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(Chest. 2006;129:1683-1692.)
© 2006 American College of Chest Physicians

Do Native and Polymeric {alpha}1-Antitrypsin Activate Human Neutrophils In Vitro?

Caroline Persson, MSc; Devipriya Subramaniyam, MSc; Tim Stevens, PhD and Sabina Janciauskiene, PhD

* From the Department of Clinical Sciences, University Hospital Malmö, Lund University, Malmö, Sweden.

Correspondence to: Sabina Janciauskiene, PhD, Department of Clinical Sciences, Wallenberg Laboratory, University Hospital Malmö, S-20502 Malmö, Sweden; e-mail: Sabina.Janciauskiene{at}med.lu.se

Abstract

Background: {alpha}1-Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT.

Methods and results: The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro, were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (≤ 0.08 EU/mg protein) stimulated neutrophil chemotaxis, whereas N-formyl methionyl leucyl phenylalanine (fMLP), a positive control, increased chemotaxis fourfold. A small but nonsignificant increase in neutrophil chemotaxis, however, was observed with AAT preparations containing higher levels of endotoxin (≥ 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release.

Conclusions: The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.

Key Words: {alpha}1-antitrypsin • chemotaxis • endotoxin • neutrophils • polymers

Chronic inflammation is considered the hallmark of COPD, and collateral effects of inflammation, including proteinase/antiproteinase imbalance, oxidative stress, vascular endothelium damage, and starvation, have been adduced to explain the pathogenesis of this disease.12 {alpha}1-Antitrypsin (AAT) deficiency is a hereditary autosomal disorder characterized by an increased risk for the development of liver disease and COPD.3 Individuals with homozygous AAT-Z deficiency have serum AAT levels ≤ 10% of normal and a dramatically attenuated acute-phase AAT response.45 In these individuals, it is assumed that the development of COPD results from unopposed serine protease activity in the lung, primarily as a result of AAT deficiency.56

In severe AAT-Z deficiency, a single amino acid mutation (Glu-342 to Lys) perturbs the folding and tertiary structure of AAT, leading to spontaneous polymerization, cellular retention, and failure of secretion.78 In patients with liver disease, the accumulation of AAT-Z polymers within the endoplasmic reticulum of hepatocytes causes cellular overload leading to neonatal hepatitis, cirrhosis, and hepatocellular carcinoma.9 Studies1011 now indicate that extrahepatic as well as enterohepatic AAT polymerization may occur; for example, AAT polymers have been identified in the lungs and circulation of AAT-Z subjects.

Under conditions of optimal pH, temperature, and concentration, wild-type M AAT is reported to form polymers in vitro and generate a proinflammatory form of the molecule.121314 AAT-M is polymerized by heating stimulated neutrophil adhesion and chemotaxis,15 and AAT-Z produced in the lungs subsequently polymerizes to generate an effective neutrophil chemoattractant.16 Recently, Mahadeva and co-workers17 showed that polymeric AAT co-localizes with neutrophils in the alveoli of individuals with AAT-Z–related emphysema, and that polymers cause an influx of neutrophils when instilled into murine lungs. The latter studies suggest that the proinflammatory effects of polymeric AAT, in addition to loss of inhibitory activity, may contribute directly to the development of inflammation and COPD in individuals with AAT-Z deficiency.

In contrast to the aforementioned studies, we have been unable to demonstrate any proinflammatory effects of polymerized AAT-M on neutrophils at concentrations up to 0.5 mg/mL.18 On the contrary, we found that native and polymerized AAT-M expressed antiinflammatory activity in vitro by strongly inhibiting human monocyte activation by lipopolysaccharide (LPS).19 The discrepancies between our data and others led us to suspect that differences in qualitative properties of polymer preparations, for example, presence of contaminating proteins, bacterial endotoxins, or yeast glycoproteins (eg, zymosan), and differences in the molecular profiles of polymers may be responsible for the reported effects of AAT. So far, there are no studies that have looked at the intimate relationship between the qualitative properties of AAT polymers and their effects on various cells involved in the inflammation in terms of chemotaxis, proliferation, and activation.

In this study, we have examined the effects of native and polymerized AAT, with varying levels of endotoxin contamination, on neutrophil chemotaxis and IL-8 production. Our findings confirm previously reported antiinflammatory activity of pure (low endotoxin) AAT and suggest that at least some of the proinflammatory effects observed with AAT may be mediated by a synergy with bacterial or other contaminants. These findings also lead to the hypothesis that polymeric AAT may have an increased propensity to interact with other molecules in the inflammatory milieu in vivo, which in turn leads to the formation of new, proinflammatory molecular species. AAT polymers in complex with bacterial or other compounds may therefore represent one of the factors associated with susceptibility to and progression of the chronic inflammation in COPD. A more detailed understanding of the factors that favor AAT polymerization and polymer interaction with other molecules may offer novel prospects for understanding of COPD pathogenesis.

Materials and Methods

Determination of AAT Polymers in the Circulation
Plasma samples obtained from healthy 26-year-old individuals with AAT-Z deficiency (n = 5; follow-up study by Granquist et al20) and elderly COPD patients with AAT-Z deficiency (n = 5; Aldonyte et al21) were analyzed by 7.5% native polyacrylamide gel electrophoresis (PAGE). Electrophoretically separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore; Millipore Corporation; Bedford, MA) using semidry blot electrophoretic transfer system. Western blots were accomplished using polyclonal rabbit antibodies to human AAT (1:800; Dako; Glostrup, Denmark) or mouse monoclonal ATZ11 that specifically recognizes polymerized and AAT/elastase complexed forms of AAT (1:100). The immunocomplexes were visualized with secondary horseradish peroxidase-conjugated swine antirabbit or rabbit antimouse (1:10,000, Dako) antibodies and detection kit (ECL Plus Western blot; Amersham Biosciences; Buckinghamshire, UK).

Preparation of Native and Polymerized AAT
Purified human M and homozygous deficiency Z AAT (isolated from two different donors) were obtained from the Department of Clinical Chemistry, Malmö University Hospital, Sweden. AAT phenotyping was performed by isoelectric focusing. AAT preparations were diluted in phosphate-buffered saline solution (PBS), pH 7.4; and protein concentrations were determined according to the Lowry method. Polymeric wild-type M- and Z-deficiency AAT were produced by incubation at 60°C for 3 h or at 37°C for 6 days. In addition, AAT-M polymerization was performed at 60°C for 10 h and 24 h. Polymers were confirmed on nondenaturing 7.5% PAGE following Western blot using rabbit polyclonal and mouse monoclonal antibody ATZ11. In addition, endotoxin-purified native and polymerized AAT-M preparations were incubated up to 6 days at 37°C, and contaminating endotoxins were re-examined.

Removal and Measurement of Contaminating Endotoxin
To minimize the level of contaminating endotoxins, AAT preparations were subjected to an endotoxin removing gel (Detoxi-Gel AffinityPak columns; Pierce; Rockford, IL) according to instructions from the manufacturer, and were analyzed for endotoxin content (Limulus amebocyte lysate endochrome kit, lot No. T2092CTK4, Endosafe; Charles River Laboratories; Charleston, MA). To minimize the risk of substance interference in the enzymatic reactions in the Limulus assay as described by Milton et al,22 endotoxin determinations were performed in serially diluted AAT samples (between onefold and 60-fold). Sample dilutions were performed in depyrogenated Endosafe tubes (lot No. P02702; Charles River Laboratories). In addition, AAT samples were spiked with two known concentrations of endotoxin (0.06 endotoxin units [EU]/mL and 0.12 EU/mL) and tested for spike recovery in duplicate by the instructions of the supplier. Endotoxin assays were performed in endotoxin-free, 96-well, flat-bottom polystyrene microplates. The optical density was measured at 405 nm. After correction for the dilution, endotoxin levels were recovered in each sample; therefore, we conclude that our results are not affected by interference of nonendotoxin substances in the Limulus assay.

Neutrophil Isolation
Heparinsed blood was obtained from individuals with informed consent and approval from Lund University Ethical Committee. Neutrophils were isolated from the peripheral blood of healthy donors (Polymorphprep; Axis-Shield PoC AS; Oslo, Norway) according to the recommendations of the manufacturer. Neutrophils were harvested as the lower cellular band above the RBC pellet and washed by centrifugation with PBS. Residual erythrocytes were removed by a hypotonic lysis using ice-cold 0.2% NaCl (weight/volume for 20 s, followed by addition of an equal volume of 1.6% NaCl to restore isotonicity). Purified neutrophils were washed in PBS and then resuspended in RPMI-1640 containing GlutaMAX (Invitrogen Corporation; Carlsbad, CA). The neutrophil purity was typically 80 to 90% (AC900EO AutoCounter; Swelab Instruments; Stockholm, Sweden), and cell viability was > 95% according to Trypan blue staining.

Neutrophil Chemotaxis/Migration Assays
Neutrophil chemotaxis was assessed using 96-well chemotaxis chambers (NeuroProbe; Porvair Filtronics; Shepperton, UK) with polycarbonate filters with pores 3 µm in diameter). A suspension of neutrophils (25 µL, 5 x 106cells/mL) in RPMI-bovine serum antigen was placed in the upper wells, and 30 µL of test substance, N-formyl methionyl leucyl phenylalanine (fMLP; 100 nmol/L, used as positive control) or medium alone in the lower wells. Native and polymeric AAT-M or AAT-Z were used at varying concentrations up to 0.25 mg/mL. The chamber was then incubated in a 5% CO2 incubator at 37°C for 75 min. For each separate experiment (n = 6 in total), all tests and controls were carried out in triplicate. In some experiments, chemotaxis was measured in response to higher concentrations of various AAT polymers (up to 3 mg/mL) and low-endotoxin AAT spiked with LPS (10 ng/mL) or zymosan (1 mg/mL).

Neutrophil chemotaxis was confirmed qualitatively by microscopy (Olympus BX41, PC program; Olympus Microimage; Olympus; Hamburg, Germany). Images were taken using a digital camera (Olympus DK50) at x 10 magnification. The number of migrated cells in the lower wells was also quantified by measuring cell-associated peroxidase activity. Migrated cells were lysed with 0.5% hexadecyltrimethyl-ammonium bromide in PBS pH 6.0, and cell-associated peroxidase activity (primarily myeloperoxidase) in the lysate was measured using o-phenylendiamine dihydrochloride (Sigma-Aldrich; St. Louis, MO) as a substrate. The reaction was stopped after 5 min with 4 mol/L H2SO4, and the colored peroxidase product was measured spectrophotometrically at 492 nm. Under these conditions, product formation was linear with respect to time and enzyme concentration. Previous studies23 demonstrated that no significant peroxidase loss occurred from the cell during the course of the chemotaxis.

IL-8 Analysis
Neutrophils (107/mL) were plated out into sterile Eppendorf tubes. The zymosan was boiled, washed, and sonicated. Opsonized zymosan was prepared by incubating zymosan with serum (1:3) in 37°C water bath for 20 min. After, the zymosan was centrifuged, washed with PBS, and resuspended at 30 mg/mL. For IL-8 determinations, cells were suspended in RPMI-1640 with 10% fetal calf serum and stimulated with either AAT-M or AAT-Z preparations (0.5 mg/mL or 0.06 mg/mL, respectively) in the presence or absence of LPS (10 ng/mL) or zymosan (0.7 or 1 mg/mL) for 18 h at 37°C 5% CO2. Cell-free culture supernatants were then obtained by centrifugation at 300g for 10 min and analyzed for IL-8 using enzyme-linked immunosorbent assay (DuoSet; R&D Systems; Minneapolis, MN; detection level, 31.2 pg/mL). Neutrophil viability as assessed by exclusion of the vital dye Trypan blue was typically 92% following incubation for 18 h with either AAT, zymosan, or LPS, either alone or in combination.

Statistical Analysis
The differences in the means of experimental results were analyzed for their statistical significance with one-way analysis of variance combined with a multiple-comparisons procedure (Scheffe multiple-range test), with an overall significance level of {alpha} = 0.05. Statistical software (SPSS for Windows, release 11.0; SPSS; Chicago, IL) was used for the statistical calculations. All determinations were performed in triplicate.

Results

Plasma AAT Profile in Healthy Individuals and COPD Patients With AAT-Z Deficiency
The plasma profile of AAT-Z in healthy individuals (26 years of age) and COPD patients (mean age, 56 ± 8 years) [Fig 1 ] was determined by Western blot using a polyclonal antibody against AAT and a monoclonal antibody ATZ11 that is specific for polymerized/elastase-complexed AAT. As illustrated in Figure 1, plasma from all AAT-Z–deficient individuals contained a heterogeneous mixture of different molecular forms of AAT with immunoreactivity to both the polyclonal anti-AAT antibody and the ATZ11 antibody. No elastase-AAT complexes were detected with ATZ11 antibody, but it is noteworthy that all analyzed AAT-Z individuals contained a similar fraction and profile of plasma polymers.


Figure 1
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  		Figure 1.. Shown is 7.5% native PAGE analysis of plasma samples from clinically healthy young individuals (left, A) and COPD patients (right, B) with AAT-Z deficiency followed by Western blot. The blots were developed using rabbit polyclonal anti-AAT and mouse monoclonal ATZ11 antibodies. Left, A: lines 1 to 5 indicate healthy AAT-Z individuals (26 years of age). Right, B: Lines 1 to 5 indicate COPD patients (mean age, 56 ± 8 years).

 
Analysis of Polymeric AAT-M
AAT-M (2 to 3 mg/mL) was heated in PBS by varying time; the aliquots were removed for analysis by nondenaturing PAGE following Western blot and proteins visualized by the ECL Detection kit. As seen in Figure 2 , native AAT heated at constant temperature (60°C) for 3 h, 10 h, and 24 h appeared as a single monomer band as well as darker bands in the upper portion of the gel consistent with formation of a mixture of protein polymers or aggregates. Gel densitometry analysis revealed that AAT samples treated at 60°C for 3 h, 10 h, and 24 h contained similar amounts (approximately 13%) of monomeric AAT, suggesting that prolonged heating had no additive influence on polymerization (Fig 2). Thus, the presence of high levels (approximately 70 to 80%) of polymeric AAT in relation to monomer makes it unlikely that monomer would exert significant biological activities in our experimental model.


Figure 2
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  		Figure 2.. 7.5% native PAGE analysis of 2 mg/mL AAT polymerization at 60°C for 3 h, 10 h, and 24 h. The monomer is indicated by an arrow.

 
Endotoxin Content of AAT Preparations
We have used the endotoxin content of various AAT preparations as a marker of bacterial contamination. AAT-M was contaminated with endotoxin at a level of 0.88 EU/mg (high-endotoxin AAT) protein prior to purification and 0.08 EU/mg protein (low-endotoxin AAT) following chromatography with Detoxi-gel. AAT-Z contained similar levels of endotoxin to unpurified AAT-M. Following polymerization for 3 h at 60°C, the endotoxin content of both AAT-M and AAT-Z preparations doubled. Furthermore, AAT-M and AAT-Z polymerized by incubation at 37°C for 6 days contained significantly more endotoxin than native or 3-h polymerized AAT (Table 1 ).


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  		Table 1.. Concentrations of Endotoxin in Wild-Type M and Deficiency AAT-Z Preparations*

 
Chemotactic Activity of Polymerized AAT-M and AAT-Z
Chemotactic responses of neutrophils to fMLP (100 nmol/L), a positive control, and polymerized forms of AAT-M and AAT-Z were examined using disposable, blind, 96-well chambers. Initially, we used AAT at a concentration of 0.06 mg/mL, since this has previously been reported to stimulate neutrophil chemotaxis. Microscopic analysis revealed strong directional migration when fMLP was used as a positive control (Fig 3 ). However, neither native nor polymeric AAT-M stimulated neutrophil chemotaxis above background levels (medium alone; Fig 3, top, A). In addition, Z polymers prepared by heating AAT-Z at 60°C for 3 h showed no significant chemotactic activity compared to medium alone (Fig 4 , top, A). When peroxidase activity was measured to quantify more accurately the total number of migrating cells, only fMLP significantly increased chemotaxis (p < 0.001) [Fig 3, bottom, B]. When AAT-Z (0.06 mg/mL) was polymerized by incubation at 37°C for 6 days and used as a stimulant, we did observe increased neutrophil chemotaxis (1.86-fold above basal), but this did not reach statistical significance (p = 0.058) [Fig 4, top, A] and was significantly less than the chemotaxis stimulated by fMLP (p < 0.001) [Fig 4, bottom, B]. Additional experiments using higher concentrations of native and polymerized AAT (0.25 mg/mL and 0.5 mg/mL) or polymerized AAT at 60°C for 10 h and 24 h failed to demonstrate significant neutrophil chemotaxis (data not shown).


Figure 3
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  		Figure 3.. Chemotactic response of neutrophils to native and polymeric AAT-M and fMLP. Chemotaxis of isolated neutrophils was measured in a disposable Boyden chamber. Top, A: The number of cells that migrated to the lower wells of the chemotaxis chamber was assessed qualitatively by microscope: 1, medium alone; 2, fMLP (100 nmol/L); 3, native AAT (nAAT, 0.06 mg/mL); 4, polymeric AAT (pAAT, 0.06 mg/mL), prepared by heating at 60°C for 3 h. Bottom, B: Chemotactic response of neutrophils to fMLP, native AAT, and polymeric AAT quantified by measuring the peroxidase activity in migrated cells. Each bar represents the mean ± SE of six experiments, performed in duplicate. *p < 0.001 compared to median alone. O.D. = optical density.

 

Figure 4
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  		Figure 4.. Chemotactic response of neutrophils to fMLP (100 nmol/L) and to polymeric AAT-Z chemotaxis of isolated neutrophils was measured in a disposable Boyden chamber. Top, A: The number of cells that migrated to the lower wells of the chemotaxis chamber was assessed qualitatively by microscope: 1, medium alone; 2, fMLP (100 nmol/L); 3 and 4, polymeric AAT-Z (0.06 mg/mL), prepared by heating at 60°C for 3 h and by incubating at 37°C for 6 days, respectively. Bottom, B: Chemotactic response of neutrophils to fMLP, and polymeric AAT-Z quantified by measuring the peroxidase activity in migrated cells. Each bar represents the mean ± SE of six separate experiments, performed in duplicate. *p < 0.001 compared with median alone. See Figure 3 legend for expansion of abbreviations.

 
Contamination-Dependent Effects of Native and Polymeric Forms of AAT on Neutrophil Chemotaxis
We used low-endotoxin (≤ 0.03 EU/mL) preparations of native and polymerized AAT alone or supplemented with known concentrations of endotoxin (LPS, 10 ng/mL) or zymosan (1 mg/mL, cell wall preparation derived from Saccharomyces cerevisiae) in neutrophil chemotaxis assays. In order to evaluate the effects of bacterial contamination on neutrophil chemotaxis, neutrophils were stimulated with low-endotoxin native and polymerized AAT alone or preincubated with either LPS (10 ng/mL to 4.6 EU/mL) or zymosan (1 mg/mL to 2.75 EU/mL) at 37°C. Although neither AATs, LPS, nor zymosan alone stimulated neutrophil chemotaxis, both forms of AAT (native and polymeric) stimulated chemotaxis when spiked with LPS or zymosan (Fig 5 , top, A). AATs preincubated with zymosan or LPS increased neutrophil chemotaxis threefold (p < 0.001) and 1.95-fold (p < 0.01) above basal, respectively (Fig 5, bottom, B). Our findings suggest that AAT and bacterial products in combination may express biological activities that differ from protein or bacterial product alone. In vivo, this may amplify effects of endotoxins or other bacterial products. To prove such a hypothesis, further studies are needed.


Figure 5
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  		Figure 5.. Chemotactic response of neutrophils to fMLP (100 nmol/L) and to native or polymeric AAT-M (0.5 mg/mL; endotoxin < 0.03 EU/mL) supplemented with constant amount of LPS (10 ng/mL) or zymosan (1 mg/mL). Chemotaxis was measured in a disposable Boyden chamber. Top, A: The number of cells that migrated to the lower wells of the chemotaxis chamber was assessed qualitatively by microscope: 1, medium alone; 2, fMLP (100 nmol/L); 3 and 4, native and polymerized AAT, respectively; 5 and 6, native AAT preincubated with LPS (10 ng/mL) or zymosan (1 mg/mL) for 18 h at 37°C, respectively; 7 and 8, polymerized AAT preincubated with LPS (10 ng/mL) or zymosan (1 mg/mL) for 18 h at 37°C, respectively; 9, zymosan alone. Bottom, B: Chemotactic response of neutrophils to fMLP, and low-endotoxin AATs alone or supplemented with LPS or zymosan quantified by measuring the peroxidase activity in migrated cells. Each bar represents the mean ± SE of three separate experiments, performed in duplicate. *p < 0.001 and **p < 0.01 compared with median alone. See Figure 3 legend for expansion of abbreviations.

 
Contamination-Dependent Effects of Native and Polymerized Forms of AAT on Neutrophil IL-8 Release
The effects of AAT-M and AAT-Z preparations with different endotoxin concentrations (Table 1) on human neutrophil IL-8 release are shown in Figure 6 . Native and polymerized AAT-M with low endotoxin contamination had no effect on neutrophil IL-8 release. However, AAT-M (native and polymerized) and AAT-Z with high endotoxin contamination dramatically increased neutrophil IL-8 release (Fig 6, top, A).


Figure 6
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  		Figure 6.. Effects of native M and polymeric M and Z AAT on IL-8 release from neutrophils alone (top, A) and activated with opsonized zymosan (bottom, B). The release of neutrophil IL-8 was measured in cell-free supernatants as described in "Materials and Methods." Neutrophils were treated for 18 h with a constant amount of native or polymeric AATs (0.06 mg/mL) containing low and high endotoxin concentrations, 0.08 EU/mg protein and 0.88 EU/mg protein, respectively. Polymers of M and Z AAT were prepared by incubating protein for 3 h at 60°C. Each bar represents the mean ± SE of three separate experiments. *p < 0.001 compared with median alone. See Figure 3 legend for expansion of abbreviations.

 
Bacterial Contamination of AAT Masks Its Antiinflammatory Activity on Human Neutrophils
Neutrophils stimulated with opsonized zymosan release significant amount of IL-8. The effects of AAT-M and AAT-Z preparations with low and high endotoxin content on zymosan-stimulated IL-8 release were therefore investigated. Low-endotoxin preparations of native and polymeric AAT-M (0.08 EU/mg protein) abolished IL-8 release by neutrophils stimulated with opsonized zymosan (Fig 6, bottom, B). By contrast, AAT preparations with high endotoxin contamination were without effect (Fig 6, bottom, B). AAT-Z and AAT-M polymerized for 6 days at 37°C and containing high levels of endotoxin (2.55 EU/mg protein and 1.62 EU/mg protein, respectively) also markedly induced the generation of IL-8 with a magnitude similar to that observed with zymosan (Fig 7 ).


Figure 7
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  		Figure 7.. Effects of opsonized zymosan and polymeric AAT-Z on IL-8 release from neutrophils. The release of neutrophil IL-8 was measured in cell-free supernatants as described in "Materials and Methods." Neutrophils were treated for 18 h with constant amount of zymosan (0.7 mg/mL) or polymeric Z or M AAT (0.06 mg/mL) containing high endotoxin concentrations, 2.55 EU/mg protein and 1.62 EU/mg protein, respectively. Polymers of AAT were prepared by incubating protein for 6 days at 37°C. Each bar represents the mean ± SE of three separate experiments. *p < 0.001 compared with median alone. See Figure 3 legend for expansion of abbreviations.

 
Discussion

It is now established that Z homozygous AAT-deficient individuals have greatly reduced circulating levels of AAT compared to normal AAT-M individuals due to intracellular AAT-Z polymerization and retention. In addition, extracellular AAT polymers have been observed in the lungs of patients with AAT-Z deficiency-related emphysema.10 The formation of polymers within the lungs is predicted to cause additional loss of AAT and further exacerbate the local deficiency of inhibitor and inflammation. Until now, however, characterization of AAT polymers in Z-deficiency individuals and polymer association with disease has not been fully addressed. The important questions of how polymers are generated, what their biological activities are, and if they are directly linked to COPD and/or may be useful markers for characterization of disease state remain to be answered.

In the present study, we have demonstrated that both elderly COPD patients and healthy individuals (at 26 years of age) with Z homozygous AAT deficiency have remarkable levels of polymeric AAT of relatively similar profile; therefore, it is unlikely that the presence of AAT polymers per se is a factor contributing to the increased inflammation and disease progression in AAT-Z individuals. There is remarkable heterogeneity among AAT-Z individuals with regard to the severity, rate of progression, and clinical manifestations of COPD. It is well known that environmental factors, including smoking, air pollution, and bacterial infection, amplify the effects of AAT deficiency and lead to COPD development in early years of life.16 We hypothesize that in susceptible individuals, polymerization and other posttranslational modifications of AAT-Z molecule may favor an interaction of AAT with other molecules, including bacteria products, and these new byproducts may stimulate inflammatory processes and lead to COPD development.

In the present study, we examined the possibility that bacterial (LPS) and/or yeast (zymosan) contamination may account for the observed biological activity of M and Z polymerized AAT preparations in human neutrophils. We showed that polymeric AAT-M with low endotoxin contamination was either antiinflammatory or inactive, while AAT contaminated with endotoxin or spiked with endotoxin demonstrated significant proinflammatory activity. This was particularly noticeable when we used the 6-day polymerization protocol, in which the highest levels of endotoxin content were measured.

Under similar conditions, Parmar and coworkers15 reported that polymerized AAT-M stimulated human neutrophil chemotaxis. In that study,15 measures were also taken to remove LPS, but no indication was given whether differences in biological activity were observed prior to or after removal of endotoxin. Using a 10-day polymerization protocol, Mulgrew et al16 clearly demonstrated a strong chemotactic response to polymerized, but not to nonpolymerized, AAT-Z. Unfortunately, the authors16 did not comment on whether AAT-M was also chemotactic following their 10-day polymerization protocols. Since in our studies we noted increased bacterial contamination, in conjunction with enhanced proinflammatory activity, in the prolonged polymerization protocols, we suspected that bacterial or other contaminants may be responsible for the observed effects of AAT on neutrophil chemotaxis.

Endotoxins are immunostimulatory LPSs liberated by Gram-negative bacteria, are frequent contaminants of protein solutions, and often give rise to false-positive proinflammatory effects.242526 In neutrophils, LPS is a powerful priming agent, synergizing with other agonists to elicit full functional activity.27 Zymosan, also a powerful neutrophil activator, is a ß-glucan polysaccharide widely found in nature from many sources, including yeast, bacteria, and algae.282930 Although we cannot claim that endotoxin or zymosan contamination per se was responsible for the observed proinflammatory effects of polymerized AAT-M and AAT-Z in this and other studies,15–17 it was clear from our experiments that their presence either as contaminant or when spiked into preparations of AAT resulted in proinflammatory activity. It is possible that AAT delivers a proinflammatory signal or in some way facilitates the activation of neutrophil chemotaxis by these agonists. Alternatively, AAT polymers may represent a template for the binding of bacterial or yeast products and allow the generation of new proinflammatory molecular species. Although we cannot explain mechanistically how AAT and either LPS or zymosan interact, these observations do help to clarify the discordant observations between different studies. Clearly, additional investigations are required to examine more closely the effect of polymeric AAT on neutrophil activation in the presence of other stimuli and with the measurement of different aspects of functional activity.

The finding that native and polymeric AAT inhibited human neutrophil IL-8 release extends our previous findings of the inhibitory effects of native, oxidized, and polymeric AAT on LPS-stimulated cytokine and chemokine release in human monocytes,19 and suggests that AATs exerts broad antiinflammatory activity in different cells types and under different conditions of stimulation. These data also indicate that the antiproteinase, but not the antiinflammatory, effects of AAT are abolished by oxidation or polymerization. Similarly, the oxidized (noninhibitory) form of AAT has been shown to be just as effective as the native (inhibitory) form in reducing lavage neutrophils to control levels and preventing increase in plasma tumor necrosis factor-{alpha} in cigarette smoke-induced emphysema in the mouse,31 and in suppressing the acute inflammatory response to silica in a mouse model of acute silica-induced inflammation.32

To summarize, out findings suggest that AATs may exert both proinflammatory and antiinflammatory activities in vitro, and highlight the potentially complex nature of the regulatory role of endogenous AATs in inflammatory diseases. Thus, a comprehensive understanding of AAT properties and functional mechanisms may have a bearing on our understanding and treatment of COPD, arising from both natural point mutations and from post-synthetically modified, noninhibitory byproducts of AAT.

Footnotes

Abbreviations: AAT = {alpha}1-antitrypsin; EU = endotoxin unit; fMLP = N-formyl methionyl leucyl phenylalanine; IL = interleukin; LPS = lipopolysaccharide; PAGE = polyacrylamide gel electrophoresis; PBS = phosphate-buffered saline solution

Received for publication September 14, 2004. Accepted for publication August 6, 2005.

References

  1. Lee, P, Gildea, TR, Stoller, JK (2002) Emphysema in nonsmokers: {alpha}1-antitrypsin deficiency and other causes. Cleve Clin J Med 69,928-936[ISI][Medline]
  2. Sandford, AJ, Silverman, EK Chronic obstructive pulmonary disease: 1. Susceptibility factors for COPD the genotype-environment interaction. Thorax 2002;57,736-741[Abstract/Free Full Text]
  3. Crystal, RG The {alpha}1-antitrypsin deficiency gene and its deficiency states. Trends Genet 1989;5,411-417[CrossRef][ISI][Medline]
  4. Luisetti, M, Seersholm, N {alpha}1-Antitrypsin deficiency: 1. Epidemiology of {alpha}1-antitrypsin deficiency. Thorax 2004;59,164-169[Abstract/Free Full Text]
  5. Carrell, RW, Lomas, DA {alpha}1-Antitrypsin deficiency. N Engl J Med 2002;346,45-53[Free Full Text]
  6. Brantly, M {alpha}1-Antitrypsin genotypes and phenotypes. Crystal, RG eds. {alpha}1-Antitrypsin deficiency 1996,45-59 Marcel Dekker. New York, NY:
  7. Carrell, RW, Lomas, DA, Sidhar, S, et al {alpha}1-Antitrypsin deficiency: a conformational disease. Chest 1996;110,243S-247S[Medline]
  8. Janciauskiene, S Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles. Biochim Biophys Acta 2001;1535,221-235[Medline]
  9. Qu, D, Teckman, JH, Perlmutter, DH Review: {alpha}1-antitrypsin deficiency associated liver disease. J Gastroenterol Hepatol 1997;12,404-416[ISI][Medline]
  10. Elliott, PR, Bilton, D, Lomas, DA Lung polymers in Z {alpha}1-antitrypsin deficiency-related emphysema. Am J Respir Cell Mol Biol 1998;18,670-674[Abstract/Free Full Text]
  11. Janciauskiene, S, Dominaitiene, R, Sternby, NH, et al Detection of circulating and endothelial cell polymers of Z and wild type {alpha}1-antitrypsin by a monoclonal antibody. J Biol Chem 2002;277,26540-26546[Abstract/Free Full Text]
  12. Purkayastha, P, Klemke, JW, Lavender, S, et al {alpha}1-Antitrypsin polymerization: a fluorescence correlation spectroscopic study. Biochemistry 2005;44,2642-2649[CrossRef][Medline]
  13. Devlin, GL, Chow, MK, Howlett, GJ, et al Acid denaturation of {alpha}1-antitrypsin: characterization of a novel mechanism of serpin polymerization. J Mol Biol 2002;324,859-870[CrossRef][ISI][Medline]
  14. Devlin, GL, Parfrey, H, Tew, DJ, et al Prevention of polymerization of M and Z {alpha}1-antitrypsin ({alpha}1-AT) with trimethylamine N-oxide: implications for the treatment of {alpha}1-AT deficiency. Am J Respir Cell Mol Biol 2001;24,727-732[Abstract/Free Full Text]
  15. Parmar, JS, Mahadeva, R, Reed, BJ, et al Polymers of {alpha}(1)-antitrypsin are chemotactic for human neutrophils: a new paradigm for the pathogenesis of emphysema. Am J Respir Cell Mol Biol 2002;26,723-730[Abstract/Free Full Text]
  16. Mulgrew, AT, Taggart, CC, Lawless, MW, et al Z {alpha}1-antitrypsin polymerizes in the lung and acts as a neutrophil chemoattractant. Chest 2004;125,1952-1957[CrossRef][Medline]
  17. Mahadeva, R, Atkinson, C, Li, Z, et al Polymers of Z {alpha}1-antitrypsin co-localize with neutrophils in emphysematous alveoli and are chemotactic in vivo. Am J Pathol 2005;166,377-386[Abstract/Free Full Text]
  18. Janciauskiene, S, Zelvyte, I, Jansson, L, et al Divergent effects of {alpha}1-antitrypsin on neutrophil activation, in vitro. Biochem Biophys Res Commun 2004;315,288-296[CrossRef][ISI][Medline]
  19. Janciauskiene, S, Larsson, S, Larsson, P, et al Inhibition of lipopolysaccharide-mediated human monocyte activation, in vitro, by {alpha}1-antitrypsin. Biochem Biophys Res Commun 2004;321,592-600[CrossRef][ISI][Medline]
  20. Granquist, A, Bredberg, A, Sveger, T, et al A longitudinal cohort study on the prevalence of Helicobacter pylori antibodies in Swedish children and adolescents. Acta Paediatr 2002;91,636-640[CrossRef][ISI][Medline]
  21. Aldonyte, R, Jansson, L, Piitulainen, E, et al Circulating monocytes from healthy individuals and COPD patients. Respir Res 2003;4,11[CrossRef][Medline]
  22. Milton, DK, Johnson, DK, Park, JH Environmental endotoxin measurement: interference and sources of variation in the Limulus assay of house dust. Am Ind Hyg Assoc J 1997;58,861-867[ISI][Medline]
  23. Nagase, H, Yamaguchi, M, Jibiki, S, et al A rapid and simple photometric assay for quantification of eosinophil chemotaxis. Int Arch Allergy Appl Immunol 2000;49,10-14
  24. Bito, LZ Inflammatory effects of endotoxin-like contaminants in commonly used protein preparations. Science 1977;196,83-85[Abstract/Free Full Text]
  25. Hirayama, C, Sakata, M Chromatographic removal of endotoxin from protein solutions by polymer particles. J Chromatogr B Analyt Technol Biomed Life Sci 2002;781,419-432[ISI][Medline]
  26. Geisler, F, Algul, H, Riemann, M, et al Questioning current concepts in acute pancreatitis: endotoxin contamination of porcine pancreatic elastase is responsible for experimental pancreatitis-associated distant organ failure. J Immunol 2005;174,6431-6439[Abstract/Free Full Text]
  27. Tsukahara, Y, Lian, Z, Zhang, X, et al Gene expression in human neutrophils during activation and priming by bacterial lipopolysaccharide. J Cell Biochem 2003;89,848-861[CrossRef][ISI][Medline]
  28. DiCarlo, FJ, Fiore, JV On the composition of zymosan. Science 1957;127,756-758
  29. Barrios, R, Santos, GG, Figueroa, J, et al Zymosan-induced experimental hypersensitivity pneumonitis in rabbits. Am J Pathol 1980;99,731-740[Abstract]
  30. Young, SH, Ye, J, Frazer, DG, et al Molecular mechanism of tumor necrosis factor-{alpha} production in 1->3-ß-glucan (zymosan)-activated macrophages. J Biol Chem 2001;276,20781-20787[Abstract/Free Full Text]
  31. Churg, A, Wang, RD, Xie, C, et al {alpha}-1-Antitrypsin ameliorates cigarette smoke-induced emphysema in the mouse. Am J Respir Crit Care Med 2003;168,199-207[Abstract/Free Full Text]
  32. Churg, A, Dai, J, Zay, K, et al {alpha}-1-Antitrypsin and a broad spectrum metalloprotease inhibitor, RS113456, have similar acute anti-inflammatory effects. Lab Invest 2001;81,1119-1131[ISI][Medline]




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