Skip to main content
Download PDF
  • Original Articles
  • Open access
  • Published:

α-Linoleic Acid Enhances the Capacity of α1-Antitrypsin to Inhibit Lipopolysaccharide-Induced IL-1β in Human Blood Neutrophils

Molecular Medicine volume 22, pages 680–693 (2016)Cite this article

Abstract

Alphal-antitrypsin (A1AT, SERPINA1), a major circulating inhibitor of neutrophil elastase (NE) and protelnase-3 (PR3), has been proposed to reduce the processing and release of IL-1β. Since the antiinflammatory properties of A1AT are influenced by the presence of polyunsaturated fatty acids, we compared the effects of fatty acid-free (A1AT-0) and α-linoleic acid (LA)-bound (A1AT-LA) forms of A1AT) on lipopolysaccharide (LPS)-induced synthesis of the IL-1β precursor and the release of IL-1β from human blood neutrophils. The presence of A1AT-LA or A1AT-0 significantly reduced LPS-induced release of mature IL-1β. However, only A1AT-LA reduced both steady-state mRNA levels of IL-1β and the secretion of mature IL-1β. In LPS-stimulated neutrophils, mRNA levels of TLR2/4, NFKBIA, P2RX7, NLRP3, and CASP1 decreased significantly in the presence of A1AT-LA but not A1AT-0. A1AT-0 and A1AT-LA did not inhibit the direct enzymatic activity of caspase-1, but we observed complexes of either form of A1AT with NE and PR3. Consistent with the effect on TLR and IL-1β gene expression, only A1AT-LA inhibited LPS-induced gene expression of NE and PR3. Increased gene expression of peroxisome proliferator-activated receptor (PPAR)-γ was observed in A1AT-LA-treated neutrophils without LPS stimulation, and the selective PPAR-γ antagonist (GW9662) prevented a reduction in IL-1β by A1AT-LA. We conclude from our data that the ability of A1AT to reduce TLR and IL-1β gene expression depends on its association with LA. Moreover, the antiinflammatory properties of A1AT-LA are likely to be mediated by activation of PPARγ.

Introduction

Acute-phase proteins constitute an essential component of the innate immune response during infection or inflammation. Human alpha1-antitrypsin (A1AT) is one of the major acute-phase proteins found in the circulation. So far, the best-known and best-understood function of A1AT is rapid inhibition of neutrophil-released elastase (NE) and proteinase 3 (PR3). (1) Some studies, however, suggest that A1AT facilitates the concomitant inhibition of NE and PR3 inside and outside the cell. (2) PR3 and NE are closely related enzymes that enhance neutrophil-dependent inflammation not only by degrading components of the extracellular matrix but also by other deleterious effects, such as activation of IL-1β. (3) Hence, tight control of NE and PR3 activity by A1AT confirms its fundamental role in controlling and resolution of inflammation. More recently, A1AT has been shown to have independent antiinflammatory/immunomodulatory properties. A1AT protects against cell apoptosis, (4) inhibits neutrophil adhesion, chemotaxis, and superoxide production, (5–8) induces IL-1 receptor antagonist expression, (9) and regulates heme oxygenase-1 activity. (10) Moreover, A1AT purified from human blood modulates and/or prevents tissue injury in experimental animal models of human diseases, including graft-vs-host disease, inflammatory bowel disease, rheumatoid arthritis, acute liver failure, autoimmune diabetes, gouty arthritis, and ischemia-reperfusion injuries. (11–17) Despite growing reports on the antiinflammatory properties demonstrated for A1AT, the mechanism of a direct effect of A1AT on cells remains to be defined. Both in vitro and in vivo studies provide evidence that the beneficial effects of A1AT are mostly related to suppression of Toll-like receptor (TLR) agonist-induced innate immune cell activation. (18) Recent studies support the concept that some of the pleiotropic activities of A1AT are linked to the specific molecular form of the protein. For example, biological activities of A1AT can be modified due to interactions with lipid moieties, in which A1AT was found in association with cell membrane lipid rafts (19,20) and was detected in complexes with low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs). (21,22) The binding of A1AT to HDL augments its protective effect in a mouse model of elastase-induced pulmonary emphysema. (22) We recently reported that A1AT purified from human plasma binds polyunsaturated fatty acids (FAs) like α-linoleic and oleic acid, and that only FA-bound forms of A1AT increase the expression and release of ANGPTL4 in human blood-adherent monocytic cells and in primary human lung microvascular endothelial cells. (23,24)

The above functional differences between FA-free and FA-bound forms of A1AT led us to hypothesize that these 2 forms may express distinct immunomodulatory activities during neutrophil activation. Neutrophils account for about 50–75 % of circulating leukocytes, and during inflammation they are the first immune defense cells to arrive and function via multiple intra- and extracellular mechanisms. (25) Antiinflammatory actions of A1AT as well as FAs in neutrophils have previously been observed. (5,6,26–28) For example, short-chain FAs reduced TNF-α production by lipopolysaccharide (LPS)-stimulated human neutrophils, (29) and also inhibited the expression of TNF-α and NO in rat neutrophils via attenuation of NF-κB activation. (30) Polyunsaturated FAs have been reported to decrease expression of adhesion molecules on the surface of neutrophils (31) and to inhibit neutrophil chemotaxis. (32) Thus, the goal of the present study was to compare effects of FA-free (A1AT-0) and α-linoleic acid (LA)-bound (A1AT-LA) forms of A1AT on LPS-induced levels of the IL-1β precursor and the release of processed IL-1β in freshly isolated human blood neutrophils in vitro.

Materials and Methods

A1AT Preparations

Lipidomic analysis of A1AT Prolastin (Grifols) revealed that it consists of protein-lipid complexes, whereas A1AT (Zemaira, CSL Behring) is lipid-free, termed as A1AT-0. For experiments, A1AT-0 was mixed with α-linoleic acid (LA) (Sigma Aldrich) at a molar ratio of 1:4.8 and incubated for 3 h at 37°C in a water bath as previously described. (23) After the incubation, unbound LA was removed and A1AT-LA complexes were recovered in sterile phosphate buffered saline (PBS). A1AT-LA preparations were used immediately or were kept no longer than 48 h at 4°C.

A1AT-0 and A1AT-LA complex formation with NE and proteinase-3 (PR3) in vitro.

Preparations of A1AT-0 and A1AT-LA were assessed for their ability to form complexes with active pancreatic elastase (Sigma-Aldrich) and PR3 (Enzo Life Sciences). A1ATs plus NE or PR3 (both at a molar ratio of 1.2:1) were incubated at room temperature for increasing periods of time up to 5 h. The reaction was stopped by adding 2 × SDS loading buffer and boiling for 5 min. Samples were analyzed by electrophoresis on 12.5% sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE).

Caspase-1 Inhibition Assay

A1AT-0 and A1AT-LA were tested for inhibition of caspase-1 activity in vitro by using Caspase-1 Inhibitor Drug Detection kit as recommended by the manufacturer (Abcam). In brief, increasing concentrations of A1AT-0 and A1AT-LA were pre-incubated with active caspase-1 and mixed with reaction buffer containing fluorogenic substrate, YVAD-AFC. Fluorescence readings were then taken at 37°C using 400 nm excitation and 505 nm emission wavelengths on Tecan Infinite M200 plate reader (Männedorf,) every 10 min for 90 min. The synthetic caspase-1 inhibitor z-VAD-FMK was used as a positive control for the assay.

Human Blood Neutrophil Isolation

Neutrophils were isolated from freshly obtained peripheral blood of 50 healthy donors (under 30 years of age) using Polymorphoprep (Axis-Shield PoC AS) as described previously. (6) This protocol routinely yields more than 97% cell purity as assessed by flow cytometry and Wrights-Giemsa stain (Sigma-Aldrich).

Human Neutrophil Culture and Treatments

Neutrophils (5 × 106 per well) were suspended in RPMI-1640 medium (Gibco, Life Technologies) and allowed to stand for 30–40 min in cell culture plates precoated with fetal calf serum. In some experiments, cells were preincubated with 10 µM GW9662 (Sigma-Aldrich), a selective irreversible PPAR-γ antagonist, for 30 min at 37°C. Neutrophils were then stimulated with 20 ng/mL LPS (Escherichia coli LPS 055:B5, Sigma-Aldrich) alone or in a combination with 1 mg/mL A1AT-0 or A1AT-LA at 37°C and placed in 5% CO2. In parallel, neutrophils were incubated with PBS or LA, A1AT-0 or A1AT-LA alone. In some experiments, 1 mg/mL A1AT (Prolastin) was used alone or together with LPS. Following incubations with LPS and/or A1AT for 5 h, the supernatants and cells were analyzed for protein release and gene expression, respectively.

Microscopic Analysis of Interaction of A1AT-0 and A1AT-LA with Neutrophils

Human neutrophils (3 × 106) were plated onto glass coverslips alone or in the presence of A1AT-0 (0.5 mg/mL) or A1AT-LA (0.5 mg/mL) for 5 min and 1 h at 37°C CO2, or for 5 min at 4°C. Cells were then washed with PBS and fixed with 100% methanol for 10 min at −20°C. Fixed cells were washed 3 times with PBS and incubated for 1 h at room temperature without or with anti-human A1AT mouse monoclonal antibody (Santa Cruz), diluted 1:50 in PBG (PBS containing 0.5% BSA and 0.2% fish gelatin). Coverslips were washed 3 times with PBS, followed by incubation with AlexaFluor-488-labeled goat anti-mouse IgG (1:400 dilution, Thermo Fisher Scientific) for 1 h at room temperature. The cells were then washed in PBS and mounted on microscope slides using ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific). Images were made using an Olympus FluorVIew 1000 scanning confocal microscope equipped with a 60 × oil immersion objective.

RNA Isolation, cDNA Synthesis and Real-Time PCR

Total RNA was prepared using RNeasy Micro kit (Qiagen Sample and Assay Technologies). For cDNA synthesis, 1 µg of total RNA was transcribed using high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific). mRNA levels of selected genes (Table 1) were determined using TaqMan Gene Expression Assays (Applied Biosystems, Life Technologies) on Step One Plus real time PCR machine (Applied Biosystems). Threshold cycle (Ct) value for each sample was calculated by determining the point at which the ßuorescence exceeded a threshold limit. GAPDH, HPRT, and glucuronidase beta (GUSB) were used as reference genes. Relative gene expression was calculated according to the ΔΔCT method. Basal expression of TLR2, TLR4, P2RX7, and SERPINA1 genes was calculated according to ΔCT method: 2^(Ct value of target gene — Ct value of reference gene).

Table 1 List of the primers. All primers were purchased from Life Technologies.
Full size table

Cytoplasm and Nuclei Isolation

Cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Briefly, neutrophils (1.5 × 107 per well) were incubated with LPS, A1AT-0, or A1AT-LA alone and in combinations for 5 h. Afterward, cells were pelleted and cell membranes were disrupted by addition of ice-cold CER I and CER II. Cytoplasmic content was collected post centrifugation at 17 000 g for 10 min at 4°C. After recovering the intact nuclei from the cytoplasmic extract by centrifugation, the nuclei were lysed with a third reagent, NER, to yield the nuclear extract. Nuclear lysates were centrifuged at 17 000 g for 10 min and supernatants were collected. Cytoplasmic and nuclear fractions were analyzed by electrophoresis and western blot.

Electrophoresis and western Blot Analysis

Cell pellets were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (SC-24948, Santa Cruz) for 30 min on ice. Following centrifugation at 15 000 g for 10 min, the protein concentration in the supernatants was determined by Bradford assay (Thermo Fisher Scientific). In some experiments (immunoblots for NE and PR3), neutrophils were sonicated in PBS. The sonicated preparations were centrifuged at 10 000g for 5 min. Following centrifugation, replicate aliquots from the supernatants were immediately analyzed. Equal amounts of protein were loaded on 7.5%, 10%, or 12.5% SDS-PAGE gels. Elecrophoretically separated proteins were stained with 0.1% Coomassie Blue R250. From the parallel gels, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore) using semidry blot transfer. Blots were blocked with 5% bovine serum albumin (BSA, Calbiochem) or milk (5%, Roth) for 1 h at room temperature and then probed with specific primary antibodies (Table 2). The immune complexes were visualized with horseradish peroxidase-conjugated antibodies (DAKO A/S) and ECL western blotting substrate (Thermo Fisher Scientific). The density of the specific bands was quantified using ImageJ software (http://imagej.nih.gov/ij).

Table 2 List of the antibodies used in western blotting.
Full size table

Analysis of Surface Expression of TLR2 and TLR4 by Flow Cytometry

Neutrophils (1 × 106 per condition) were incubated for 5 h with LPS and LPS plus either A1AT-0 or A1AT-LA. Neutrophils were then labeled with fluorescein conjugated monoclonal anti-human TLR2 or TLR4 (eBioscience) and the corresponding isotype controls (BD Pharmingen) for 35 min at 4°C. After labeling, cells were washed, resuspended in FACS buffer (PBS containing 0.5% BSA) and examined using FACS Calibur (Becton Dickinson) or FACS Canto (BD Biosciences). Data were analyzed using FACS Diva software (BD Biosciences).

NF-κB Pathway Array for Neutrophils

Neutrophils were incubated for 4 h with PBS, Prolastin, and 1 mg/mL LPS (10 ng/mL) separately or in combination. Total RNA was prepared using the RNeasy Mini kit (Qiagen) and quantified with NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific). RNA purity was analyzed on 1% agarose gels. cDNA was synthesized from 1 µg of RNA with RT2 First Strand kit (Biosciences, Qiagen) and analyzed for expression of NFκB-pathway related genes using Human Angiogenesis RT2 Profiler PCR Array (Biosciences, Qiagen) according to the manufacturer’s protocol. Quantitative real-time PCR was conducted on an ABI 7500 machine (Applied Biosystems). β2-microglobulin, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a, GAPDH, and β-actin were measured at the same time and used as internal controls. Relative gene expression (fold change) was determined using the ΔΔCT method on the SAB web portal (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).

Lactate Dehydrogenase Assay

Treatment-associated cytotoxicity was determined using the Cytotoxicity Detection Kit (LDH, Roche) according to the manufacturer’s protocol. In brief, the assay quantifies lactate dehydrogenase (LDH) released from ruptured or dead cells into the culture supernatant by a colorimetric reaction. Neutrophils were treated according to the experimental setting and cell supernatants were collected at the end of the incubation time. Total cell lysate was used for 100% control value. For low control and background control, supernatant from untreated cells and assay medium alone were used, respectively. Absorbance of the colorimetric product of LDH reaction was measured at 490 nm using an Infinite M200 microplate reader (Tecan). Measurements were carried out in triplicate.

Myeloperoxidase Assay

For quantitative determination of supernatant myeloperoxidase (MPO), we used a commercially available ELISA kit (Human DuoSet R&D Systems). Samples were diluted 1:500 with dilution buffer and measured in duplicate using the Infinite M200 microplate reader. Detection limit was 62.5 pg/mL.

IL-1β ELISA Assay

Cell culture supernatants and lysates from different experimental conditions were examined for IL-1β concentration using the DuoSet ELISA kit. Detection limit was 4 pg/mL.

Statistical Analysis

Statistical Package (SPSS for Windows, release 23.0) was used for statistical calculations. The differences in the means of experimental results were analyzed for their statistical significance using one-way ANOVA combined with a multiple-comparison procedure (Scheffe multiple range test), with an overall significance level of p = 0.05. An independent 2-sample t-test was also used.

All supplementary materials are available online at www.molmed.org .

Results

IL-1β Release and IL-1β Precursor Expression Levels in LPS- and LPS/A1AT-0- or A1AT-LA-Treated Neutrophils

LPS induces the processing and release of high levels of mature IL-1β by neutrophils. (33,34) As expected, neutrophils incubated for 5 h with LPS significantly increased IL-1β release (Figure 1A) and IL-1β precursor expression (Figure 1B) relative to controls. We next investigated the capacity of non-lipidated A1AT-0 and lipidated A1AT-LA to alter the production of IL-1β by neutrophils in response to LPS stimulation. As shown in Figure 1A, LPS-stimulated IL-1β release was reduced by about 50% in both the LPS/A1AT-0 (p = 0.006) and LPS/A1AT-LA (p < 0.001) treated cells compared with LPS. The levels of IL-1β in the supernatants of control neutrophils and treated with A1AT-0 or A1AT-LA were below detection. However, mRNA expression of IL-1β precursor decreased significantly only in neutrophils stimulated with LPS in the presence of A1AT-LA compared with neutrophils stimulated with LPS/A1AT-0 (Figure 1B). In parallel, higher levels of mature IL-1β protein were detected in LPS- and LPS/A1AT-0 stimulated cells as compared with controls or LPS/A1AT-LA (Figure 1C). Total concentration of IL-1β in cell lysates was higher in LPS- and LPS/A1AT-0-treated than in LPS/A1AT-LA-treated cells (Figure 1D). Similarly, when human neutrophils were stimulated with LPS in the presence of commercially available lipidated A1AT (Prolastin) for 4 h, significant inhibition of LPS-induced IL-1β release and IL-1β mRNA levels was observed (Supplementary Figures S1A and B). LDH levels revealed no toxicity after neutrophil stimulation with LPS and A1AT preparations alone or in combinations (Supplementary Figure S2). Expression of IL-18, another proinflammatory cytokine of the IL-1 family, was undetectable (data not shown).

Figure 1
figure 1

Effects of A1AT-0 and A1AT-LA on LPS-induced IL-1β precursor and IL-1β released. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). Released IL-1β levels (A), IL-1β gene expression (B), cell-associated profile, and levels of IL-1β protein were determined by western blotting (C) and ELISA (D). GUSB and GAPDH were used as housekeeping genes in qPCR experiments. Box plots represent data from 5 individual blood donors; n = number of replicates for each experiment. Blot is representative of 3 independent donors. P value indicates significant differences compared with the values seen in LPS-activated cells.

Full size image

Expression Levels of TLRs in LPS- and LPS/A1AT-Treated Neutrophils

Human peripheral blood neutrophils express TLR2 and TLR4, albeit at lower levels than monocytes. (35) As expected, TLR2 and TLR4 mRNA levels were low in resting neutrophils [TLR2 and TLR4, mean (SD): 0.023 (0.005), n = 9; and 0.103 (0.05), n = 15, respectively]. Commercial LPS contains TLR2-stimulating substances, lipopeptides, (36) and indeed, Figures 2A and B demonstrate an increase in TLR2 and TLR4 mRNA expression after LPS stimulation of neutrophils for 5 h. Stimulation of neutrophils with LPS in the presence of A1AT-0 did not change TLR mRNA expression when compared with the LPS-treated cells. In contrast, TLR2 and TLR4 expression was reduced by two-fold (p < 0.001) and 1.6-fold (p < 0.05), respectively, in LPS-stimulated neutrophils in the presence of A1AT-LA (Figures 2A and B). By flow cytometry, the levels of TLR2 and TLR4 expression on the cell surface were low and did not differ significantly between experimental conditions (Supplementary Figure S3).

Figure 2
figure 2

Effects of A1AT-0 and A1AT-L on LPS-induced TLR2 and TLR4 mRNA expression. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). Expression of TLR2 (A) and TLR4 (B) was determined by real-time PCR as described in Materials and Methods. GAPDH was used as a housekeeping gene in this experiment. Box plots represent data from 4 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in LPS-activated cells.

Full size image

Effect of A1AT on LPS-Induced NF-κB Pathway Gene and Protein Expression

The main NF-κB subunits RelA/p65 and c-Rel/p50 and the inhibitor (I) κB, an NF-κB-inactivating factor, are constitutively expressed in neutrophils. (37) Scheibel et al. (38) demonstrated that the transcription of IL-1β depends on a positively acting p65-c-Rel-IκB complex. As expected, RelA/p65, NFKBIA, and NFKBIB mRNA levels were significantly enhanced in the LPS-stimulated neutrophils in comparison to controls (Figures 3A, B, and C). LPS-stimulated expression of these genes was not significantly different from neutrophil-stimulated LPS in the presence of A1AT-0. In contrast, in neutrophils treated with A1AT-LA, LPS-induced expression of RelA/p65 and NFKBIA but not NFKBIB was reduced (RelA/p65 was reduced by 1.48-fold, p < 0.05, and NFKBIA by three-fold, p < 0.001) (Figures 3A, B, and C). Lipid-containing A1AT (Prolastin) added to neutrophil cultures for 4 h also lowered LPS-induced expression of RelA/p65 and other genes of the NF-κB pathway (Table 3). However, after 5 h profiles of cytosolic p65, IκBβ, and IκBα proteins did not differ markedly between LPS and LPS/A1ATs. In the nuclear fraction, only IκBα protein was detected but did not differ between treatments (Figures 3D, E, and F).

Figure 3
figure 3

Effects of LPS and LPS/A1AT-0 and LPS/A1AT-LA on Re/A/p65, NFKBIA/IκBα and NFKBIB/IκBβ expression and protein levels. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of Re/A/p65 (A), NFKBIA/IκBα (B) and NFKBIA/IκBβ (C) genes was analyzed as described in Materials and Methods. GAPDH and GUSB were used as housekeeping genes in qPCR experiments. Box plots represent data from 3 to 5 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in LPS-activated cells. Subcellular fractions were analyzed by western blotting with anti-p65 (D), anti-NFKBIA/IκBα (E) and anti-NFKBIA/IκBβ (F) antibodies. The blots were reprobed with anti-β-actin and anti-histone H1 antibodies as loading controls for cytosolic and nuclear fractions, respectively. Each blot is representative of 3 independent donors.

Full size image
Table 3 Fold changes in NF-κB pathway mRNA gene expression following neutrophil treatment with LPS and LPS/A1AT (Prolastin®) for 4 h.
Full size table

A1AT-LA Inhibits the Ability of LPS to Upregulate NLRP3 and CASP-1 mRNA Levels

Neutrophil IL-1β processing and secretion in response to LPS requires activation of the inflammasome containing the components NLRP3, pro-caspase-1, and apoptosis-associated speck-like protein containing a CARD (ASC), encoded by the PYCARD gene. Human peripheral blood neutrophils constitutively contain NLRP3 and ASC protein, and NLRP3 gene expression also increases following TLR2 stimulation. (39) After 5 h, neutrophils treated with LPS significantly increased NLRP3 mRNA expression (Figure 4A). A1AT-LA but not A1AT-0 lowered LPS-induced NLRP3 mRNA. Under the same experimental conditions, we found a marginal increase in the level of NLRP3 protein in LPS-stimulated neutrophils, but this was not observed in LPS/A1AT-LA or LPS/A1AT-0 treatments (Figure 4B). The expression levels of ASC in LPS-treated neutrophils were low, and A1AT-LA and A1AT-0 had no specific effect [0.21-fold (0.1) (LPS), 0.17-fold (0.04) (LPS + A1AT-0), and 0.17-fold (0.04) (LPS + A1AT-LA) against the controls, n.s.] (data not shown). The expression of caspase-1 (CASP-1) was also significantly increased by LPS treatment compared to controls (Figure 5A). Treating neutrophils with A1AT-0 did not alter LPS-induced CASP-1 expression compared with LPS. However, treatment with A1AT-LA reduced LPS-induced CASP-1 mRNA levels (by 1.4-fold, p = 0.032) compared with LPS (Figure 5A). The molecular forms of caspase-1 protein were lower in neutrophils treated with A1AT-LA or A1AT-0 relative to controls. Furthermore, neutrophils stimulated with LPS plus A1AT-0 or A1AT-LA showed a shift toward the precursor form (45 kDa) of caspase-1 compared with neutrophils stimulated with LPS alone (Figure 5B). As illustrated in Figure 5C, neither A1AT-0 nor A1AT-LA inhibited caspase-1 activity in vitro.

Figure 4
figure 4

Effects of A1AT-0 and A1AT-LA on LPS-induced NLRP3 expression. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of the NLRP3 gene (A) was analyzed by real-time qPCR as described in Materials and Methods. GAPDH and GUSB were used as housekeeping genes. Box plots represent data from 4 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in LPS-activated cells. Total cell lysates were analyzed for NLRP3 protein levels by western blotting (B). For loading control, blots were reprobed with antibodies to β-actin. Each blot is representative of 3 independent donors with similar results.

Full size image
Figure 5
figure 5

Effect of A1AT-0 and A1AT-LA on LPS-induced caspase-1 expression and activity. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of the CASP1 gene (A) was analyzed by real-time qPCR as described in Materials and Methods. GAPDH and HPRT were used as housekeeping genes. Box plots represent data from 4 individual experiments; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in LPS-activated cells. Total cell lysates were analyzed for the caspase-1 protein profile characterization by western blotting (B). For loading control, blots were reprobed with antibodies to β-actin. The blot is representative of 3 independent donors with similar results. Caspase-1 activity (C) was analyzed in vitro by using caspase-1 fluorogenic tetrapeptide substrate (YVAD-AFC). Data represents caspase activity (in relative fluorescence units) in the presence of either 1 mg/mL A1AT-0 or A1AT-LA or 20 µM z-VAD-FMK (generic caspase inhibitor). Measurements were taken every 10 min. Each curve represents data from 2 independent experiments, each performed in triplicate.

Full size image

A1AT-LA Reduces LPS-Induced P2RX7 Expression in Human Neutrophils

P2RX7 expression is reported in human neutrophils (40) and found to be involved in NLRP3 inflammasome activation and IL-1β secretion. The basal expression of P2RX7 (relative to GUSB, used as a housekeeping gene) was low in neutrophils [mean (SD): 0.068 (0.02), n = 15]. However, as shown in Figure 6, in LPS-stimulated neutrophils, P2RX7 mRNA levels increased by 30-fold, p < 0.001, compared with unstimulated cells. Compared with A1AT-LA- but not A1AT-0-treated neutrophils, LPS-induced P2RX7 mRNA levels were reduced by 45%, p < 0.01 (Figure 6).

Figure 6
figure 6

Effects of A1AT-0 and A1AT-LA on LPS-induced P2RX7 expression Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of the P2RX7 gene was analyzed by real-time qPCR as described in Materials and Methods. GAPDH and GUSB were used as housekeeping genes. Box plots represent data from 4 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in control cells.

Full size image

Effects of A1AT-LA and A1AT-0 on LPS-Induced Expression of SERPINA1, ELANE, and PRTN3 Genes

As expected, gene expression of SERPINA1 was significantly induced following LPS treatment as compared with control neutrophils [mRNA relative expression, fold changes, mean (SD): 5.0 (0.98) vs 1.0 (0.13), respectively, n = 18, p < 0.001]. However, treatment of neutrophils with A1AT-0 or A1AT-LA did not significantly alter LPS-induced SERPINA1 expression as compared with LPS-treated neutrophils in the absence of LPS stimulation [five-fold (1.4) and 4.2-fold (0.9), respectively, n = 15 vs five-fold (0.98) LPS] (data not shown). As illustrated in Figures 7A and B, LPS stimulated expression of PRTN3 and ELANE remained enhanced in A1AT-0-treated neutrophils (PRTN3, p < 0.001; ELANE, p = 0.008) but not in A1AT-LA-treated cells. Analysis of cell lysates by western blots using specific antibodies against human A1AT (as a control) and PR3 detected multiple forms of high-molecular-size bands representing enzyme-inhibitor complexes in A1AT-0 or A1AT-LA-treated samples but not in controls or LPS-treated cells (Figures 7C and D). Concomitantly, the level of PR3 protein alone (29 kDa) was lower in A1AT- and LPS/A1AT-treated cells relative to controls and LPS-stimulated cells. Regarding NE, similar profiles of protein were detected in all experimental conditions (Figure 7E). In support, A1AT-0 and A1AT-LA formed similar complexes with pancreatic elastase and PR3 in vitro (Supplementary Figures S4A and B). Under these experimental conditions, neither A1AT-0 nor A1AT-LA had a significant effect on LPS-induced MPO release (Supplementary Figure S5).

Figure 7
figure 7

Effects of A1AT-0 and A1AT-LA on LPS-induced NE and PR3 expression and protein profile. Human neutrophils (5 × 106) were incubated for 5 h in either medium alone or medium containing LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). mRNA expression of PRTN3 (A) and ELANE (B) were analyzed by real-time qPCR as described in Materials and Methods. GAPDH and HPRT were used as housekeeping genes. Box plots represent data from 5 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in control cells. Cell lysates were analyzed for A1AT (C), PR3 (D) and NE (E) protein profile characterization by 12.5% SDS-PAGE following western blotting. M-molecular size marker (kDa). Each blot is representative of 3 independent donors.

Full size image

Interaction of A1AT-LA and A1AT-0 with Neutrophils

To compare cellular localization of exogenous AAT-0 and A1AT-LA, neutrophils were incubated with each form of A1AT for 5 min at 4°C or 37°C, or for 1 h at 37°C, and then subjected to immunofluorescence microscopy. Cells without A1AT treatment were used as controls (Figure 8A). Microscopic analysis revealed a marked difference in fluorescence intensity. To appreciate A1AT distribution in the samples with the weaker signal, such as the control and 4°C-incubated cells, the settings were optimized for the weak fluorescence, resulting in overexposed signals in the cells incubated for 1 h at 37°C. As expected, A1AT-treated cells showed enrichment of the A1AT-positive signal at the cell surface, as compared with control cells (Figure 8). Notably, on cells incubated at 4°C, which inhibits A1AT uptake, finely dispersed immunoreactivity was observed, whereas cells incubated at 37°C revealed large A1AT-positive granules or aggregates, especially prominent after 1 h of incubation. As shown in Figure 8C, if compared with A1AT-LA-treated neutrophils, cells treated with AAT-0 formed larger granules with stronger fluorescence. Apart from that, neutrophils incubated with AAT-0 or A1AT-LA, in general, showed a similar pattern of A1AT distribution on the cell surface and/or within the cells (Figure 8). Analysis of cytoplasmic A1AT-0 and A1AT-LA by western blots confirmed the similar pattern between both forms of A1AT (Supplementary Figure S6).

Figure 8
figure 8

Immunofluorescence microscopy analysis of A1AT-0 and A1AT-LA interaction with neutrophils. Neutrophils were incubated alone as controls for 5 min at 37°C (A), or with either A1AT-0 or A1AT-LA for 5 min at 4°C (B), or for 5 min and 1 h at 37°C (C). Cells were stained with anti-A1AT antibody (green) and DAPI (blue). Arrows point to the larger A1AT-reactive granules formed upon A1AT-0 treatment as compared with those of A1AT-LA (arrowheads). Images were acquired using Olympus FluorView 1000 scanning confocal microscope equipped with a 60 × oil immersion objective. Scale bar: 10 µm.

Full size image

Effects of A1AT-LA and A1AT-0 on Antiinflammatory Transcription Factor PPAR-γ

As demonstrated in Figures 9A and B, neutrophils incubated with A1AT-LA or lipid-containing A1AT (Prolastin) significantly increased PPAR-γ gene expression relative to untreated cells. AIAT-0 had no effect on PPAR-γ mRNA levels. Similarly, treatment with A1AT-LA but not A1AT-0 strongly enhanced LPS-induced PPAR-γmRNA levels compared with LPS-treated cells without A1AT (Figures 9 C and D). Neutrophils treated with GW9662 reversed the ability of A1AT-LA to inhibit LPS-induced IL-1β mRNA levels (Figure 9E).

Figure 9
figure 9

A1AT and LPS/A1AT induce PPAR-γ expression; effect of GW9662 on A1AT-LA ability to inhibit LPS-induced IL-1β expression Human neutrophils (5 × 106) were incubated for 5 h in either medium alone, medium containing A1AT (Prolastin, 1 mg/mL) or LPS (20 ng/mL) in the absence or presence of A1AT-0 (1 mg/mL) or A1AT-LA (1 mg/mL). Expression of PPAR-γ (A–D) was analyzed by real-time qPCR as described in Materials and Methods. GAPDH and HPRT were used as housekeeping genes. Box plots represent data from 3 individual donors; n = number of replicates for each experiment. P value indicates significant differences compared with the values seen in control cells (A and B) or LPS (C and D). (E) Cells were pretreated for 30 min with 10 µM GW9662, an irreversible PPAR-γ antagonist, prior to addition of LPS or LPS/A1ATs. Data are expressed as percentage of IL-1β gene expression in cells activated with LPS (100%) and show means (SD) of 3 independent donors; n = number of replicates for each experiment. P < 0.001 represents significant difference compared with the values seen in LPS-activated cells.

Full size image

Discussion

IL-1β is a highly inflammatory cytokine and is implicated in several pathological conditions. (41) Neutrophils are a major source of IL-1β (39,42,43); moreover, neutrophil-derived IL-1β mediates further neutrophil recruitment and activation. Synthesis of the IL-1β precursor is considered as a priming step, whereas intracellular cleavage of the IL-1β precursor and secretion of active IL-1β is a key step in the inflammatory response. Therefore, understanding the molecular regulation of these steps is of critical importance for the design of IL-1β-based modulatory therapies.

Human-plasma-purified A1AT used as therapy not only inhibits neutrophil elastase and proteinase 3 but also expresses broad antiinflammatory and immunomodulatory effects, including modulation of IL-1β levels. (11,39–41) This latter observation prompted us to investigate the effect of currently used preparations of A1AT in treating humans on processing secretion of active IL-1β in LPS-activated human blood neutrophils in vitro. It is important to point out that therapeutic preparations of human A1AT may contain lipid-free (38) and polyunsaturated fatty acid (LA) bound forms of A1AT. (42) Therefore, we investigated whether lipid-free (A1AT-0) and LA-bound (A1AT-LA) forms of A1AT differ in their effects on LPS-induced IL-1β.

Our data demonstrate a significant inhibitory effect of A1AT-LA and A1AT-0 on LPS- induced IL-1β release from human blood neutrophils in vitro. Regarding IL-1β mRNA and protein levels, the findings were unexpected. When compared with LPS, there was a marked decrease of IL-1β mRNA and IL-1β immunoreactivity in cell lysates following LPS/A1AT-LA stimulation. However, under the same experimental conditions, A1AT-0 did not affect LPS-induced IL-1β gene expression and protein levels. This latter finding illustrates a fundamental role of the lipid content in A1AT therapeutic preparations. We therefore investigated the mechanism for the effects of the 2 forms of A1AT on LPS-induced inflammatory gene expression. To generate activated neutrophils, we treated them for 5 h with a low concentration of LPS (20 ng/mL) in the absence or presence of A1AT (1 mg/mL). LDH release as a measure of cytotoxicity confirmed that our experimental settings did not affect the viability of neutrophils.

In neutrophils, LPS triggers synthesis of the IL-1β precursor through the TLR-NFκB pathway (44) and induces release of mature IL-1β. (33,34) As expected, LPS-treated neutrophils showed higher expression of NF-κB pathway genes compared with nontreated controls and A1AT-treated cells. Concomitantly, we observed a several-fold increase in IL-1β precursor and supernatant levels of IL-1β. Compared with neutrophils treated with LPS, treatment with LPS/A1AT-LA but not A1AT-0 significantly reversed LPS-induced TLRs, RelA/p65, and IκBα mRNA levels. Interestingly, A1AT-LA had no significant effect on LPS-induced IκBβ expression. Considering protein levels, we found no differences in cytosolnuclear profiles of RelA/p65, IκBα, and IκBβ between LPS and LPS/A1AT- or LPS/A1AT-LA-treated neutrophils. As all experiments were performed within 5 h of incubation time, we avoid making firm conclusions based on the protein data. Different incubation periods may be required to monitor changes at the protein level. Several studies provide evidence that in neutrophils, the relationship between gene transcription and translation is far from linear. (42,45,46) Notably, we did not detect p65 and IkBβ proteins in the nuclear fraction. This observation is in line with the finding that the IκBα-NF-κB complex shuttles between cytoplasm and nucleus while the IκBβ-NF-κB complex is cytosolic and does not undergo shuttling at all. (47–49) IκBα and IκBβ are cytoplasmic isoforms of IκB responsible for keeping NF-κB inactive (50,51); however, signaling that proceeds through these proteins has unique implications for target gene expression. (52) For example, in response to LPS, IκBβ is required for IL-1β transcription, (53) whereas IκBα is associated with caspase-3 activation and increased apoptosis. (54)

A detailed investigation of how A1AT-LA affects LPS-induced expression of IκB isoforms was beyond the scope of this work. However, the data suggest that A1AT-LA reduces LPS-induced synthesis of the IL-1β precursor by impairing the ability of LPS to upregulate the TLR-NFκB pathway.

Recent findings have revealed the pivotal role of NLRP3 in IL-1β production by human peripheral blood neutrophils. (55) Human neutrophils constitutively express NLRP3, whereas NF-κB activation downstream of TLRs can increase the expression of NLRP3 required to activate caspase-1. (39) Thus, in neutrophils, LPS alone can upregulate caspase-1 and generate a release of small amounts of IL-1β. (34,56,57) Therefore, we next investigated NLRP3 inflammasome components in neutrophils stimulated with LPS compared with neutrophils treated with LPS and A1AT combinations. The mRNA and protein expression of NLRP3 and CASP1 increased following LPS treatment as compared with cells not exposed to LPS. Under the same experimental conditions, A1AT-LA but not AAT-0 significantly lowered LPS-induced NLRP3 and CASP1 mRNA levels. Protein levels of NLRP3 were marginally lower in LPS/A1AT and LPS/A1AT-LA compared with LPS-treated cells. ASC acts as a bridge to recruit NLRP3 using the PYD-PYD interaction, and to recruit caspase-1 via the CARD-CARD interaction. (58) In the present experimental setting, in which neutrophils were treated for 5 h, the levels of ASC mRNA did not change in response to LPS and LPS/A1AT-LA. Indeed, earlier studies on ASC reported a transient induction (at 2 h of LPS stimulation). (59–61) Ionotropic P2RX7 expression on neutrophils is required for NLRP3 inflammasome assembly and IL-1β secretion. (39) In the present experimental model, basal expression of P2RX7 was low but increased after treatment with LPS. When neutrophils were treated with LPS in the presence of A1AT-LA, no significant induction of P2RX7 expression was observed. These data further support the notion that A1AT-LA but not A1AT-0 inhibits LPS-induced synthesis of the IL-1β precursor by reducing gene expression of the TLR/inflammasome pathway.

Proteolytic Cleavage of the IL-1β Precursor Is Required for Its Activity and Secretion

Caspase-1-mediated cleavage of the IL-1β precursor following inflammasome complex formation is suggested as the major mechanism responsible for the secretion of IL-1β. (62,63) Previous studies reported conflicting results regarding the ability of A1AT to inhibit caspase-1. (64–66) Data from our in vitro experiments show that neither A1AT-0 nor A1AT-LA inhibits active caspase-1. Neutrophil-derived serine proteases can activate TLR4 (67) and can also be responsible for processing the IL-1β precursor. (68–73) A mouse model of arthritis identified that IL-1β processing by neutrophils is independent of caspase-1 but is dependent on NE. (74) Some studies, however, suggest that PR3 has a greater role than NE in IL-1β processing and secretion. (3) PR3 is localized in the same lipid raft domains (75,76) as A1AT (5,20) and LA (77). Bilayer-bound PR3 has a reduced catalytic efficiency, whereas its inhibition by A1AT is more important than that observed for the soluble form of the enzyme. (78) We previously published results showing that A1AT-0 and A1AT-LA have similar inhibitory activity against elastase. (23) Here we confirm that A1AT-LA and A1AT-0 can form complexes equally well with NE and PR3 in vitro and in neutrophil lysates. In support of this, both forms significantly inhibited LPS-induced IL-1β release, although the effect of A1AT-LA was more pronounced relative to A1AT-0. Noticeably, when compared with LPS, treatment with A1AT-LA but not A1AT-0 inhibited ELANE and PRTN3 mRNA levels. This further favors A1AT-LA as a more effective inhibitor of LPS-induced IL-1β release. Assuming that NE and PR3 play a critical role in LPS-induced IL-1β release, the above results would explain why both A1AT-LA and A1AT-0 inhibited LPS-induced IL-1β release.

Taken together, A1AT complexed with unsaturated fatty acids such as LA reduces LPS-induced synthesis of the IL-1β precursor and release of active IL-1β, whereas the fatty acid-free form of A1AT only reduces LPS-induced release of IL-1β. The molecular mechanism behind the observed differences between the 2 forms of A1AT appears to be dependent on PPARs as sensors for fatty acids. (79) We previously found that A1AT-LA requires the activity of PPARs to induce expression of ANGPTL4 and related genes in adherent peripheral blood mononuclear cells. (23) PPAR-γ is expressed in neutrophils, and the ligand-dependent activation of this receptor results in suppression of cytokine production. (80) The present data confirm that A1AT-LA independent of LPS stimulation upregulates PPAR-γ mRNA in neutrophils, while A1AT-0 has no effect. Furthermore, neutrophil pretreatment with GW9662, a specific inhibitor of PPAR-γ, markedly diminished the ability of A1AT-LA to inhibit LPS-induced synthesis of the IL-1β precursor. Hence, A1AT-LA-induced PPAR-γ appears to control synthesis of the IL-1β precursor in activated neutrophils.

Conclusion

We speculate that LA in complex with A1AT results in a novel form of A1AT having a strong ability to regulate inflammation-induced synthesis, processing, and release of active IL-1β. These observations may be of value in evaluating the biological effects and therapeutic efficiency of A1AT preparations.

Disclosure

The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.

References

  1. Ehlers MR. (2014) Immune-modulating effects of alpha-1 antitrypsin. Biol. Chem. 395:1187–93.

    Article  CAS  Google Scholar 

  2. Hajjar E, et al. (2010) Structures of human proteinase 3 and neutrophil elastase — so similar yet so different. FEBS Journal. 277:2238–54.

    Article  CAS  Google Scholar 

  3. Coeshott C, et al. (1999) Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc. Natl. Acad. Sci. U.S.A. 96:6261–66.

    Article  CAS  Google Scholar 

  4. Zhang B, et al. (2007) Alpha1-antitrypsin protects beta-cells from apoptosis. Diabetes. 56:1316–23.

    Article  CAS  Google Scholar 

  5. Bergin DA, et al. (2010) Alpha-1 antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8. J. Clin. Invest. 120:4236–50.

    Article  CAS  Google Scholar 

  6. Al-Omari M, et al. (2011) Acute-phase protein alpha1-antitrypsin inhibits neutrophil calpain I and induces random migration. Mol. Med. 17:865–74.

    Article  CAS  Google Scholar 

  7. Tuder RM, Janciauskiene SM, Petrache I. (2010) Lung Disease Associated with α(1)-Antitrypsin Deficiency. Proc. Am. Thorac. Soc. 7:381–86.

    Article  CAS  Google Scholar 

  8. Bucurenci N, Blake DR, Chidwick K, Winyard PG. (1992) Inhibition of neutrophil superoxide production by human plasma α1-antitrypsin. FEBS Lett. 300:21–24.

    Article  CAS  Google Scholar 

  9. Tilg H, et al. (1993) Anti-inflammatory properties of hepatic acute phase proteins: preferential induction of interleukin 1 (IL-1) receptor antagonist over IL-1 beta synthesis by human peripheral blood mononuclear cells. J. Exp. Med. 178:1629–36.

    Article  CAS  Google Scholar 

  10. Maes OC, et al. (2006) Characterization of alpha1-antitrypsin as a heme oxygenase-1 suppressor in Alzheimer plasma. Neurobiol. Dis. 24:89–100.

    Article  CAS  Google Scholar 

  11. Marcondes AM, et al. (2011) Inhibition of IL-32 activation by α-1 antitrypsin suppresses allore-activity and increases survival in an allogeneic murine marrow transplantation model. Blood. 118:5031–39.

    Article  CAS  Google Scholar 

  12. Tawara I, et al. (2012) Alpha-1-antitrypsin monotherapy reduces graft-versus-host disease after experimental allogeneic bone marrow transplantation. Proc. Natl. Acad. Sci. U.S.A. 109:564–69.

    Article  CAS  Google Scholar 

  13. Daemen MA, et al. (2000) Functional protection by acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by preventing apoptosis and inflammation. Circulation. 102:1420–26.

    Article  CAS  Google Scholar 

  14. Lewis EC. (2012) Expanding the Clinical Indications for α(1)-Antitrypsin Therapy. Mol. Med. 18:957–70.

    Article  CAS  Google Scholar 

  15. Grimstein C, et al. (2011) Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model. J. Transl. Med. 9:21.

    Article  CAS  Google Scholar 

  16. Jedicke N, et al. (2014) Alpha-1-antitrypsin inhibits acute liver failure in mice. Hepatology. 59:2299–308.

    Article  CAS  Google Scholar 

  17. Lu Y, et al. (2006) Alpha1-antitrypsin gene therapy modulates cellular immunity and efficiently prevents type 1 diabetes in nonobese diabetic mice. Hum. Gene Ther. 17:625–34.

    Article  CAS  Google Scholar 

  18. Jonigk D, et al. (2013) Anti-inflammatory and immunomodulatory properties of alpha1-antitrypsin without inhibition of elastase. Proc. Natl. Acad. Sci. U.S.A. 110:15007–12.

    Article  CAS  Google Scholar 

  19. Slaughter N, et al. (2003) The flotillins are integral membrane proteins in lipid rafts that contain TCR-associated signaling components: implications for T-cell activation. Clin. Immunol. 108:138–51.

    Article  CAS  Google Scholar 

  20. Subramaniyam D, et al. (2010) Cholesterol rich lipid raft microdomains are gateway for acute phase protein, SERPINA1. Int. J. Biochem. Cell Biol. 42:1562–70.

    Article  CAS  Google Scholar 

  21. Mashiba S, et al. (2001) In vivo complex formation of oxidized alpha(1)-antitrypsin and LDL. Arterioscler. Thromb. Vasc. Biol. 21:1801–08.

    Article  CAS  Google Scholar 

  22. Moreno JA, et al. (2014) High-density lipoproteins potentiate alpha1-antitrypsin therapy in elastase-induced pulmonary emphysema. Am. J. Respir. Cell Mol. Biol. 51:536–49.

    Article  Google Scholar 

  23. Frenzel E, et al. (2015) Alpha1-Antitrypsin Combines with Plasma Fatty Acids and Induces Angiopoietin-like Protein 4 Expression. J. Immunol. 195:3605–16.

    Article  CAS  Google Scholar 

  24. Schumacher A, et al. (2015) Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelo-suppressive therapy. J. Hematol. Oncol. 8:1–16.

    Article  CAS  Google Scholar 

  25. Kolaczkowska E, Kubes P. (2013) Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13:159–75.

    Article  CAS  Google Scholar 

  26. Lockett AD, et al. (2013) Alpha(1)-Antitrypsin modulates lung endothelial cell inflammatory responses to TNF-alpha. Am. J. Respir. Cell Mol. Biol. 49:143–50.

    Article  CAS  Google Scholar 

  27. Petrache I, et al. (2006) Alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis. Am. J. Pathol. 169:1155–66.

    Article  CAS  Google Scholar 

  28. Janciauskiene SM, et al. (2011) The discovery of α1-antitrypsin and its role in health and disease. Respir. Med. 105:1129–39.

    Article  Google Scholar 

  29. Tedelind S, Westberg F, Kjerrulf M, Vidal A. (2007) Anti-inflammatory properties of the short-chain fatty acids acetate and propionate: a study with relevance to inflammatory bowel disease. World J. Gastroenterol. 13:2826–32.

    Article  CAS  Google Scholar 

  30. Vinolo MA, et al. (2011) Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils. J. Nutr. Biochem. 22:849–55.

    Article  CAS  Google Scholar 

  31. Sanderson P, Calder PC. (1998) Dietary fish oil diminishes lymphocyte adhesion to macrophage and endothelial cell monolayers. Immunology. 94:79–87.

    Article  CAS  Google Scholar 

  32. Calder PC. (2013) Omega-3 polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology? Br. J. Clin. Pharmacol. 75:645–62.

    Article  CAS  Google Scholar 

  33. Cassatella MA. (1995) The production of cytokines by polymorphonuclear neutrophils. Immunol. Today 16:21–26.

    Article  CAS  Google Scholar 

  34. Rowe SJ, et al. (2002) Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged inflammatory response to lipopolysaccharide-induced acute lung injury. J. Immunol. 169:6401–07.

    Article  CAS  Google Scholar 

  35. Hayashi F, Means TK, Luster AD. (2003) Toll-like receptors stimulate human neutrophil function. Blood. 102:2660–69.

    Article  CAS  Google Scholar 

  36. Kurt-Jones EA, et al. (2002) Role of Toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophils. Blood. 100:1860–08.

    CAS  PubMed  Google Scholar 

  37. McDonald PP, Bald A, Cassatella MA. (1997) Activation of the NF-kappaB pathway by inflammatory stimuli in human neutrophils. Blood. 89:3421–33.

    CAS  PubMed  Google Scholar 

  38. Scheibel M, et al. (2010) IκBβ is an essential co-activator for LPS-induced IL-1β transcription in vivo. J. Exp. Med. 207:2621–30.

    Article  CAS  Google Scholar 

  39. Karmakar M, Katsnelson MA, Dubyak GR, Pearlman E. (2016) Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1[beta] secretion in response to ATP. Nat. Commun. 7:10555.

    Article  CAS  Google Scholar 

  40. Suh BC, et al. (2001) P2X7 nucleotide receptor mediation of membrane pore formation and superoxide generation in human promyelocytes and neutrophils. J. Immunol. 166:6754–63.

    Article  CAS  Google Scholar 

  41. Dinarello CA, Simon A, van der Meer JW. (2012) Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nat. Rev. Drug. Discov. 11:633–52.

    Article  CAS  Google Scholar 

  42. Karmakar M, et al. (2015) Neutrophil IL-1beta processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on K+ efflux. J. Immunol. 194:1763–75.

    Article  CAS  Google Scholar 

  43. Chen Kaiwen W, et al. (2014) The Neutrophil NLRC4 Inflammasome Selectively Promotes IL-1β Maturation without Pyroptosis during Acute Salmonella Challenge. Cell Reports 8:570–82.

    Article  CAS  Google Scholar 

  44. Ben-Neriah Y, Karin M. (2011) Inflammation meets cancer, with NF-[kappa]B as the matchmaker. Nat. Immunol. 12:715–23.

    Article  CAS  Google Scholar 

  45. Rorvig S, Ostergaard O, Heegaard NH, Borregaard N. (2013) Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors. J. Leukoc. Biol. 94:711–21.

    Article  Google Scholar 

  46. Koussounadis A, et al. (2015) Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system. Sci. Rep. 5:10775.

    Article  Google Scholar 

  47. Huang TT, Kudo N, Yoshida M, Miyamoto S. (2000) A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc. Natl. Acad. Sci. U.S.A. 97:1014–19.

    Article  CAS  Google Scholar 

  48. Birbach A, et al. (2002) Signaling molecules of the NF-kappa B pathway shuttle constitutively between cytoplasm and nucleus. J. Biol. Chem. 277:10842–51.

    Article  CAS  Google Scholar 

  49. Tam WF, Wang W, Sen R. (2001) Cell-Specific Association and Shuttling of IκBα Provides a Mechanism for Nuclear NF-κB in B Lymphocytes. Mol. Cell. Biol. 21:4837–46.

    Article  CAS  Google Scholar 

  50. Oeckinghaus A, Ghosh S. (2009) The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb. Perspect. Biol. 1:a000034.

    Article  Google Scholar 

  51. Tang J-R, et al. (2013) The NF-κB iInhibitory Proteins IκBα and IκBβ Mediate Disparate Responses to Inflammation in Fetal Pulmonary Endothelial Cells. J. Immunol. 190:2913–23.

    Article  CAS  Google Scholar 

  52. Thompson JE, et al. (1995) I kappa B-beta regulates the persistent response in a biphasic activation of NF-kappa B. Cell. 80:573–82.

    Article  CAS  Google Scholar 

  53. Scheibel M, et al. (2010) IkappaBbeta is an essential co-activator for LPS-induced IL-1beta transcription in vivo. J. Exp. Med. 207:2621–30.

    Article  CAS  Google Scholar 

  54. Castro-Alcaraz S, et al. (2002) NF-kappa B regulation in human neutrophils by nuclear I kappa B alpha: correlation to apoptosis. J. Immunol. 169:3947–53.

    Article  CAS  Google Scholar 

  55. Tamassia N, Zimmermann M, Cassatella MA. (2012) An additional piece in the puzzle of neutrophil-derived IL-1beta: the NLRP3 inflammasome. Eur. J. Immunol. 42:565–68.

    Article  CAS  Google Scholar 

  56. Watson RW, et al. (1998) The IL-1 beta-converting enzyme (caspase-1) inhibits apoptosis of inflammatory neutrophils through activation of IL-1 beta. J. Immunol. 161:957–62.

    CAS  PubMed  Google Scholar 

  57. Lord PC, Wilmoth LM, Mizel SB, McCall CE. (1991) Expression of interleukin-1 alpha and beta genes by human blood polymorphonuclear leukocytes. J. Clin. Invest. 87:1312–21.

    Article  CAS  Google Scholar 

  58. Narayanan KB, Park HH. (2015) Purification and analysis of the interactions of caspase-1 and ASC for assembly of the inflammasome. Appl. Biochem. Biotechnol. 175:2883–94.

    Article  CAS  Google Scholar 

  59. Masumoto J, et al. (2006) Regulation of the ASC expression in response to LPS stimulation is related to IL-8 secretion in the human intestinal mucosa. Biochem. Biophys. Res. Commun. 346:968–73.

    Article  CAS  Google Scholar 

  60. Gurung P, et al. (2015) Chronic TLR Stimulation Controls NLRP3 Inflammasome Activation through IL-10 Mediated Regulation of NLRP3 Expression and Caspase-8 Activation. Sci. Rep. 5:14488.

    Article  CAS  Google Scholar 

  61. Shiohara M, et al. (2002) ASC, which is composed of a PYD and a CARD, is up-regulated by inflammation and apoptosis in human neutrophils. Biochem. Biophys. Res. Commun. 293:1314–18.

    Article  CAS  Google Scholar 

  62. Martinon F, Burns K, Tschopp J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell. 10:417–26.

    Article  CAS  Google Scholar 

  63. Franchi L, Eigenbrod T, Muñoz-Planillo R, Nuñez G. (2009) The Inflammasome: A Caspase-1 Activation Platform Regulating Immune Responses and Disease Pathogenesis. Nature Immunol. 10:241.

    Article  CAS  Google Scholar 

  64. Rahman MA, Mitra S, Sarkar A, Wewers MD. (2015) Alpha 1-Antitrypsin Does Not Inhibit Human Monocyte Caspase-1. PLoS One. 10:e0117330.

    Article  Google Scholar 

  65. Wang Y, et al. (2012) Cytosolic, autocrine alpha-1 proteinase inhibitor (A1PI) inhibits caspase-1 and blocks IL-1beta dependent cytokine release in monocytes. PLoS One. 7:e51078.

    Article  CAS  Google Scholar 

  66. Toldo S, et al. (2011) Alpha-1 antitrypsin inhibits caspase-1 and protects from acute myocardial ischemia-reperfusion injury. J. Mol. Cell Cardiol. 51:244–51.

    Article  CAS  Google Scholar 

  67. Pham CT. (2006) Neutrophil serine proteases: specific regulators of inflammation. Nat. Rev. Immunol. 6:541–50.

    Article  CAS  Google Scholar 

  68. Karmakar M, et al. (2012) Cutting edge: IL-1beta processing during Pseudomonas aeruginosa infection is mediated by neutrophil serine proteases and is independent of NLRC4 and caspase-1. J. Immunol. 189:4231–35.

    Article  CAS  Google Scholar 

  69. Hazuda DJ, et al. (1990) Processing of precursor interleukin 1 beta and inflammatory disease. J. Biol. Chem. 265:6318–22.

    CAS  PubMed  Google Scholar 

  70. Mizutani H, Black R, Kupper TS. (1991) Human keratinocytes produce but do not process pro-interleukin-1 (IL-1) beta. Different strategies of IL-1 production and processing in monocytes and keratinocytes. J. Clin. Invest. 87:1066–71.

    Article  CAS  Google Scholar 

  71. Black RA, et al. (1988) Generation of biologically active interleukin-1 beta by proteolytic cleavage of the inactive precursor. J. Biol. Chem. 263:9437–42.

    CAS  PubMed  Google Scholar 

  72. Irmler M, et al. (1995) Granzyme A is an interleukin 1 beta-converting enzyme. J. Exp. Med. 181:1917–22.

    Article  CAS  Google Scholar 

  73. Dinarello CA. (1996) Biologic basis for interleukin-1 in disease. Blood. 87:2095–147.

    CAS  PubMed  Google Scholar 

  74. Guma M, et al. (2009) Caspase-1 Independent IL-1β Activation in Neutrophil Dependent Inflammation. Arthritis and Rheum. 60:3642–50.

    Article  CAS  Google Scholar 

  75. David A, Fridlich R, Aviram I. (2005) The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcgammaRIIIb and cytochrome b558. Exp. Cell Res. 308:156–65.

    Article  CAS  Google Scholar 

  76. Fridlich R, David A, Aviram I. (2006) Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes. J. Cell. Biochem. 99:117–25.

    Article  CAS  Google Scholar 

  77. Czysz AH, Rasenick MM. (2013) G-Protein Signaling, Lipid Rafts and the Possible Sites of Action for the Antidepressant Effects of n-3 Polyunsaturated Fatty Acids. CNS Neurol. Disord. Drug Targets 12:466–73.

    Article  CAS  Google Scholar 

  78. Goldmann WH, Niles JL, Arnaout MA. (1999) Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers. Eur. J. Biochem. 261:155–62.

    Article  CAS  Google Scholar 

  79. Georgiadi A, Kersten S. (2012) Mechanisms of gene regulation by fatty acids. Adv. Nutr. 3: 127–34.

    Article  CAS  Google Scholar 

  80. Standiford TJ, Keshamouni VG, Reddy RC. (2005) Peroxisome proliferator-activated receptor-{gamma} as a regulator of lung inflammation and repair. Proc. Am. Thorac. Soc. 2:226–31.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to express their gratitude to the Polish Foundation for Patients with α-1 Antitrypsin Deficiency. RM is supported by the Cambridge NIHR Biomedical Research Unit. These studies are also supported by NIH grant AI-15614 (to CAD).

Author information

Authors and Affiliations

  1. Department of Respiratory Medicine, German Center for Lung Research, Hannover Medical School, Feodor-Lynen Str. 23, 30625, Hannover, Germany

    Nupur Aggarwal, Tobias Welte & Sabina Janciauskiene

  2. Institute of Biophysical Chemistry, Hannover Medical School, University of Cambridge, Cambridge, UK

    Elena Korenbaum

  3. Cambridge NIHR Biomedical Research Centre, Department of Respiratory Medicine, University of Cambridge, Cambridge, UK

    Ravi Mahadeva

  4. Institute for Transfusion Medicine, Hannover Medical School, Justus-Liebig-University Giessen, Giessen, Germany

    Stephan Immenschuh

  5. Department of General and Thoracic Surgery, Laboratory of Experimental Surgery, Justus-Liebig-University Giessen, Giessen, Germany

    Veronika Grau

  6. Department of Medicine, University of Colorado Denver, Denver, Aurora, Colorado, USA

    Charles A Dinarello

  7. Radboud University Medical Center, Nijmegen, The Netherlands

    Charles A Dinarello

Authors
  1. Nupur Aggarwal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elena Korenbaum
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ravi Mahadeva
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stephan Immenschuh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Veronika Grau
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Charles A Dinarello
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tobias Welte
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sabina Janciauskiene
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sabina Janciauskiene.

Electronic supplementary material

10020_2016_2201680_MOESM1_ESM.pdf

α-Linoleic Acid Enhances the Capacity of α1-Antitrypsin to Inhibit Lipopolysaccharide-Induced IL-1β in Human Blood Neutrophils

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aggarwal, N., Korenbaum, E., Mahadeva, R. et al. α-Linoleic Acid Enhances the Capacity of α1-Antitrypsin to Inhibit Lipopolysaccharide-Induced IL-1β in Human Blood Neutrophils. Mol Med 22, 680–693 (2016). https://doi.org/10.2119/molmed.2016.00119

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2119/molmed.2016.00119

Keywords

Molecular Medicine

ISSN: 1528-3658