Effects of Noninhibitory α-1-Antitrypsin on Primary Human Monocyte Activation in Vitro

Archives of Biochemistry and Biophysics Vol. 386, No. 2, February 15, pp. 221–226, 2001 doi:10.1006/abbi.2000.2211, available online at http://www.idealibrary.com on

Effects of Noninhibitory ␣-1-Antitrypsin on Primary Human Monocyte Activation in Vitro Fabian Moraga, Stefan Lindgren, and Sabina Janciauskiene 1 Department of Medicine, University Hospital Malmo¨, 20502 Malmo¨, Sweden

Received August 11, 2000, and in revised form October 19, 2000; published online January 19, 2001

A major function of ␣-1-antitrypsin (AAT) is the inhibition of overexpressed serine proteinases during inflammation. However, it is also known that the biological activity of AAT is affected by chemical modifications, including oxidation of the reactive-site methionine, polymerization, and cleavage by unspecific proteases, all of which will result in AAT inactivation and/or degradation. All inactive forms of AAT can be detected in tissues and fluids recovered from inflammatory sites. To test for a possible link between the inflammation-generated, noninhibitory, cleaved form of AAT and cellular processes associated with inflammation, we studied the effects of this form at varying concentrations on human monocytes in culture. We found that cleaved AAT at concentrations ranging between 1 and 10 ␮M in monocyte cultures over 24 h induces elevation in monocyte chemoattractant protein-1 (MCP-1) and pro-inflammatory cytokines such as TNF␣ and IL-6 and also increases production of interstitial collagenase (MMP-1) and gelatinase B (MMP-9), members of two different classes of matrix metalloproteinase. Moreover, monocytes stimulated with higher doses of cleaved AAT show an increase in cellular oxygen consumption by about 30%, while native AAT under the same experimental conditions inhibits oxygen consumption by about 50%. These results indicate that the cleaved form of AAT may play a role in monocyte recruitment and pro-inflammatory activation during inflammatory processes, and also suggest that changes in structure occurring upon AAT cleavage could alter its functional properties with potential pathological consequences. © 2001 Academic Press Key Words: ␣-1-antitrypsin; inflammation; monocytes; pro-inflammatory molecular species; metalloproteinases.

1 To whom correspondence should be addressed at Gastroenterology-Hepatology Division, Department of Medicine, Wallenberg Laboratory, Ing.46, MAS, S-20502, Malmo¨, Sweden. Fax: ⫹46-40-33-4071. E-mail: [email protected].

0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

The primary function of plasma serine proteinase inhibitors (serpins) is regulation of proteolytic enzymes under both physiological and pathological conditions (1). The local balance between proteinases and endogenous inhibitors like ␣-1-antitrypsin (AAT) 2 is an important factor in determining whether inflammation results in connective tissues damage. AAT is a member of the serine proteinase inhibitor system in humans and has the physiological function of inhibiting target proteinases such as neutrophil elastase and proteinase 3 (2– 4). It also inhibits other serine proteinases, including cathepsin G, thrombin, trypsin, and chymotrypsin. However, the degree of inhibition of these proteinases is less than that for neutrophil elastase (5). AAT is synthesized predominantly in the liver, but also in extrahepatic tissues and cells, including neutrophils, monocytes and macrophages, alveolar macrophages, intestinal epithelial cells, breast carcinoma cells and the cornea (6 –9). The concentration of AAT during acute phase processes rises by three- to fourfold above normal (1.34 mg/mL) (2). Human neutrophils, monocytes, and alveolar macrophages are also known to increase expression of AAT in response to inflammatory mediators, such as IL-6 and endotoxins, or in response to AAT itself when complexed with neutrophil elastase (10 –12). A number of studies have shown that AAT in vivo may exist not only in a native, functionally active form, but also in several other noninhibitory molecular forms, such as complexed with protease, cleaved, polymerized, and oxidized (13–16). Abbreviations used: AAT, ␣-1-antitrypsin; cAAT, cleaved ␣-1antitrypsin; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline, EDTA, ethylenediaminetetraacetic acid; MCP-1, monocyte chemoattractant protein-1; fluorescein isothiocyanate; IL, interleukin; LDL, low-density lipoprotein; SEC, serpin– enzyme complex; VLDL, very-low-density lipoprotein; LRP, lowdensity lipoprotein receptor; IDDM, insulin-dependent diabetes mellitus; LPS, lipopolysaccharide; ROS, reactive oxygen species. 2

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AAT, like most inhibitory serpins, inhibits proteinases through the formation of a serpin– enzyme complex. AAT contains a single reactive site, centred at a Met-Ser sequence 36-amino-acid residues from the Cterminus (17). Formation of a 1:1 molar complex between serpin and enzyme is accompanied by cleavage in the reactive site of the AAT and an irreversible conformational transition to a stable, inactive form (18). Studies by many groups have shown that under normal physiological conditions, the liver mediates rapid clearance of serpin-proteinase complexes, including AAT-proteinase complexes, from the circulation via several receptors, including the serpin-enzyme complex (SEC) receptors (19), low-density lipoprotein receptor (LRP) (20) and very-low-density lipoprotein receptor (VLDL) (21). However, high levels of circulating AAT-elastase complexes have been detected during acute processes such as myocardial infarction and acute leukaemia, in patients with lung emphysema, rheumatic diseases, and elevated levels of AAT–trypsin complexes have been found in patients with biliary tract cancer (13, 22–24). It was shown that these complexes stimulate the biosynthesis of AAT in cell cultures and are chemotactic for neutrophils (25). Moreover, in vitro studies have shown that serpin–proteinase complexes are unstable, their rate of breakdown apparently dependent on their structure and environmental conditions (26). Dissociation of AAT–proteinase complex is one possible source for the degraded forms of AAT found in vivo. Cleaved and/or degraded forms of AAT are know to occur when AAT forms an inhibitor complex with serine proteinase or when it is cleaved by nontarget proteinases, usually at sites in its reactive site, without formation of stable inhibitor complexes. Human cathepsin L, collagenase, and stromelysin (27, 28) and bacterial proteinases from Staphylococcus aureus (29), Serratia marcescens metalloproteinase (30), Pseudomonas aeruginosa elastase (31), and Porphyromonas gingivalis (32) all fall into the latter class and exhibit efficient AAT degrading activity. In some pathological circumstances, the inhibitory activity of AAT may be considerably reduced due to oxidative damage of a critical methionine at the reactive center of AAT. Recently it has been shown that a number of metalloproteinases cleave oxidized AAT faster than native, suggesting that oxidative and proteolytic processes of AAT inactivation may work synergistically to create a local depletion of inhibitory activity and excessive connective tissue degradation (33). Such oxidative inactivation with subsequent enhanced proteolysis of AAT, particularly by neutrophil elastase, has been invoked in the pathogenesis of rheumatoid arthritis, pulmonary emphysema, and several respiratory disorders (34 –37).

Cleaved fragments of AAT arising from nontarget proteolytic cleavage were found to be present in biological fluids and in a variety of human tissues, such as placenta, pancreas, stomach, and small intestine (8, 38, 39). Increased levels of fragmented AAT in oral tissue and fluids were found in subjects with insulindependent diabetes mellitus (IDDM) who had been clinically diagnosed as having periodontal disease (40). Also, the C-terminal fragment of AAT created by reactive site cleavage was found to be associated with extracellular matrix proteins such as collagen and/or laminin-1, and it was suggested that this AAT peptide plays an important role in the protection of these proteins from inappropriate enzyme digestion (41). Cleaved, noninhibitory form(s) of AAT are found in extracellular fluids from patients with a variety of inflammatory diseases which suggests that these forms of AAT may also have yet undefined biological activities in inflammatory processes. We have examined the effects of proteolytically cleaved AAT, containing Cterminal peptide noncovalently bound to the large Nterminal fragment of the protein, the form of AAT which commonly occurs in vivo, and amyloidogenic Cterminal fragment of AAT on hepatoma cells and monocyte cultures, respectively (42– 44), and showed that these forms of AAT exert significant effects on cellular lipid catabolism. Based on previous observations that inflammatory processes, which are mediated by a complex network of molecular interactions, are often associated with elevated levels of native and modified (including cleaved) forms of AAT, we suggest that AAT may play multiple roles in inflammatory processes determined by its conformational state. To test this, we investigated the effects of cleaved AAT on primary human monocyte activation in culture. This study demonstrates that cleaved AAT induces expression of pro-inflammatory molecular species in monocytes and suggests that functional activity of AAT at sites of inflammation is not limited to the inhibition of overexpressed proteinase activity. MATERIALS AND METHODS Native, purified AAT was a gift from Prof. C.-B. Laurell (Department of Clinical Chemistry, UMAS, Malmo¨, Sweden). Porcine pancreatic elastase, lipopolysaccharide (LPS), and TNF␣ were obtained from Sigma and R&D, respectively. Preparation of cleaved form of AAT. Stock solution of AAT prepared in sterile, endotoxin-free Tris-buffered saline (0.015 M Tris, 0.15 M NaCl, pH 7.4) (TBS) at a concentration of 18 mg/mL was used. Lyophilized elastase was dissolved in 0.9% NaCl at a concentration of 5 mg/ml. Cleaved AAT was prepared as previously described (44). Briefly, native AAT was incubated with porcine pancreatic elastase, at a 1:5 molar ratio, for 15 min at 37°C. The cleaved AAT was separated from the enzyme using a centrifugal microconcentrator Centricon-30 (Amicon). The concentration of cleaved AAT was determined from absorbance at 280 nm and the quality of the preparations was analyzed using 10% SDS–PAGE electrophoresis. Prepara-

MONOCYTE ACTIVATION BY CLEAVED ␣-1-ANTITRYPSIN tions of cleaved AAT did not contain complexed form of AAT. The endotoxin content in the cleaved AAT preparations used was tested by limulus Amebocyte Lysate, Coamatic Chromo-LAL assay (Chromogenix, AB, Sweden) according to the manufacturer’s instructions. Endotoxin standard concentrations (from 50 to 0.005 EU/mL) and tested samples were placed into the microplate (preincubated at 37°C), mixed with substrate, and incubated in a reader (ThermoMax, Molecular Devices, Inc) at 37°C for 1 h. Negative controls (endotoxin free water) were included in every set of assays. Absorbance measurements at 405 nm were performed with time after addition of chromo-LAL and analyzed with the software provided by the manufacturer. Assay sensitivity was 0.005 EU/mL. According to this assay the endotoxin levels ranged between 0.015 and 0.045 EU/mL in all preparations used in our experiments. (According to international standards not more than 0.25 EU/mL of endotoxin can be found in water solutions.). Isolation and culture of monocytes. Human monocytes were isolated from buffy coats from different donors by the Ficoll-Hypaque procedure. A monocyte isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to obtain a highly pure monocyte population. Cell purity was ⬎97% as determined on an AC900EO AutoCounter (Swelab Instruments, AB); cell viability was analyzed by 0.4% trypan blue staining. Monocytes were plated at a density of 4 ⫻ 10 6 cells/mL into plastic plates or dishes. After removal of nonadhering cells, the remaining adherent monocytes were cultured in RPMI 1640 (Gibco, Life Technologies, Paisley, Scotland) supplemented with 2 mM N-acetyl-L-alanyl-L-glutamine, 100 U/mL penicillin, 100 ␮g/mL streptomycin, 1% nonessential amino acid, 2% sodium pyruvate and 20 mM Hepes (Fluka, Chemie AG) without serum at 37°C in a 5% CO 2. Experiments were performed within 24 h after plating of monocytes. Cultured monocytes were exposed to various concentrations of cleaved AAT (from 0 to 10 ␮M) for 24 h. For the negative control, cultured monocytes were also exposed to a buffer solution without addition of protein or to the native AAT under the same experimental conditions. For positive controls, cells were stimulated with TNF␣ (10 ng/mL) or LPS (1 ␮g/mL). In some experiments synthetic C-terminal fragment of AAT (C-36) was used. Cytokine release determination. Cell culture supernatants from monocytes treated with cleaved AAT for 24 h were analyzed to determine human IL-6 and TNF␣ release. A quantitative sandwich enzyme immunoassay (Quantikine, R&D Systems, Minneapolis, MN) technique sensitive to pg/mL assay levels was used according to manufacturer’s instructions. Samples were analyzed at 490 nm (wavelength correction at 650 nm) using a microplate reader (Labsystems, USA). Monocyte chemoattractant protein-1 (MCP-1) expression assay. Monocytes were cultured for various time points alone or with addition of cleaved AAT. Culture medium was collected and MCP-1 expression was assayed by a quantitative sandwich immunoassay technique according to manufacturers instructions (R&D Systems Europe Ltd., Abingdon, UK). The optical density was determined using a microplate reader at 450 nm. The readings at 570 nm were subtracted from the readings at 450 nm for wavelength correction. The duplicate readings for each standard, control, and sample were averaged and the average zero standard optical density was subtracted. Human gelatinase B (MMP-9) and collagenase (MMP-1) determination. Cell culture supernatants from monocytes treated with cleaved or native AAT or TNF␣ (10 ng/mL) for 24 h were analyzed to determine gelatinase B (MMP-9) and collagenase (MMP-1) secretion. The immunoassays are designed to measure total MMPs (active MMP-9 plus pro-MMP-9) activity in biological fluids and cell culture supernatantes. A quantitative sandwich enzyme immunoassay (Quantikine, R&D Systems) technique sensitive to pg/mL assay levels was used according to manufacturer’s instructions. Samples were analyzed at 450 nm (wavelength correction at 540 nm) using a microplate reader (Labsystems, USA).

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Measurement of oxygen consumption. Freshly isolated monocytes were treated with cleaved AAT. Oxygen consumption was measured polarographically with a Clark-type oxygen electrode (CB1-D3, Techtum Lab., AB) in a water-jacketed chamber connected to a circulating bath at 37° C. The instrument was calibrated prior to each assay according to the manual instructions. Reaction mixtures containing 2 ⫻ 10 5cells/mL in air saturated 0.5 mL monocyte culture medium were used. Statistical analysis. Data are presented as the mean ⫾ SD. Statistical comparisons of differences between mean values were performed by using the Student‘s t test and/or one-way analysis of variance (ANOVA) combined with a multiple comparisons procedure (Scheffe multiple range test) with the overall significance level of ␣ ⫽ 0.05.

RESULTS AND DISCUSSION

Cleaved forms of AAT can be detected in extracellular fluids of patients with inflammatory diseases such as pulmonary emphysema and rheumatoid arthritis, and from plasma of cancer patients, but the biological role and significance of these forms have not been well characterized. Proteolytic cleavage of AAT at its target cleavage site between the C-terminal peptide and the rest of the molecule results in conformational changes at the two liberated termini. Cleavage of AAT at sites upstream from the specific target cleavage site induce AAT polymer formation in vitro through intermolecular strand insertion of the aberrantly cleaved reactive site loop into ␤-sheet A (45, 46). Joslin et al. showed that proteolytically cleaved AAT binds to receptors on HepG2 cells and that the binding is mediated by a segment near the amino terminal end of the C- terminal fragment of AAT, which is available for receptor binding only when AAT is proteolytically modified (25). In our previous study we have described the ability of the cleaved form of AAT to stimulate low density lipoprotein (LDL) binding and internalization in HepG2 cell culture (44). These studies together prompted us to further investigate the effects of the cleaved molecular form of AAT on human monocyte activation in culture. Recent data have shown that chemokines are involved in the pathogenesis of various inflammatory diseases through promotion of direct migration of inflammatory cells (47). Monocyte chemoattractant protein (MCP-1) is one such chemokine, and its increased expression has been associated with infiltration of circulating monocytes in inflammatory conditions (48). In this study we found that cleaved AAT is a strong activator of MCP-1 protein expression in monocyte cultures. As shown in Fig. 1, cells stimulated with various concentrations of cleaved AAT increased MCP-1 protein expression from 13.9- to 23-fold (P ⬍ 0.01) compared to control, nonstimulated cells. In contrast, native AAT had no significant effect on MCP-1 expression in our experimental model (Fig. 1). MCP-1 has been implicated in disease characterized by monocyte-rich infiltrates including atherosclerosis, rheumatoid arthritis, and multiple sclerosis (48 –50), which are also

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MORAGA, LINDGREN, AND JANCIAUSKIENE TABLE I

Cytokines Produced by Monocytes Incubated Alone and with Addition of Various Concentrations of Cleaved ␣-1-Antitrypsin for 24 h IL-6 (pg/mL) Cleaved ATT (␮M) 0 1 5 10 a

FIG. 1. MCP-1 protein concentration measured in monocyte culture supernatants stimulated with native or cleaved AAT for 24 h. Each point represents the average the triplicate readings for the controls and samples.

related to increased levels of native and modified forms of AAT. In this study we for the first time show that cleaved AAT, like that produced in inflammation, could be an important factor mediating acute inflammatory responses. Specifically, it induces expression of MCP-1, which is known to play a central role in monocyte trafficking and activation. Monocytes also influence inflammation through production of multiple inflammatory cytokines. It is widely believed that tumor necrosis factor (TNF␣) and interleukins such as IL-6 are the main pro-inflammatory mediators induced in host cells. Induction of MCP-1, like other chemokines, has also been suggested to be mediated by cytokines such as IL-6 and TNF␣ (47). Therefore, in this study we also investigated the effects of cleaved AAT on pro-inflammatory cytokine production in monocyte cultures. Monocytes were incubated for 24 h in control or medium supplemented with various concentrations of cleaved AAT. Both cytokines tested showed significant dose-dependent stimulation in response to cleaved AAT (Table I). In contrast, native AAT had no effect on cytokine levels (data not shown). Thus, the previously observed effects of cleaved AAT on MCP-1 expression may be mediated directly or via induction of pro-inflammatory cytokine levels. Pro-inflammatory cytokines have been reported to induce production of matrix metalloproteinases in human monocytes (51). Since exposure of monocytes to cleaved AAT led to increased pro-inflammatory cytokine levels, we also determined expression of two metalloproteinases, collagenase (MMP-1) and gelatinase B (MMP-9). As shown in Fig. 2, monocytes incubated for

Mean

a

SD

5.9 ⫾ 0.1 17.7 ⫾ 0.9 84.6 ⫾ 4.1 244 ⫾ 5.5

TNF␣ (pg/mL) Mean

SD

19.6 ⫾ 1.3 57.2 ⫾ 2.9 118.7 ⫾ 6.8 147.4 ⫾ 8.2

Mean and standard error of four experiments.

24 h with various concentrations ranging between 1 and 10 ␮M of cleaved AAT increased MMP-1 expression up to 6.4-fold (P ⬍ 0.05) compared to controls. MMP-9 levels increased by 4.3-fold (P ⬍ 0.05) in monocytes treated with cleaved AAT at concentrations of 1 and 5 ␮M, while exposure of the cells to higher concentrations of cleaved AAT (10 ␮M) did not change MMP-9 expression levels compared to controls. The differences in MMP-9 and MMP-1 activation in response to varying concentrations of cleaved AAT suggests the involvement of other pathways in their induction. Recent studies by Chizzoline et al. have similarly shown that IL-4 enhances MMP-1 while decreasing MMP-9 production by mononuclear phagocytes (52). TNF␣ and

FIG. 2. MMP-1 and MMP-9 release from the monocytes nonstimulated or stimulated for 24 h with cleaved AAT. Each bar represents the mean ⫾ SD of four repeats. One-way ANOVA and the Scheffe multiple-comparison test (␣ ⫽ 0.05) show that cleaved AAT increases total MMP-1 levels in a dose-dependent manner, while cleaved AAT increased MMP-9 to the similar magnitude only at lower used concentration.

MONOCYTE ACTIVATION BY CLEAVED ␣-1-ANTITRYPSIN

FIG. 3. Mitochondrial oxygen consumption by monocytes stimulated with cleaved or native AAT. Mitochondrial oxygen consumption was measured polarographically following incubation of the cells with the indicated concentrations of the proteins. Data represent mean values ⫾ SE from four independent experiments.

IL-1 have been shown to enhance the production of MMP-9 by monocytes, while having no effect on MMP-1 (53). These data suggest that MMP-1 and MMP-9 are differently regulated by the various types and amounts of cytokines. Therefore, it is reasonable to believe that monocyte stimulation with increasing concentrations of cleaved AAT might result in a change of cytokine profile, leading first to the induction and than to the inhibition of increased levels of MMP-9, but not MMP-1. Additional studies will be required to further clarify our observation. The inflammatory response involves the release of a number factors including reactive oxygen species (ROS). Most of the ROS produced as part of the inflammatory response comes from various phagocytic cells which, when activated, are capable of producing large amounts of superoxide and hydrogen peroxide (54). Activated phagocytic cells exhibit a marked increase in oxygen consumption during their rapid production of superoxide from oxygen (55). Mitochondria generate reactive oxygen species as by-products of molecular oxygen consumption in the electron transport chain (56). Cytokines, especially TNF␣, are known to induce respiratory burst, and we found that cleaved AAT elevates TNF␣ levels. Therefore we exposed monocytes to various amounts of cleaved AAT and measured cellular respiration rates polarographically with a Clark-type oxygen electrode. We found that cleaved AAT enhances oxygen consumption in monocytes up to 30%, while increasing amounts of native AAT rapidly inhibited oxygen consumption in nonstimulated cells (Fig. 3). Under inflammatory conditions oxygen-dependent mechanisms are implicated in several cell-damaging processes, including protein and lipid oxi-

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dation, mitochondrial respiratory chain injury, and ablation of the anti-proteolytic defense provided by AAT (57, 58). Based on our data, we suggest that cleaved AAT is a potent activator of mitochondrial respiration in intact cells and may thereby cause ROS-generated tissue injury during the inflammatory response. On the other hand, observed relatively high drop in oxygen consumption in cells treated with native AAT suggests that two different molecular forms of the same protein may have opposite effects on monocyte activation. The data presented in this study strongly implicate the inhibitory serpin AAT as a multiple regulator of inflammatory processes. Dependent on which molecular form of AAT is dominant in inflammatory loci, AAT may act either as an anti-inflammatory or pro-inflammatory protein. AAT is a natural inhibitor of serine proteinases, particularly neutrophil elastase, which regulates overexpressed proteinase activity and protects against connective tissue destruction. However, in vivo AAT can be rendered inactive as an inhibitor by at least two known mechanisms: either oxidation of the reactive center or cleavage by proteinases, including metalloproteinases. Previously we have shown that in contrast to the native AAT, oxidized AAT added to human monocyte cultures induces pro-inflammatory activation and intracellular cholesterol efflux (59). In the present study we show that another modified form of AAT, namely cleaved form of AAT, also has pro-inflammatory activity toward cultured primary human monocytes, and induces production of several pro-inflammatory factors, such as chemokine MCP-1, cytokines, IL-6 and TNF␣, and metalloproteinases, MMP-9 and MMP-1. It has been shown that expression of native AAT in monocytes is specifically regulated by cytokines (10, 11), which implies that chemically modified forms of AAT such as cleaved AAT could induce synthesis of native AAT and thereby contribute to a self-perpetuating cycle of inflammation via stimulation of cytokine and other pro-inflammatory molecular species production. We hypothesize that under certain inflammatory conditions, the critical balance between the anti-inflammatory, active inhibitory form of AAT and inactive cleaved form(s) is disrupted. Imbalance in favor of the latter induces inflammatory cell activation and may promote complications of the inflammatory process. These data establish a new link between various molecular forms of the acute phase serine proteinase inhibitor, AAT, and inflammation, which may be important for understanding the multiple roles of AAT in inflammatory processes. ACKNOWLEDGMENTS The authors thank Camilla Orbjo¨rn and Birgitta Tenngart for excellent technical assistent. The authors also thank The Faculty of Medicine Lund University and the Swedish Medical Research Foundation (K99-72X-13140-01A and K1999-03P-013008-01A).

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