02). Moreover, the chemotactic index of ZZ neutrophils in response to soluble immune complex (sIC) (10% v/v) was 25% lower than that of MM control cells when performed in PBS (P = 0.001), yet 3.6-fold higher when experiments were repeated in the presence of respective ZZ and MM sera (Figure 1B).

Figure 1

Chemotactic analysis of neutrophils in response to AAT. (A) Increased mean chemotactic index of ZZ-AATD neutrophils (n = 6) compared with healthy control (MM) cells (n = 6) in response to IL-8 (10 ng/2 × 10⁷ cells). Assays were preformed in PBS or serum (50% v/v) from respective ZZ or MM individuals. (B) Decreased mean chemotactic index of ZZ-AATD neutrophils (n = 6) in PBS in response to sIC (10% v/v), yet increased mean chemotactic index of ZZ-AATD neutrophils in serum (50% v/v) compared with healthy control cells (n = 6). (C) An increase in chemotactic inhibition efficiency of increasing concentration of AAT (0.2–27.5 μM). Black bar, positive IL-8 (10 ng) control; white bar, negative HSA (27.5 μM) control. *P < 0.05 versus IL-8 control. (D) Neutrophil chemotaxis in a linear gradient of IL-8 (1, 10, 20, or 40 ng) or complex gradient in the presence of inhibitory AAT (27.5, 6.9, 0.9, 0.2 μM) (*P < 0.05 compared with AAT-untreated cells). (E) Increased chemotactic inhibition efficiency of increasing concentrations of AAT (0.4–27.5 μM). Black bar, positive sIC (10% v/v) control; white bar, negative HSA (27.5 μM) control. *P < 0.05 versus sIC control. Experiments illustrated in A–E were performed in triplicate on 3 consecutive days, and for comparative analysis the PBS control was set at a chemotactic index of 1. Each measurement is the mean ± SEM.

To investigate whether the increased chemotactic activity of ZZ neutrophils in response to IL-8 was a result of AATD, we explored the effect of exogenous AAT on neutrophil chemotaxis. As illustrated in Figure 1C, the chemotactic index of MM neutrophils in response to IL-8 (10 ng) was 6.05 ± 0.66. AAT inhibited MM neutrophil chemotaxis in a dose-dependent manner, with an IC₅₀ of 0.5 μM. HSA (control plasma protein, 27.5 μM) did not affect neutrophil chemotaxis. At a higher concentration, AAT (6.9 μM) reduced the MM neutrophil chemotactic index produced by 10 ng or 20 ng IL-8 by 91.5% (P = 0.002) and 46.8% (P = 0.07), respectively (Figure 1D). Biologically relevant serum concentrations of AAT (27.5 μM) abolished neutrophil chemotaxis in response to 1 ng or 10 ng IL-8 and reduced the cellular chemotaxis index to 20 ng and 40 ng IL-8 by approximately 72% and 52% (P = 0.009 and P = 0.09, respectively). To investigate the inhibitory effect of AAT on IL-8’s chemotactic activity (versus chemokinesis), we also used checkerboard analysis with serial dilutions of IL-8 above and below the filter. Neutrophil migration depended on the presence of an IL-8 gradient, implying that AAT inhibits directed cell migration rather than chemokinesis (Supplemental Figure 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI41196DS1). The effect of exogenous AAT (0.4–27.5 μM) on the chemotactic index of MM neutrophils in response to sIC (10% v/v) was explored next. As illustrated in Figure 1E, AAT inhibited MM neutrophil chemotaxis in a dose-dependent manner, with an IC₅₀ of 8.2 μM, while HSA (27.5 μM) had no effect on sIC-mediated chemotaxis.

To understand (a) how AAT inhibited both IL-8 and sIC mediated MM neutrophil chemotaxis, (b) why ZZ cells demonstrated an increased chemotactic response to IL-8, and (c) why ZZ neutrophils illustrated conflicting chemotactic responses to sIC in PBS and serum was the goal of subsequent experiments.

AAT is localized to the neutrophil membrane. To establish how AAT impacted on neutrophil chemotaxis, it was necessary to pinpoint its location within the cell. By subcellular fractionation of neutrophils, AAT was defined as a component of the cell membrane (Figure 2A). Successful separation of plasma membranes from secretory vesicles was validated by Western blotting for HLA class I and HSA, and 83% ± 1.5% of AAT was localized within the plasma membrane fraction (Figure 2B). Confocal laser scanning microscopy was employed to confirm localization of AAT to the membrane and showed bright foci of anti-AAT labeling in the membrane region of isolated cells (Figure 2C). In addition, AAT was found in association with detergent-free low-density sucrose fractions (15% w/w) containing membrane lipid rafts (Figure 2D). A similar distribution of flotillin-1 was observed, and treatment with the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD) diminished AAT and reduced the level of flotillin-1 in the low-density sucrose fractions. 2D electrophoresis of isolated MM neutrophil membranes (Supplemental Figure 2A) and purified membrane lipid rafts (Figure 2E) revealed 4 isoforms of AAT of similar molecular mass (~52 kDa). By employing FACS analysis of nonpermeabilized cells, we detected AAT on the plasma membrane of unstimulated resting MM healthy neutrophils (Figure 2F). In contrast, neutrophils isolated from ZZ-AATD individuals illustrated significantly lower levels of AAT (mean fluorescence: control MM cells, 31.41; ZZ-AATD cells, 17.52; P = 0.001). Fractionation of ZZ neutrophil cytosol, membrane, and granule compartments localized Z-AAT to neutrophil membranes, with reduced levels (~50%) confirmed by Western blotting (Supplemental Figure 2B). Collectively, these findings support localization of AAT to the neutrophil membrane and reveal that ZZ-AATD cells express significantly lower levels of AAT.

Figure 2

Localization of AAT in peripheral blood neutrophils. (A) Coomassie blue–stained gel of normal MM neutrophils subjected to subcellular fractionation yielding a cytosol fraction (lane 1), combined secretory vesicle/plasma membrane fraction (lane 2), and primary and secondary granule fraction (lanes 3 and 4, respectively). Western blotting employed antibodies to MPO and PR3 as markers of primary granules and antibodies against p22^phox of cytochrome b₅₅₈ as a marker of secondary granules and combined secretory vesicle/membrane fractions. AAT was localized to the secretory vesicle/membrane fraction at the interface between the 17.5/35% (w/w) sucrose. (B) By Percoll gradient fractionation, approximately 83% of AAT was localized to the plasma membrane (PM) compared with secretory vesicles (SV). As controls, HLA served as a marker of PM and HSA as a marker of SV. (C) By confocal microscopy, the distribution of AAT in non-permeabilized MM neutrophils was predominantly localized to the membrane margin of the cell. Cell nuclei are stained red. Scale bar: 10 μm. (D) Detection of AAT and flotillin-1 in the low-density lipid raft fractions (15% w/w sucrose) of human MM neutrophil lysates: untreated (–) or treated with MβCD (10 mM) (+). Immunoblots were probed with polyclonal anti-AAT and monoclonal anti–flotillin-1. (E) Coomassie blue–stained 2D SDS-PAGE gel of isolated MM neutrophil membrane lipid rafts (top panel). A Western blot probed with polyclonal anti-AAT revealed 4 associated AAT isoforms (lower panel). (F) Flow cytometry analysis of membrane-bound AAT in isolated resting MM (green) and ZZ-AATD (red) neutrophils. The level of AAT was found to be significantly higher on resting MM neutrophils. The isotype control antibody is illustrated in black (filled). Experiments in A, D, and E are each representative gels and blots of 3 separate experiments. Results in B represent experiments performed in triplicate on 3 consecutive days, and each bar is the mean ± SEM. Confocal analysis in C and FACS analysis in F are one illustrative result from 3 independent experiments.

Origin of membrane-associated AAT. To clarify whether AAT was being produced by the neutrophil, as has been described by others (15), or whether it was being absorbed from the serum, we compared the phenotype of MM and ZZ-AATD neutrophil membrane-associated AAT with that of a ZZ-AATD individual after liver transplantation. Figure 3A shows the different patterns of phenotypes frequently observed for serum of MM and ZZ-AATD individuals (16). The phenotype of the ZZ-AATD serum sample after liver transplantation was entirely MM, a result confirmed by the use of an ELISA specific for the ZZ protein (17, 18) (Figure 3B). In contrast, neutrophil membranes of the ZZ-AATD liver transplant recipient contained detectable levels of Z-AAT protein (Figure 3B). Additionally, although these cells contained lower levels of membrane Z-AAT compared with a non-transplant patient, greater levels of total AAT (Z and M isoforms) were detected by ELISA (Figure 3C) and immunoblotting (Figure 3D), most likely due to absorption of MM AAT from the serum. Collectively, these experiments suggest that both serum- and neutrophil-derived AAT binds the cell membrane.

Figure 3

Serum- and neutrophil-derived AAT binds the neutrophil membrane. (A) Isoelectrofocusing patterns for ZZ, heterozygous MZ, or MM serum compared with serum of a ZZ-AATD individual after liver transplantation (ZZ+LT). The isoform numbers for the M and Z variants are indicated. The AAT pattern of the ZZ+LT sample was of the M variant. (B) ELISA for detection of the Z-form of AAT in serum and neutrophil membranes. Results in arbitrary fluorescence units (FU) indicate positive detection of Z-AAT in serum and neutrophil membranes of a ZZ-AATD patient but only in neutrophil membranes after LT (P = 0.2 between ZZ and ZZ+LT neutrophil membranes). An ELISA for detection of total AAT (M and Z variants) (C) and Western blots probed with goat (Gt) polyclonal anti-AAT (D) revealed significantly higher levels of total AAT on ZZ-AATD neutrophil membranes after liver transplantation (ZZ+LT) compared with a non-transplant ZZ-AATD patient (*P = 0.01, **P = 0.001). Results represent experiments performed in triplicate, and data in B and C are the mean ± SEM.

Proinflammatory cytokine exposure causes release of AAT within a protein complex. The effect of proinflammatory stimuli (IL-8 or TNF-α) on release/shedding of AAT from the neutrophil membrane was investigated. MM neutrophils were treated with TNF-α (10 ng), a known potent inducer of L-selectin (CD62-L) shedding (Figure 4A). After 10 minutes of treatment, a significant decrease in cell surface AAT was detected by flow cytometry (mean fluorescence: control untreated cells, 20.7; TNF-α–treated cells, 10.28; P < 0.001) (Figure 4A). A corresponding increase in released AAT in the surrounding medium was detected by Western blot analysis, with a maximum increase in extracellular released AAT at 40 ng TNF-α and 20 ng IL-8 (Figure 4B). To assess the kinetics of shedding, we treated neutrophils with 10 ng IL-8 or TNF-α and assessed extracellular released AAT at 2, 5, and 10 minutes. As depicted in Figure 4C, an immunoreactive band corresponding to released AAT was detected at 5 and 10 minute after TNF-α treatment and at an earlier time point of 2 minutes in response to IL-8. Maximum release of AAT from neutrophil membranes exposed to either IL-8 or TNF-α occurred at 10 minutes (Supplemental Figure 3, A–D). ELISA was performed to quantify levels of released AAT, and at the 2- and 5-minute time points, neutrophils released 4.46 ± 1.13 and 5.25 ± 0.44 ng AAT per 1 × 10⁷ neutrophils in response to IL-8 (10 ng) (Figure 4D). In contrast, and at the same time points, neutrophils released significantly lower levels of AAT in response to TNF-α treatment (P = 0.02 and P = 0.014 for 2 and 5 minutes, respectively). To investigate whether neutrophils synthesized AAT to substitute for membrane-released AAT, we exposed cells to IL-8 (10 ng) and quantified levels of extracellular and membrane-associated AAT for up to 16 hours. Results revealed no increase in either membrane-associated or newly released levels of AAT (Supplemental Figure 3E), suggesting that de novo protein synthesis of AAT did not occur.

Figure 4

Release of biologically active AAT from TNF-α– or IL-8–treated MM neutrophils. (A) Membrane expression of L-selectin or AAT on neutrophils at baseline (green) or in response to TNF-α (red). (B) Western blot showing extracellular released AAT (52 kDa) following treatment with TNF-α or IL-8 in a dose- (B) and time-dependent manner (C). Serum AAT (52 kDa) was loaded as a positive antibody control, and untreated cells are in lanes labeled as control (Con). (D) Time course analysis of the release of AAT (*P < 0.05). (E) Gel filtration chromatography of neutrophil-released AAT. The start material (St) was released AAT from IL-8–treated neutrophils (10 ng). Inset: Western blot analysis illustrating altered migration of purified serum AAT (arrow) compared with neutrophil-released AAT (arrowheads). By Western blot analysis, fraction 28 contained neutrophil-released AAT and chromatographed at a molecular mass of 100–110 kDa. Fractions 30–33 were run on separate gels. (F) Formation of AAT-protease complexes. Samples were immunoblotted employing a rabbit anti-human AAT antibody. Lanes 1 and 3 show serum purified AAT and AAT released from neutrophils, respectively. Lanes 2 and 4 illustrate serum and neutrophil-released AAT incubated with NE. FACS analysis illustrated in A is a representative result of 3 independent experiments. B, C, and F are each representative Western blots of 3 separate experiments. Results in D represent experiments performed in triplicate. Each measurement is the mean ± SEM. FPLC analyses (E) were performed in triplicate on 2 consecutive days.

Native gel electrophoresis was employed to examine potential conformational differences between serum purified AAT and IL-8–induced neutrophil-released AAT. Serum AAT migrated faster and ran as a predominant single band (Figure 4E, inset). In contrast, neutrophil-released AAT migrated differently from serum AAT, showing a smeared, retarded mobility suggestive of a significant degree of conformational heterogeneity and/or protein-protein complexation. For assessment of the complexed state of the AAT species present, neutrophil-released AAT was chromatographed by gel filtration on a Superose 6 column. Protein elution was monitored by Western blot analysis, and as can be seen in Figure 4E, neutrophil AAT protein eluted at a position corresponding to a complex molecular mass of approximately 100–110 kDa. To exclude the possibility that neutrophil-released AAT eluted at increased molecular mass due to a protease-inhibitor complex, we confirmed the affinity of neutrophil-released AAT for neutrophil elastase (Figure 4F).

AAT interacts with the neutrophil membrane via the GPI-anchored lipid raft membrane protein FcγRIIIb. AAT was immunoprecipitated from IL-8–induced neutrophil-shedded material by rabbit polyclonal anti-AAT and the immunoprecipitates resolved by denaturing and reducing SDS-PAGE. A Coomassie blue stained gel is illustrated in Figure 5A, and a putative 45- to 50-kDa AAT binding partner was identified by LC-MS/MS as FcγRIIIb (CD16b). FcγRIIIb is a GPI-linked low-affinity IgG receptor, and to exclude the possibility of experimental binding artefact, we performed reciprocal immunoprecipitation of FcγRIIIb and subsequent identification of AAT by LC-MS/MS (Figure 5B). This finding was further supported by the presence of FcγRIIIb and AAT on Western blots of an immunoprecipitation for AAT (Figure 5C, top panel) and for FcγRIIIb, respectively (Figure 5C, lower panel). By expanding the gel filtration result illustrated in Figure 4E by Western blot analysis for FcγRIIIb, we observed the coelution of AAT and FcγRIIIb in fraction 28 (Supplemental Figure 4A). Moreover, native gel electrophoresis and Western blot analysis of fraction 28 supported the possibility of an AAT-FcγRIIIb interaction, while excluding formation of AAT polymers or neutrophil elastase (NE) and PR3 as possible binding partners within this complex (Supplemental Figure 4B). The exposure of AAT-coated surfaces to FcγRIIIb revealed that FcγRIIIb bound to AAT in a concentration-dependent manner but not to a second non-GPI-linked IgG receptor, FcγRIIa (Figure 5D). In addition, an ELISA utilizing mouse capture antibody to FcγRIIIb and goat detection antibody for AAT quantified significant levels of cell-free serum FcγRIIIb-AAT complex in serum of CF patients (Figure 5E). Reciprocal ELISA utilizing goat capture antibody to AAT and mouse detection antibody for FcγRIIIb quantified considerably higher levels of AAT-FcγRIIIb complex in CF serum, compared with MM and ZZ-AATD serum (Supplemental Figure 4C). Since AAT and FcγRIIIb coimmunoprecipitated, it also appeared logical that AAT and FcγRIIIb would colocalize on the neutrophil membrane. Analysis by confocal microscopy indicated that, as expected, AAT (red) and FcγRIIIb (green) stained the plasma membrane (ring-like staining at the edge of the cells) (Figure 5F), and superimposition of the two images clearly identified colocalization (yellow) of the two signals. We next tested the hypothesis that enzymatic cleavage of the GPI-anchored FcγRIIIb with phosphatidylinositol-specific phospholipase C (PI-PLC) would also displace the associated AAT binding partner (Figure 5G). Western blot analysis of PI-PLC–treated (1 U/1 × 10⁶ cells) neutrophil membranes resulted in release of AAT and FcγRIIIb, but not p22^phox, a control non-GPI membrane component. Further treatment of membranes with NaCl (350 mM) caused release of AAT but not FcγRIIIb (Figure 5G). In addition, in comparison to MM donors, neutrophils of ZZ-AATD patients showed a significant decrease in the level of FcγRIIIb protein (Figure 5H and Supplemental Figure 4D). The observation that levels of FcγRIIIb were significantly elevated in serum of ZZ-AATD patients compared with normal MM controls (P = 0.01) (Figure 5I) contrasts results illustrating similar levels of FcγRIIIb-AAT complex (Figure 5E), possibly indicating that a proportion of FcγRIIIb in ZZ-AATD serum is AAT unbound. Collectively, these experiments further suggest FcγRIIIb as a binding partner that is necessary for AAT membrane presentation and suggest that AAT may functionally impact on maintaining FcγRIIIb membrane expression.

Figure 5

AAT binds FcγRIIIb on the neutrophil membrane. (A) Immunoprecipitate (IP) employing rabbit (Rb) antibody to AAT. The AAT binding partner (labeled 1) was identified as FcγRIIIb (40–60 kDa). (B) Reciprocal immunoprecipitate with FcγRIIIb goat (Gt) antibody. Coimmunoprecipitated proteins were identified as FcγRIIIb (labeled 1; accession number AA128563) and as AAT (labeled 2; accession number CAJ15161). (C) Western blots of immunoprecipitate reactions were performed with goat antibody against AAT (top panel) and mouse (Mo) antibody against FcγRIIIb (bottom panel). Serum purified AAT and recombinant human (Rh) FcγRIIIb are included as positive controls. (D) Binding of Rh FcγRIIIb or Rh FcγRIIa to AAT (*P = 0.02 compared with control containing no FcγRIIIb). (E) FcγRIIIb-AAT complex in serum of CF (n = 4) individuals was significantly higher than that of normal MM (Con; n = 6) or AATD (n = 6) subjects (*P < 0.05, CF versus control or ZZ). (F) Colocalization (merged image in yellow) of rhodamine labeling for AAT and FITC for FcγRIIIb, at the membrane margin of cells (×64 magnification, ×4 zoom). (G) NaCl and PI-PLC treatment of neutrophil membranes. Immunoblots included untreated membranes (Con) and antibodies against p22^phox. (H) Flow cytometry analysis of membrane-bound FcγRIIIb (mean fluorescence: MM, 41.93; ZZ, 15.44). The isotype control antibody is in black (filled). (I) Levels of FcγRIIIb in serum of ZZ-AATD individuals and MM controls (Con) (*P = 0.01). In A–C and G, images are representative results from 1 of 3 separate experiments. Lanes in A and C were run on the same gel but were noncontiguous. D, E, H, and I represent results performed in triplicate. Images in F are representative results of 2 independent experiments.

AAT regulates release of FcγRIIIb by inhibiting metalloprotease ADAM-17 activity. Evidence exists that neutrophil chemotaxis requires activity of surface sheddases, including ADAM-17, which has been implicated in the shedding of FcγRIIIb (19), a prerequisite for neutrophil chemotaxis (20–23). This was confirmed when cells were exposed to sIC (10% v/v) and the extracellular supernatants analyzed by Western blot for the presence of released FcγRIIIb (Figure 6A). Release of FcγRIIIb was observed at 5 and 10 minutes after exposure, an effect greatly reduced by the specific ADAM-17 inhibitor TAPI-1 (10 μM) (24). To investigate the possibility that AAT regulated sIC-induced chemotaxis by modulating shedding of FcγRIIIb, we exposed cells to sIC (10% v/v) in the presence or absence of AAT and monitored the kinetics of FcγRIIIb shedding by immunoblotting. As illustrated in Figure 6B, AAT inhibited the release of FcγRIIIb elicited by sIC at 5 and 10 minutes by 58% ± 1.2% and 69% ± 5.9%, respectively (P < 0.05). The ability of AAT to directly inhibit ADAM-17 activity was analyzed fluorometrically, and the results indicated that AAT inhibited ADAM-17 enzymatic activity with a K_i value of 5.7 μM (Figure 6C). In addition, an equimolar concentration of AAT competitively inhibited ADAM-17, with an association rate constant of 2.23 × 10⁴ ± 0.37 × 10⁴ M^–1s^–1, although this may underestimate the physiological inhibitory capacity, as AAT is one of the most abundant proteins in the circulation. As polymerization and oxidation have previously been shown to alter enzymatic activity of AAT against NE, we tested whether an intact reactive center loop was also essential for inhibiting ADAM-17 activity. As illustrated in Figure 6D, a loss of anti–ADAM-17 activity was observed following oxidation of AAT with N-chlorosuccinimide, heat-induced polymerization, or by the AAT cleavage product C-36. Additionally, results revealed a significant 68% reduction in ADAM-17 activity in the presence of 27.5 μM AAT (P = 0.001), a value comparable to inhibition of ADAM-17 activity by TAPI-1 (10 μM) (Figure 6D). Moreover, the exposure of AAT-coated surfaces to ADAM-17 (250 ng) together with native AAT (250 ng) reduced the level of detectable bound ADAM-17 by approximately 36% (P = 0.005), a competitive effect not observed with either polymerized AAT or the C-36 cleaved fragment of AAT (Figure 6E). From these experiments, we conclude that neither polymers nor the C-terminal fragment of AAT can inhibit binding of native AAT to ADAM-17.

Figure 6

AAT prevents neutrophil chemotaxis in response to sIC through inhibition of ADAM-17 activity. (A) Western blots showing time course of sIC-induced release of FcγRIIIb from MM neutrophils with or without TAPI-1 (10 mM). (B) Quantification of FcγRIIIb release from MM neutrophils treated with sIC (10% v/v) with or without AAT (27.5 mM) (*P < 0.05). Inset: Western blot illustrating FcγRIIIb release at the 10-minute time point. (C) Competitive inhibition of ADAM-17 by AAT. (D) The effect of oxidation (Ox), polymerization (Poly), or the cleaved AAT peptide (C-36 fragment) on TACE inhibition (percent inhibition of maximum activity) was compared with native AAT. *P < 0.05, native AAT or TAPI-1 versus maximal activity; ^§P = 0.008, polymerized AAT versus native AAT (27.5 mM). (E) Binding of ADAM-17 (250 ng) to immobilized AAT detected in the presence or absence of equimolar concentration of polymerized, C-36 fragment, or native AAT (*P = 0.005). Levels of membrane-bound AAT (F) and FcγRIIIb (G) on isolated resting control MM (green) and ZZ-AATD neutrophils before (red) and 2 days after augmentation therapy (blue). (H) The mean chemotactic index of ZZ-AATD neutrophils (black bars) toward sIC (10% v/v) in serum (50% v/v) was significantly reduced on day 2 when compared with day 0 and with day 7 after augmentation therapy. For comparative analysis, MM neutrophils treated with sIC (white bar) were set at a chemotactic index of 1. Immunoblots in A and B are representative results from 1 of 3 separate experiments. B–E and H are the mean ± SEM of triplicate experiments. F and G are representative results from 1 of 3 separate experiments.

We next explored the effect of intravenous augmentation therapy on FcγRIIIb expression in vivo. Two days after treatment, ZZ-AATD (n = 4) patients possessed serum AAT concentrations slightly elevated above MM control serum levels (30.51 ± 1.9 μM and 24.8 ± 0.68 μM AAT, respectively) (Supplemental Figure 5) but also significantly greater than on day 0 (pretreatment) (6.3 μM AAT, P < 0.0001) and 7 days after treatment (13.72 μM AAT, P < 0.0001). Results revealed that infused AAT bound to circulating neutrophils (Figure 6F), as a significant increase in the level of membrane-bound AAT was detected on day 2 after therapy (mean fluorescence, 43) compared with day 0 (mean fluorescence, 17.23; P < 0.001). In addition, downregulation of FcγRIIIb was observed on ZZ compared with MM control cells before therapy (FcγRIIIb mean fluorescence: ZZ-AATD day 0, 16.94; control MM, 29.35; P < 0.001) (Figure 6G). However, on day 2 after treatment, an increase in the level of FcγRIIIb-expressing ZZ neutrophils was observed, comparable to that in MM controls and significantly greater than on day 0 (FcγRIIIb mean fluorescence: ZZ-AATD day 2, 30.41; P < 0.001). A chemotaxis assay toward sIC (10% v/v) in the presence of respective ZZ and MM sera revealed that ZZ neutrophils on day 2 after augmentation therapy had a chemotactic index similar to that of the MM neutrophils and which was significantly reduced compared with day 0 and with day 7 after treatment (P = 0.001 and P = 0.03, respectively) (Figure 6H).

Collectively, these experiments provide evidence for the mechanism by which native AAT modulates neutrophil chemotaxis in response to sIC and illustrate the ability of AAT to bind the circulating neutrophil in vivo and prevent release of FcγRIIIb from the neutrophil membrane, via inhibitory activity against ADAM-17.

AAT regulates IL-8–induced chemotaxis by modulating interaction with CXCR1. To understand the possible mechanism by which AAT inhibits IL-8–induced chemotaxis, we first explored the ability of AAT to modulate ligand binding to CXCR1. The exposure of CXCR1-coated surfaces to IL-8 in the presence or absence of AAT (27.5 μM) revealed that AAT blocked the ability of IL-8 (10 ng) to engage with CXCR1 (Figure 7A). This result implied direct binding of AAT to IL-8, and as IL-8 interacts with glycosaminoglycans with high affinity (24–26), we hypothesized that binding of IL-8 to glycosylated AAT may modulate IL-8–induced chemotaxis. To challenge this hypothesis, we exposed surfaces coated with AAT at a concentration of 27.5 μM to increasing concentrations of IL-8, and the plateau level of maximum binding observed was approximately 20 ng IL-8/cm² (Figure 7B). In contrast, binding of IL-8 to HSA (control protein) remained linear. The importance of glycosylated moieties of AAT for successful IL-8 binding was confirmed, as nonglycosylated recombinant AAT produced in E. coli failed to bind IL-8 (Figure 7C). The impact of IL-8 binding to AAT or HSA on its ability to stimulate Akt Ser473 phosphorylation, a key step in IL-8–mediated neutrophil chemotaxis (27), was explored. Figure 7D shows that IL-8 (1 ng) stimulated adequate Akt phosphorylation after just 10 minutes and that this effect was unaffected by pre-binding of IL-8 to HSA (27.5 μM). In contrast, pre-binding of IL-8 to AAT (27.5 μM) inhibited IL-8–mediated Akt Ser473 phosphorylation to the level observed after the inclusion of the phosphatidylinositol 3-kinase inhibitor wortmannin. Additionally, binding of IL-8 to AAT (27.5 μM) significantly reduced IL-8 neutrophil Akt phosphorylation in response to both 10 and 20 ng IL-8 by approximately 81% and 62%, respectively (P < 0.05) (Figure 7E). Conversely, however, AAT had no effect on Akt Ser473 phosphorylation stimulated by 40 ng IL-8, illustrating that the described inhibitory effect can be overcome. In addition, an increase in cytoplasmic Ca²⁺ (calcium flux) is central to the transduction of neutrophil response to IL-8 (28), and changes in average Fluo-4 NW emission in IL-8–stimulated (10 ng) cells was measured over the course of 2 minutes (Figure 7F). IL-8 caused a robust increase in intracellular calcium that reached a peak after 50 seconds, and in comparison the level of intracellular calcium release in the presence of AAT was greatly diminished.

Figure 7

AAT binds IL-8 and modulates neutrophil chemotaxis by controlling CXCR1 binding. (A) Binding of IL-8 (10 ng) to immobilized CXCR1 was inhibited in the presence of AAT (27.5 mM) (*P = 0.002). (B) Comparative binding of IL-8 to AAT and HSA. AAT (27.5 mM) bound approximately 20 ng IL-8. (C) Comparative binding of IL-8 to serum purified AAT and recombinant non-glycosylated (Rec non-glycos) AAT (*P = 0.001). (D) Expression levels of phospho-Akt Ser473 after IL-8 (1 ng/1 × 10⁷ cells) treatment. Wortmannin (Wort, 100 nM) and AAT (27.5 mM), but not HSA, inhibited Akt activity efficiently (*P < 0.05 versus IL-8 control). (E) AAT (27.5 mM) significantly inhibited phosphorylation of Akt induced by 10 ng and 20 ng IL-8 (*P < 0.05 versus IL-8 control). (F) Time course of changes in Ca²⁺ intensity in response to IL-8 (10 ng) with or without AAT (27.5 mM). (G) AAT suppression of IL-8–induced actin cytoskeletal rearrangements. Immunoblot with anti-actin antibody for the distribution of G-actin (in the supernatant fraction [S]) and F-actin (in the pellet fraction [P]) in untreated or AAT-treated (27.5 mM) cells (top panel). Lower panel: IL-8–treated (10 ng) MM neutrophils with or without AAT or control Wort (100 nM). Distribution ratios are shown. (H) Confocal images of neutrophils (MM) undergoing chemotaxis in response to IL-8 (10 ng). F-actin at the leading edge of neutrophils (arrows) was not apparent in resting cells (Un) or cells treated with IL-8 plus AAT (27.5 mM) (×40 magnification, ×10 zoom). (I) IL-8–induced (10 ng) mean chemotactic index of ZZ-AATD neutrophils (black bars) on day 2 after therapy compared with day 0 and with day 7 after augmentation therapy. All experiments were performed in triplicate on 3 consecutive days, and each measurement is the mean ± SEM. Images in D-H are representative results of 1 of 3 separate experiments.

Remodeling of the actin cytoskeleton is a prerequisite for the chemotactic process, and to analyze the effect of AAT on the redistribution of F- versus G-actin after IL-8 exposure, we employed a biochemical assay to analyze in situ F-actin levels. While AAT (27.5 μM) on its own had no effect, IL-8 (10 ng) induced a substantial change in the ratio of G-actin (supernatant fraction) versus F-actin (pellet fraction) (Figure 7G). The presence of AAT or wortmannin (100 nM) as a control suppressed IL-8–induced F-actin formation. Since coronin is an F-actin–interacting protein solubilized upon neutrophil stimulation (29), we analyzed whether AAT could impact on the solubilization of coronin-1 in response to IL-8. As illustrated in Supplemental Figure 6, AAT (27.5 μM) significantly reduced the level of soluble coronin-1 by 40% (P = 0.01). In addition, polarization of F-actin to the leading edge during neutrophil chemotaxis was greatly diminished in the presence of a physiological concentration of AAT (Figure 7H). Confocal microscopy images illustrated that resting neutrophils had punctuate F-actin staining intensity similar to that of cells treated with IL-8 in the presence of AAT, indicative of sites of contact between cell and substratum (30), rather than polarization. Results also revealed that in response to IL-8 (10 ng), in the presence of respective ZZ and MM sera, ZZ neutrophils on day 2 after augmentation therapy showed a chemotactic index that was similar to the MM controls and was also significantly reduced compared with ZZ neutrophils on day 0 and on day 7 after treatment (P = 0.02 and P = 0.003, respectively) (Figure 7I). Taken together, these data suggest that glycosylated AAT–IL-8 complex formation determines CXCR1 engagement, and in this process AAT functions as a negative regulator of cell chemotaxis.

In patients with AATD, the development of emphysema is believed to be caused by the unchecked action of proteases on lung tissue. However, this long-standing dogma on the sole function of AAT as an anti-serine protease has been questioned in the last several years, with a number of antiinflammatory roles being described for AAT (1–3, 5). Studies have shown that native AAT may modulate immune cell function of neutrophils (4, 6, 31), monocytes (1, 3, 32), and T cells (33), and the results of this study are analogous to data demonstrating the inhibitory effect of AAT on fMLP-induced neutrophil migration in vitro (4). Moreover, this study elucidates what we believe to be a novel role for AAT and has uncovered the ability of AAT to inhibit chemotactic response of both CXCR1 and FcγRIIIb receptor signaling by divergent mechanisms. The first involves AAT–IL-8 complex formation consequently inhibiting IL-8–CXCR1 interaction, and the second concerns the ability of AAT to control sIC-induced neutrophil chemotaxis by modulating ADAM-17 activity, thus impeding FcγRIIIb release from the cell membrane (Figure 8).

Figure 8

AAT regulates neutrophil chemotaxis through two different mechanisms. (A) In response to IL-8, cell activation is accompanied by Akt phosphorylation and Ca²⁺ flux, leading to F-actin formation and cytoskeletal rearrangements. In vivo, the resting circulating MM neutrophil is surrounded by a high concentration of AAT (27.5 μM). AAT binds IL-8 and modulates CXCR1 engagement. (B) In response to sIC (i), ADAM-17 activity is increased (ii), causing shedding of the FcγRIIIb from the membrane (iii). Possible signaling mechanisms involve crosslinking with CR3, CD32a, or the fMLP receptor (iv) and migration of cells to the site of inflammation. AAT modulates chemotaxis by inhibiting ADAM-17 activity, resulting in diminished release of FcγRIIIb from the neutrophil membrane.

AAT has been shown to reduce neutrophil infiltration into kidneys during ischemia/reperfusion (34), and this essential inhibitory effect of AAT on circulating neutrophils prompted us to investigate the chemotactic response of AATD neutrophils donated by clinically stable ZZ-AATD patients compared with normal MM control cells. The findings of enhanced IL-8–induced chemotaxis are in keeping with a pivotal role for AAT in modulation of cell activity. In support of this theory, increasing concentrations of AAT exhibited significant inhibition of neutrophil migration toward IL-8. The observed reduction in chemotaxis by AAT was, however, alleviated by increasing the concentration of chemoattractant, suggesting that this inhibitory effect may be overcome in the presence of sufficient chemokine. In agreement with the results of others (35), we found that fMLP alone only poorly activated the NADPH oxidase of isolated MM neutrophils, and pronounced oxidase activity was only noted after TNF-α priming (Supplemental Figure 7), thus confirming the isolation of unprimed cells within this study. Isolation procedures used to obtain purified neutrophils and priming agents such as GM-CSF and TNF-α can enhance neutrophil activity, leading to an increased respiratory burst and elevated levels of superoxide production (36). This may explain the variance between our findings and a previous study that reported comparable activity between control and ZZ-AATD neutrophils (37), a disparity possibly arising due to the use of an alternate cell purification protocol and the successful isolation of nonprimed cells within our study.

Our results suggest a sensing mechanism in cells that has not to our knowledge been previously described, and experiments demonstrate that neutrophils can migrate down a functional gradient of AAT in response to an increasing gradient of IL-8. The interaction of inflammatory molecules with glycosaminoglycans has been hypothesized to provide a mechanism for establishing chemokine gradients in order to provide directional signals for migrating cells (38). Most chemokines, including IL-8, interact with glycosaminoglycans via charge complementation (26), as they are basic proteins, while glycosaminoglycans are acidic polymers. Moreover, it has previously been shown that IL-8 binds heparan sulfate and that this interaction facilitates binding of the cytokine to its specific receptor, thus prolonging IL-8 activity (39). CXCR1 is specific for IL-8 (40, 41), and within this study we have found that IL-8 interacts with glycosylated AAT, but rather than facilitating IL-8 activity, physiological concentrations of AAT inhibit CXCR1 engagement of up to 20 ng of IL-8, thereby preventing downstream signaling events including Akt phosphorylation, calcium flux, and chemoattractant-induced cytoskeletal rearrangements. This antiinflammatory effect is not exclusive to AAT, as it has also been reported that the plasma glycoprotease inhibitor α-2 macroglobulin creates a complex with IL-8, resulting in reduced neutrophil infiltration into the lungs compared with IL-8 controls (42). AAT and α-2 macroglobulin are acute-phase reactants, and as both proteins show a rise in mean value after infection, it is tempting to speculate that the increased levels of both serum proteins function to dampen down the inflammatory response after infection by binding and inactivating IL-8 activity. With regard to AATD, this AAT gradient is grossly disrupted, impacting on both interstitium and lung concentrations of AAT and the potential for neutrophil-mediated interstitial emphysema in AATD (43). In addition, the inability of E. coli–derived nonglycosylated AAT to bind and modulate the activity of IL-8 is of clinical importance. Indeed, while aerosolized nonglycosylated AAT may boost the natural antiprotease within the AATD lung (44), the results of the present study suggest added beneficial therapeutic effects of infused glycosylated AAT. The major outcome of augmentation therapy is a rise in blood AAT to levels usually referred to as being at the “protective” threshold. Studies have demonstrated that infusions of AAT are safe and can reverse the biochemical abnormalities in serum and lung fluid (12). In addition, there have been some recent studies on the clinical efficacy of AAT augmentation therapy beyond these biochemical considerations (45–48), although the way in which AAT confers these clinical benefits is not fully known. The present study illustrates that AAT augmentation therapy impacts on the circulating neutrophil and demonstrates that infused AAT fulfills an antiinflammatory role in vivo by modulating neutrophil chemotaxis.

This investigation elucidates a hitherto undescribed localization of neutrophil-associated AAT. AAT has previously been localized within primary granules containing neutrophil elastase and proteinase 3 (PR3) (15, 49); however, there remained a paradox as to why an enzyme and cognate inhibitor would simultaneously compartmentalize, potentially impeding protease-dependent antimicrobial activity. Our study reports the finding that both neutrophil-derived and serum-absorbed AAT bind the circulating neutrophil and that approximately 80% of cellular AAT is localized to the plasma membrane within lipid rafts and to a lesser extent within secretory vesicles. Further experiments employing confocal fluorescence microscopy and flow cytometry confirmed that AAT in resting MM neutrophils is bound to the outer membrane of the cell. Slight heterogeneity in molecular mass of membrane-associated isoforms of AAT was revealed by 2D electrophoresis, possibly relating to the described variable composition of sialic acids on various glycosylation sites (50). In addition, low circulating serum levels of AAT correlated with a concomitant reduction in the AAT membrane protein composition of ZZ-AATD neutrophils.

Rather than secretion of AAT from internal stores following full cell activation with combined cytochalasin B and fMLP stimuli (49), flow-cytometric analysis clearly showed that the inflammatory mediators IL-8 and TNF-α, in a concentration-dependent manner, induced the release of membrane-associated AAT by neutrophils. Gel filtration chromatography and immunoprecipitation analysis revealed that AAT was released from the cell membrane in a protein complex with its membrane-binding partner FcγRIIIb. In addition, released AAT was capable of forming irreversible complexes with purified NE. This last result is important, as it specifically shows that degranulation of primary granules did not occur during IL-8 treatment and also that AAT did not interact with the neutrophil membrane via NE, PR3, or cathepsin G, which have previously been proposed to bind hydrophobic or low-affinity proteoglycan moieties of neutrophil membranes (51). FcγRIIIb (40–60 kDa) is expressed by neutrophils and basophils (52) and is unique in that it is the only Fc receptor linked to the plasma membrane by a GPI anchor. In line with our results for AAT, FcγRIIIb has also been found associated with membrane lipid rafts (53), and in our study confocal laser microscopy showed membrane colocalization of AAT and FcγRIIIb in intact cells. Of note, a substantial amount of AAT was removed from the MM neutrophil membrane following treatment with either PI-PLC or NaCl, indicating that AAT interacts electrostatically with this GPI-linked low-affinity IgG receptor. Of relevance, within this study we found that low membrane-associated AAT correlated with a significant reduction in the FcγRIIIb membrane protein composition of ZZ-AATD neutrophils, most likely due to ADAM-17 activity. This latter result may explain the observed reduced ZZ-AATD cell chemotaxis in response to sIC when assays were performed in PBS, as the importance of normal FcγRIIIb expression for sIC-mediated neutrophil recruitment has been previously shown (20). In vivo, however, signaling and chemotaxis through FcγRIIIb has been shown to occur by shedding of this mobile receptor and co-capping via lectin-saccharide binding or cross-linking of FcγRIIIb with CR3, CD32a (54), or the fMLP receptor (21). For this reason, reduced chemotactic capacity of ZZ-AATD neutrophils suspended in PBS in response to sIC in vitro does not reflect in vivo conditions. To more closely imitate in vivo conditions, we evaluated the chemotactic index of MM and ZZ-AATD neutrophils toward sIC in the presence of respective MM and ZZ sera. The mean chemotactic index value of ZZ-AATD neutrophils was found to be significantly higher than that of MM control cells. When AATD serum with sIC was employed, soluble FcγRIIIb re-bound the ZZ-AATD neutrophil membrane (Supplemental Figure 8), initiating a signaling cascade leading to increased chemotaxis. sIC does not bind AAT and AAT does not inhibit binding of sIC to the cell surface (our unpublished observations), suggesting that AAT inhibition of ADAM-17 enzymatic activity is central to its inhibitory effect on neutrophil chemotaxis to sIC.

Shedding of FcγRIIIb from the neutrophil membrane (55) is involved in immune complex–stimulated neutrophil recruitment in vivo (20). Both metalloproteases and serine proteases have been implicated in the cleavage of FcγRIIIb (56), but the most likely candidates are members of the ADAM family of metalloproteases (57). Evidence exists that neutrophil chemotaxis requires surface sheddase activity, and ADAM-17 has been implicated in the shedding of FcγRIIIb (58). Our results indicate that the anti-chemotaxis effects of AAT in response to sIC may be mediated by inhibition of ADAM-17, resulting in decreased shedding of FcγRIIIb. In support of this theory, neutrophils isolated from ZZ-AATD individuals before augmentation therapy possessed low levels of membrane-associated FcγRIIIb, and results after augmentation therapy showed that newly produced FcγRIIIb-expressing cells contained an optimal concentration of bound AAT. Inhibition of ADAM-17 activity by serine-protease inhibitors (tosyl-l-lysine chloromethyl ketone) has previously been recognized (59) and further confirmed by this study. Native AAT, but not oxidized or polymerized AAT, inhibited ADAM-17 activity, indicating that an intact serpin-reactive center loop was required for the AAT–ADAM-17 inhibition. In addition, neither polymerized AAT nor the C-36 cleaved fragment of AAT could inhibit the AAT–ADAM-17 binding event. Such broad-spectrum activity of AAT is supported by its ability to abrogate cysteine–aspartic acid protease activity (5). In addition, AAT illustrates acute antiinflammatory effects similar to that of the metalloprotease inhibitor RS113456 (60). In support of our results, AAT has been shown to reduce neutrophil infiltration into lung tissue after intratracheal silica administration and after cigarette smoke–induced emphysema (61). Furthermore, the ability of AAT to inhibit ADAM-17 activity has been postulated in studies investigating shedding of TNF-α from islet cells (62).

In summary, our study has found that AAT modulates the chemotactic response of CXCR1 signaling by binding IL-8 and inhibiting receptor engagement. Additionally, AAT modulates neutrophil chemotaxis in response to sIC by inhibiting ADAM-17 activity, thereby decreasing release of FcγRIIIb from the neutrophil membrane. After augmentation therapy in ZZ-AATD individuals, increased serum levels of AAT function to bind IL-8– and FcγRIIIb-expressing neutrophils, thereby inhibiting ADAM-17, and in turn normalizing excessive AATD neutrophil chemotactic responses. Consequently, this study delineates a key antiinflammatory role for AAT in neutrophil chemotaxis and supports the possibility of additional benefits of AAT augmentation therapy in AATD.

View Supplemental data

We thank B.J. Harvey, W. Thomas, and O. McCabe, RCSI Molecular Medicine Laboratories, for providing support on the Zeiss LSM710 confocal immunofluorescence microscope purchased through the National Biophotonics Imaging Platform funding; the Children’s Research Centre, Crumlin Hospital, Dublin, for FPLC facilities; G. Mullins and C. O’Connor, Alpha One Foundation (Ireland) for collection of AATD patient samples; E. Hayes for collection of CF patient serum samples; R. O’Kennedy and B. Byrne, Applied Biochemistry Group, School of Biotechnology, Dublin City University, for protein binding studies. We thank all AATD and CF patients who participated in this study. This work was supported by the Alpha-1 Foundation (U.S.), the Medical Research Charities Group/Health Research Board, and the Programme for Research in Third-Level Institutes (PRTLI) administered by the Higher Education Authority.

Address correspondence to: Emer P. Reeves, Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland. Phone: 353.1.8093877; Fax: 353.1.8093808; E-mail: emerreeves@rcsi.ie.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2010;120(12):4236–4250. doi:10.1172/JCI41196.

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