Evidence for Unfolded Response Activation in Monocytes from Individuals with α-1 Antitrypsin Deficiency

This information is current as Tomás P. Carroll, Catherine M. Greene, Catherine A. of September 24, 2021. O'Connor, Áine M. Nolan, Shane J. O'Neill and Noel G. McElvaney J Immunol 2010; 184:4538-4546; Prepublished online 12 March 2010;

doi: 10.4049/jimmunol.0802864 Downloaded from http://www.jimmunol.org/content/184/8/4538

References This article cites 63 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/184/8/4538.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 24, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Evidence for Unfolded Protein Response Activation in Monocytes from Individuals with a-1 Antitrypsin Deficiency

Toma´s P. Carroll, Catherine M. Greene, Catherine A. O’Connor, A´ ine M. Nolan, Shane J. O’Neill, and Noel G. McElvaney

The hereditary disorder a-1 antitrypsin (AAT) deficiency results from mutations in the SERPINA1 gene and presents with emphysema in young adults and liver disease in childhood. The most common form of AAT deficiency occurs because of the Z mutation, causing the protein to fold aberrantly and accumulate in the endoplasmic reticulum (ER). This leads to ER stress and contributes significantly to the liver disease associated with the condition. In addition to hepatocytes, AAT is also synthesized by monocytes, neutrophils, and epithelial cells. In this study we show for the first time that the unfolded protein response (UPR) is activated in quiescent monocytes from ZZ individuals. Activating transcription factor 4, X-box binding protein 1, and a subset of genes involved in the UPR are increased in monocytes from ZZ compared with MM individuals. This contributes to an in- Downloaded from flammatory phenotype with ZZ monocytes exhibiting enhanced cytokine production and activation of the NF-kB pathway when compared with MM monocytes. In addition, we demonstrate intracellular accumulation of AAT within the ER of ZZ monocytes. These are the first data showing that Z AAT protein accumulation induces UPR activation in peripheral blood monocytes. These findings change the current paradigm regarding lung inflammation in AAT deficiency, which up until now was derived from the –anti-protease hypothesis, but which now must include the exaggerated inflammatory response generated by accumulated aberrantly folded AAT in circulating blood cells. The Journal of Immunology, 2010, 184: 4538–4546. http://www.jimmunol.org/

ewly synthesized destined for secretion (such as protein synthesis, transcriptional induction of ER-resident chaper- a-1 antitrypsin [AAT]) are transported into the lumen of ones, and ER-associated degradation (ERAD) (2). Three integral ER- N the endoplasmic reticulum (ER) where they are folded and resident transmembrane sensors, protein kinase RNA-like ER kinase assembled. ER homeostasis is essential for normal cell function and (PERK), activating transcription factor 6 (ATF6), and inositol re- survival. Environmental perturbations, such as disturbances in cal- quiring kinase 1 (IRE1), form a tripartite management system that cium storage, compromise the ER protein folding capacity resulting coordinates the unfolded protein response (UPR) (3). in the accumulation of unfolded or misfolded protein within the ER. The first and immediate step in UPR engagement is translational by guest on September 24, 2021 Any imbalance between the load of unfolded proteins entering the ER attenuation. This reduces the load of host protein synthesis in the ER and the ability of the ER to process this load is termed ER stress. ER and prevents further accumulation of unfolded proteins. This is stress can also be induced by a range of pathophysiological con- carried out by PERK, through phosphorylation of eukaryotic initi- ditions, including ischemia, diabetes, viral infection, and mutations ation factor 2 (eIF2) on its a subunit (eIF2a) (4). Although it reduces that impair host protein folding (1). To maintain homeostasis, the ER global protein synthesis, eIF2a phosphorylation paradoxically has evolved to sense stress and transduce signals to the cytoplasm and promotes the translation of ATF4 mRNA, a basic leucine zipper the nucleus, striving to adapt for survival or induce apoptosis. The transcription factor. ATF4 is involved in the expression of genes protective system includes the translational attenuation of global essential for amino acid import, glutathione biosynthesis, and re- sistance to oxidative stress (5). The transcriptional arm of the UPR Respiratory Research Division, Royal College of Surgeons in Ireland, Education and uses IRE1 and ATF6, which regulate chaperone induction, ERAD, Research Centre, Beaumont Hospital, Dublin, Ireland and expansion of the ER in response to ER stress (2). IRE1 is an Received for publication August 29, 2008. Accepted for publication February 15, endoribonuclease, and ER stress causes it to oligomerize and acti- 2010. vate its RNase domain (6), and a target of IRE1 is the basic leucine This work was supported by financial assistance from the Alpha One Foundation zipper transcription factor X-box binding protein 1 (XBP-1). The (Ireland), the Alpha One Foundation (U.S.), the Health Research Board, the Medical Research Charities Group, the Children’s Research Centre Crumlin Hospital, the stress-induced spliced form of XBP-1 travels into the nucleus and Programme for Research in Third Level Institutes, Talecris Biotherapeutics, and can bind to both ER stress response elements and UPR elements, the Royal College of Surgeons in Ireland. Confocal imaging experiments were per- formed on equipment supported by the Higher Education Authority of Ireland activating the transcription of ER chaperone genes, ER quality through the National Biophotonics and Imaging Platform Ireland. T.P.C. is a recipient control genes, and folding enzymes (7, 8). In a later phase of the of the European Alpha-1-Antitrypsin Laurell’s Training Award (eALTA) 2007. UPR components of the ERAD pathway including p97/valosin- Address correspondence and reprint requests to Dr. Toma´s Carroll, Respiratory Re- containing protein (p97/VCP) and Derlin-1 are called into action. search Division, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland. E-mail address: [email protected] ERAD is the process whereby terminally misfolded ER proteins are Abbreviations used in this paper: AAT, a-1 antitrypsin; AATD, a-1 antitrypsin de- prevented from progressing along the secretory pathway, retro- ficiency; ATF, activating transcription factor; CF, cystic fibrosis; CFTR, CF trans- translocated from the ER lumen into the cytoplasm, and degraded by membrane regulator; eIF2, eukaryotic initiation factor 2; ER, endoplasmic reticulum; ERAD, ER-associated degradation; grp, glucose-regulated protein; Hsp, heat shock the ubiquitin-proteasome system (9). protein; IRE1, inositol requiring kinase 1; p97/VCP, p97/valosin-containing protein; AAT deficiency (AATD) is a genetic disorder characterized by PERK, protein kinase RNA-like ER kinase; TG, thapsigargin; TM, tunicamycin; decreased serum levels of a serine protease inhibitor and classically UPR, unfolded protein response; XBP-1, X-box binding protein 1. presents with emphysema in young to middle-aged adults and liver Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 disease in childhood and occasionally in adulthood (10, 11). www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802864 The Journal of Immunology 4539

However, AATD can be classified as an ER stress-related disease Materials and Methods as it involves mutations that affect AAT structure, resulting in re- Subjects tention of abnormal AAT cargo in the ER (12). AAT is a 52 kDa gp The study group consisted of healthy MM volunteers (n = 7, 5 males, 2 produced primarily by hepatocytes and its main site of action is the females) and asymptomatic ZZ AAT (n = 7, 4 males, 3 females, mean lung, where it serves to protect the fragile alveolar, connective, and forced expiratory volume in one second 79.9 6 8.6% predicted) in- dividuals. All ZZ patients included in the study were in a stable, non- epithelial tissues from proteolytic attack (13). AAT is the archetype exacerbated phase. The exclusion criteria were smoking, liver diseases, of the serine protease inhibitor or superfamily, inhibiting the vasculitic, or other extrapulmonary diseases. The healthy volunteers showed serine neutrophil elastase, cathepsin G, and proteinase no evidence of any disease and had no respiratory symptoms; none were on medication and all were MM phenotype with serum AAT concentrations 3 (14). The commonest cause of AATD is the Z mutation, which within the normal range (20–50 mM). The ZZ individuals were recruited differs from the normal M variant by a single glutamic acid to lysine from the Irish a-1 Antitrypsin Deficiency Registry. All individuals gave substitution at position 342 (Glu342Lys) (15). This mutation written informed consent to take part in this study, approved by the research ethics committee of Beaumont Hospital. changes the structure of the molecule leading to protein misfolding, polymerization, and accumulation of rogue Z protein within the ER Isolation, culture, and treatment of peripheral blood monocytes of cells that produce AAT. The failure in AAT secretion leads to low Mononuclear cells were isolated from heparinized venous peripheral blood circulating levels of AATand inadequate antiprotease protection in obtained from volunteers. Briefly, density gradient centrifugation was carried the lower respiratory tract, eventually causing progressive lung out using Lymphoprep (Axis Shield, Oslo, Norway) and the mononuclearcell destruction and emphysema (13). The retention and accumulation of band was aspirated and washed in HBSS (Invitrogen, Paisley, Scotland). Monocytes were purified from this mononuclear cell population using the Downloaded from aberrantly folded Z AAT in the liver, which is associated with ju- EasySep Human CD14 Selection Cocktail (StemCell Technologies, Meylan, venile hepatitis, cirrhosis, and hepatocellular carcinoma (11), im- France) and cultured in RPMI 1640 containing 1% (v/v) FCS and 1% pen- plicates a role for ER stress in the pathogenesis of this condition. icillin/streptomycin (Invitrogen) at 37˚C in a 5% CO2 atmosphere. Pseu- domonas aeruginosa LPS, DMSO, thapsigargin (TG), and tunicamycin In addition to its role in maintaining ER homeostasis, it has (TM) were purchased from Sigma-Aldrich (St. Louis, MO). become apparent that ER stress-induced UPR activation can in- Cytokine arrays fluence the expression of certain inflammatory cytokines. The http://www.jimmunol.org/ spliced form of XBP-1, a key inducer of chaperones and ERAD Monocytes were isolated as described and left untreated or stimulated with proteins, has been shown to control the production of IL-6 in the LPS (20 mg/ml) at 37˚C. After 24 h, cell supernatants were recovered, in- mouse B cell (16). XBP-1 was also shown to regulate the production cubated with the cytokine array membranes (RayBiotech, Norcross, GA) and developed according to the manufacturer’s instructions. Relative pro- of IFN-b in mouse macrophages subjected to ER stress (17). In- tein levels were quantified by densitometry and normalized for total protein terestingly, another study in a mouse model of atherosclerosis de- using the SynGene GeneSnap and GeneTools software (Cambridge, U.K.). monstrated that accumulation of free cholesterol in macrophages ELISA caused the secretion of significant amounts of TNF-a and IL-6 (18), Monocytes were isolated as described and cultured for 24 h. Supernatants illustrating a mechanism whereby cytokine production is induced by guest on September 24, 2021 were recovered and IL-8 and AAT protein concentrations determined by by intracellular events, rather than by exogenous stimuli. ELISA with specific Abs to IL-8 (R&D Systems, Minneapolis, MN) and As well as hepatocyte-derived AAT, this antiprotease is secreted AAT (ICN Biomedicals, Solon, OH). by other cell types, including monocytes, alveolar macrophages, intestinal, and bronchial epithelial cells (19–21). The production of cDNA synthesis and RT-PCR AAT in monocytes represents another source of misfolded Z AAT RNA was isolated using TRI reagent (Sigma-Aldrich) according to the (22). Although AAT production in monocytes is relatively small in manufacturer’s instructions. Equal quantities of RNA were reverse tran- scribed into cDNA using the Quantitect Reverse Transcription kit (Qiagen, comparison with hepatocytes, local regulation of the antiprotease Valencia, CA). The resulting cDNA was template for quantitative real-time shield represents an important first line of defense in times of in- PCR. Oligonucleotide primers were synthesized (MWG Biotech, Ebers- m fection and inflammation. Peripheral blood monocytes in the cir- berg, Germany) and quantitative PCR reactions performed in 20 l con- taining 2 ml template cDNA, 23 SYBR Green master mix (Roche, Basel, culation migrate through blood vessel walls into various organs and Switzerland), and 10 pmol of each primer. Amplification was performed differentiate into macrophages. Macrophages are the predominant using the LightCycler 480 PCR system (Roche) with the expression of b -DDC effector cell in the normal lung and are increased in conditions target genes relative to -actin determined using the 2 T method (27). associated with chronic inflammation, such as chronic obstructive Analysis of XBP-1 mRNA cleavage pulmonary disease and AATD-related emphysema (23, 24). It has XBP-1 mRNA splicing was analyzed using an assay described by Harding been shown that monocytes from cystic fibrosis (CF) patients are et al. (28). Briefly, RNAwas isolated using TRI reagent (Sigma-Aldrich) and intrinsically abnormal in their cytokine responses (25), and that this cDNA synthesized as described. The following primers were used to am- can be rescued by treatment with agonists designed to prevent plify XBP-1 cDNA: forward 59-AAACAGAGTAGCAGCTCAGACTGC- 9 9 9 D 3 ; reverse 5 -TCCTTCTGGGTAGACCTCTGGGA-3 . The cDNA frag- F508 cystic fibrosis transmembrane regulator (CFTR) degrada- ments were resolved on a 2.5% agarose gel with unspliced XBP-1 yielding tion by ERAD and increase CFTR secretion (26). A similar accu- a product of 480 bp, whereas spliced XBP-1 was 454 bp. mulation of Z AAT in the ER of monocytes may impact on specific phenotypes and functions of these immune cells, contributing to the Immunofluorescence and confocal microscopy overall inflammatory disease process. Monocytes were isolated and fixed in 4% paraformaldehyde in PBS. The cell We sought to investigate whether monocytes from ZZ individ- membranes were disrupted in 0.2% Triton X-100 in TBS. Nonspecific binding of Abs and fluorescent conjugates were blocked by preincubation in uals display aberrant cytokine production compared with normal 4% BSA/1% gelatin in PBS. Colocalization of AAT and ER-specific MM individuals, and whether this was influenced by intracellular chaperones was detected by immunofluorescence using goat polyclonal anti– ER stress. Our hypothesis was that intracellular accumulation of AAT-FITC (Abcam, Cambridge, U.K.) and mouse monoclonal anti-KDEL (Stressgen, Ann Arbor, MI) Abs, with an anti-mouse tetramethylrhodamine misfolded Z AAT in monocytes causes sustained activation of the isothiocyanate secondary conjugate (Abcam) for visualization of the anti- UPR with subsequent effects on immune function. KDEL Ab. Cells were mounted in Vectashield (Vector Laboratories, 4540 UPR ACTIVATION IN a-1 ANTITRYPSIN DEFICIENCY

Burlingame, CA) containing DAPI and examined using a LSM510 Meta Results laser scanning confocal microscope (Zeiss, Oberkochen, Germany). Images ZZ monocytes produce increased levels of cytokines compared 3 3 were captured at 63 magnification and 4 zoom with excitation wave- with MM monocytes lengths for FITC, tetramethylrhodamine isothiocyanate, and DAPI of 488 nm, 543 nm, and 364 nm, respectively. The images presented are single We investigated cytokine production from monocytes isolated from focal plane scans of 1-mm depth at the midsection of the fixed cells. The MM and ZZ individuals. MM (n = 3) and ZZ (n = 3) monocytes images are representative of three independent experiments and were col- were maintained for 24 h in the presence and absence of Pseu- lected using identical scanning parameters. domonas LPS. LPS is a pathogen-associated molecular pattern, which signals through the TLR 4 pathway, and regulates a pleth- Immunoblotting ora of host defense genes in immune cells, including monocytes Monocytes were pelleted by gentle centrifugation and cells resuspended in (29). The monocyte supernatants were applied to cytokine arrays 1 ml hypotonic buffer (10 mM HEPES [pH 7.9], 1.5 mM MgCl2,10mM to investigate whether there was an abnormal immune response in KCl, 0.5 mM PMSF, and 0.5 mM DTT) (Sigma-Aldrich). Cells were lysed ZZ individuals. LPS was found to induce higher levels of secreted for 30 min on ice before centrifugation at 14,000 rpm for 10 min at 4˚C. The supernatant was recovered for immunoblotting and stored at 280˚C IL-6, IL-8, and IL-10 in monocytes from ZZ individuals compared until required. Whole cell lysates were separated by electrophoresis on with MM individuals (Fig. 1A). IL-6 and IL-8 protein levels were a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose mem- 1.5-fold higher (p = 0.0015 and p = 0.0003, respectively), whereas brane (Sigma-Aldrich). Nonspecific binding was blocked with 5% BSA IL-10 production was almost 3-fold higher (p = 0.0015) in LPS- (Sigma-Aldrich) in PBS containing 0.1% Tween-20 (Sigma-Aldrich). treated ZZ monocytes compared with LPS-treated MM mono- Immunoreactive proteins were detected by incubating the membrane with a Downloaded from specific Abs to total Akt, phospho-Akt (Ser473) (Cell Signaling Tech- cytes. Significant differences in LPS-induced CXCL1 and MIP-1 nology, Beverly, MA), IkBa or b-actin (Santa Cruz Biotechnology, Santa protein production between the two groups were also observed Cruz, CA). (data not shown). To confirm the findings from the cytokine arrays, we investigated Statistical analysis the expression of IL-6, IL-8, and IL-10 genes in the same mono- cytes from Fig. 1A. We found significant differences in basal IL-6, Data were analyzed with GraphPad Prism 4.0 software package (GraphPad Software, San Diego, CA). Results are expressed as mean 6 SE and IL-8, and IL-10 levels between unstimulated MM and ZZ mono- http://www.jimmunol.org/ compared by Student t test and ANOVA. Differences were considered cytes as measured by RT-PCR. Furthermore, there were significant significant when the p value was ,0.05. differences in LPS-induced IL-6, IL-8, and IL-10 mRNA levels by guest on September 24, 2021

FIGURE 1. Monocytes from ZZ individuals produce increased levels of cytokines compared with normal MM monocytes. Monocytes from ZZ (n =3) and MM (n = 3) individuals were isolated and cultured in the presence and absence of LPS (20 mg/ml). After 24 h supernatants were recovered and RNA isolated. A, Cytokine array membranes were incubated with cell supernatants, relative cytokine levels measured by densitometry, and values normalized according to protein. Data expressed as fold induction of cytokine level. pp , 0.05. B, Cytokine mRNA expression was analyzed from the same MM and ZZ monocytes in A, normalized for b-actin, and data expressed as fold induction of gene mRNA. pp , 0.05, Student t test. The Journal of Immunology 4541 between the MM and ZZ groups (Fig. 1B). IL-6 gene expression LPS for 4 h. Basal levels of IL-6, IL-8, and IL-10 mRNA were was 8-fold higher (p = 0.0143), IL-8 gene expression 11-fold significantly higher in TG-treated monocytes compared with rest- higher (p = 0.0143), and IL-10 gene expression 5-fold higher ing cells (Fig. 2). IL-6 mRNAwas increased 7-fold (p , 0.001), IL- (p = 0.0286) in LPS-treated ZZ monocytes compared with LPS- 8 mRNA almost 4-fold (p , 0.0001), and IL-10 mRNA 8-fold treated MM monocytes. Significant differences were again ob- (p = 0.0094) in TG-treated cells compared with resting cells. In served in LPS-induced CXCL1 and MIP-1a expression between addition, LPS induced significantly higher expression of all three the two groups (data not shown). Collectively, these data demon- cytokines in monocytes undergoing ER stress when compared with strate that monocytes isolated from ZZ individuals exhibit an ex- unstressed monocytes. These data demonstrate that in ER-stressed aggerated immune response compared with monocytes from MM normal monocytes, there is an exaggerated cytokine response to individuals. bacterial stimulus. Normal monocytes undergoing ER stress produce increased Monocytes from ZZ individuals secrete less AAT protein levels of cytokines To determine whether the production of AAT was impaired in It is well-established that treatment with TG, by depleting Ca2+ monocytes from ZZ individuals, we cultured monocytes from MM stores from the ER, inhibits the activity of resident Ca2+-dependent and ZZ subjects for 24 h and quantified AAT gene expression and chaperones and thereby induces the UPR (30). To determine AAT protein secretion in these cells. We found that while there was whether ER stress can lead to an exaggerated immune response in no difference in AAT mRNA between both cell types, ZZ mon- normal monocytes, we measured IL-6, IL-8, and IL-10 gene ex- ocytes secreted 4-fold less AAT protein than the MM monocytes pression in monocytes isolated from MM individuals and pre- (p = 0.0091) (Fig. 3A). To clarify this finding was specific to AAT Downloaded from treated with TG for 1 h, followed by stimulation with Pseudomonas and not due to impairment in global protein production we quantified IL-8 mRNA and IL-8 protein produced from the same monocytes. We found that quiescent ZZ monocytes express sig- nificantly more IL-8 mRNA (p = 0.0028) and secrete twice as much IL-8 protein (p , 0.0001) compared with monocytes from

MM individuals (Fig. 3B). These findings replicate our earlier data http://www.jimmunol.org/ (Fig. 1), showing increased cytokine production from the ZZ monocyte but more importantly support our contention that mis- folded Z AAT protein is retained within these cells. TG induces the UPR in normal monocytes To investigate whether the UPR could be induced in normal mon- ocytes, we treated monocytes from MM individuals with increasing concentrations of TG. Treatment with TG caused an increase in ATF4 mRNA and caused the activation of XBP-1, transcription by guest on September 24, 2021 factors essential for UPR activation (Fig. 4A,4B). The effect of TG on the ER chaperones glucose-regulated protein (grp) 58, grp78, and grp94 and on the ERAD proteins Derlin-1 and p97/VCP was also investigated. TG dose dependently induced the expression of the chaperones grp58, grp78, grp94, and ERAD components Der- lin-1 and p97/VCP (Fig. 4C). AAT accumulates within the ER of ZZ monocytes As ZZ monocytes exhibited impaired AATsecretion we investigated whether intracellular accumulation of AAT was occurring in these cells. The subcellular location of AAT was examined by immuno- fluorescence. As shown in Fig. 5 the expression of AAT was in- creased in ZZ monocytes. Using an ER-specific marker that recognizes the KDEL motif present in ER-resident chaperones grp78 and grp94, we colocalized AAT to the ER of ZZ monocytes. In addition, the expression of KDEL was increased in ZZ mono- cytes compared with MM. Taken together, this shows that AAT protein accumulates within monocytes isolated from ZZ in- dividuals, and is localized specifically to the ER. ATF4 expression is increased and XBP-1 is spliced in ZZ monocytes Next, we explored UPR activation in vivo in monocytes isolated from MM and ZZ individuals by investigating the expression of ATF4 and splicing of XBP-1 mRNA. ATF4 expression was sig- FIGURE 2. Monocytes produce increased amounts of IL-6, IL-8, and nificantly increased in ZZ monocytes (n = 7) compared with MM IL-10 mRNA during ER stress. Monocytes were isolated from normal MM individuals (n = 3) and pretreated with TG (100 nM) for 1 h, followed by monocytes (n = 7) as measured by quantitative RT-PCR (p = 0.006) stimulation with LPS (1 mg/ml) for 4 h as indicated. Cytokine mRNA (Fig. 6A). Spliced XBP-1 mRNA was also increased in ZZ mono- expression was analyzed by real-time RT-PCR, normalized for b-actin, and cytes compared with MM monocytes (Fig. 6B). TG- and TM- expressed as relative cytokine mRNA. pp , 0.05, all by Student t test. treated normal monocytes were included as positive controls for 4542 UPR ACTIVATION IN a-1 ANTITRYPSIN DEFICIENCY

FIGURE 3. Monocytes from ZZ individuals secrete 4-fold less AAT protein but twice as much IL-8 compared with monocytes from MM individuals. Monocytes were isolated from normal MM (n = 3) and ZZ individuals (n = 3) and cultured overnight. After 24 h, supernatants were recovered and RNA isolated from these cells. AAT (A) and IL-8 (B) mRNA expression was analyzed by real-time RT- PCR, normalized for b-actin, and expressed as relative mRNA, whereas secreted AATand IL- 8 was measured by ELISA and expressed as fold induction of protein. pp , 0.05, all by Student t test. Downloaded from

ER-stress mediated splicing of XBP-1. TM induces ER stress by analysis showed that expression of chaperones grp58, grp78, and inhibiting N-linked glycosylation that causes an accumulation of grp94 is significantly increased in ZZ monocytes (n = 7) compared unfolded protein (31). This data indicates that the UPR is activated with MM monocytes (n =7)(p , 0.05), suggesting that misfolded in monocytes from ZZ individuals. Z AAT protein is inducing chaperone expression in an attempt to http://www.jimmunol.org/ refold the rogue protein (Fig. 7, top). Moreover, Derlin-1 and p97/ ER chaperone and ERAD gene expression is increased in VCP mRNA expression was also increased in ZZ monocytes monocytes from ZZ individuals compared with MM monocytes indicating that components of To further elucidate activation of the UPR in ZZ monocytes, we ERAD are upregulated in monocytes producing Z AAT protein investigated additional genes induced by ER stress. Real-time PCR (Fig. 7, bottom). by guest on September 24, 2021

FIGURE 4. TG causes ER stress and upregulates components of the UPR in normal monocytes. Monocytes isolated from healthy MM individuals (n =4) were treated with increasing concentrations of TG for 24 h and the activation of several UPR genes was investigated. A, ATF4 gene expression was analyzed by real-time RT-PCR and normalized for b-actin. B, Representative gel of PCR showing spliced XBP-1 (XBP-1s) and unspliced XBP-1 (XBP-1u) bands. C, Real-time RT-PCR analysis of five UPR genes normalized for b-actin. pp , 0.05, Student t test compared with unstimulated cells. The Journal of Immunology 4543

FIGURE 5. Intracellular accumulation of AAT in ZZ monocytes. Monocytes were isolated from MM and ZZ individuals. The subcellular distribution of AAT was determined by immunofluorescence using Abs for AAT (green) and the ER marker KDEL (red). Colocalization is indicated by white arrows (yellow). Nuclei were stained with DAPI (blue). Imaging was acquired using a Zeiss LSM 510 Meta confocal mi- croscope (original magnification 363).

Akt is phosphorylated and IkBa degraded in monocytes from viduals. Finally, we propose a mechanism linking exaggerated ZZ individuals cytokine production in ZZ individuals to UPR activation involving In the search for a mechanism linking exaggerated cytokine pro- the prosurvival serine/threonine kinase Akt and activation of the k Downloaded from duction to UPR activation, we investigated the possible involvement NF- B pathway. of Akt and NF-kB. ER stress pathways can involve Akt-dependent Two proximal indicators of UPR activation, ATF4 gene induction activation of NF-kB (32) and it has been shown that spliced XBP-1 and splicing of XBP-1 mRNA, were upregulated in ZZ monocytes. can activate Akt phosphorylation in a zebrafish embryonic cell line This UPR engagement was independent of exogenous stimulation, (33). We demonstrated that Akt phosphorylation and IkBa degra- and hints at a persistent state of UPR activation within quiescent dation, a hallmark of NF-kB pathway activation, were increased monocytes from ZZ subjects. ATF4 is selectively activated by a http://www.jimmunol.org/ in ZZ monocytes compared with MM monocytes (Fig. 8). When PERK-induced phosphorylation of eIF2 , whereas XBP-1 mRNA ER stress was induced in MM monocytes by treatment with TG, is processed by IRE1 to yield a transcription factor that regulates Akt phosphorylation was also increased. ER stress-responsive genes. Whereas IRE1 activation is difficult to reproducibly measure, processing of unspliced XBP-1 mRNA into Discussion mature XBP-1 mRNA represents a convenient, widely used in- In this study, we demonstrate for the first time that the UPR is dicator of IRE1 activation (35). The expression of several other activated in monocytes from AATD individuals and that this affects genes involved in the UPR was shown to be increased in ZZ monocyte function, specifically inflammatory gene expression. monocytes. Grp58, grp78, and grp94 mRNA expression was sig-

We have shown that monocytes from ZZ individuals exhibit an nificantly increased in ZZ monocytes. These three ER-resident by guest on September 24, 2021 exaggerated immune response, particularly in the production of proteins were first identified as grps in hamster fibroblasts (36), and IL-6, IL-8, IL-10, MIP-1a, and CXCL1, when responding to are ER-resident chaperones upregulated by ER stress (37). Grp58 bacterial challenge. IL-6 is a potent proinflammatory cytokine, (also called ERp57) is a thiol oxidoreductase, involved in protein IL-8, MIP-1a, and CXCL1 are neutrophil and monocyte che- folding and recognition of mutant proteins for ERAD (38). Grp78 moattractants, whereas IL-10 is an anti-inflammatory cytokine is a member of the heat shock protein (Hsp) 70 family, binding to (34). We found that normal monocytes subjected to ER stress misfolded proteins and also playing a crucial role in the activation display a similar enhanced immune response. In support of our of PERK, ATF6, and IRE1 (3), whereas grp 94 is a member of the intracellular AAT accumulation and ER stress hypothesis, we Hsp90 family (39). Immunofluorescence experiments also dem- have shown ZZ monocytes display impaired AAT secretion and onstrated increased grp78 and grp94 expression in resting ZZ we provide evidence of AAT accumulation within the ER of ZZ monocytes. In addition to the chaperones, the expression of ERAD monocytes. We have demonstrated that key components of the proteins Derlin-1 and p97/VCP was shown to be increased in ZZ UPR are activated in unstimulated monocytes from ZZ indi- monocytes. Derlin-1 and p97/VCP cooperate to ensure efficient

FIGURE 6. ATF4 gene expression and XBP-1 mRNA splicing are increased in ZZ monocytes compared with normal monocytes. A. Monocytes were isolated from MM (n = 7) and ZZ (n = 7) individuals and ATF4 mRNA expression was analyzed by real-time RT-PCR and normalized for b-actin. B, Monocytes were isolated from MM (n = 3) and ZZ (n = 3) individuals and XBP-1 mRNA splicing investigated by RT-PCR. Representative gel showing spliced XBP-1 (XBP-1s) and unspliced XBP-1 (XBP-1u) bands. Monocytes from MM individuals treated with DMSO (vehicle control), TM (1 mg/ml), and TG (100 nM) for 24 h are included as controls. Data was normalized for b-actin for each sample. pp , 0.05, Student t test compared with MM monocytes. 4544 UPR ACTIVATION IN a-1 ANTITRYPSIN DEFICIENCY

FIGURE 7. Genes involved in the UPR are upregulated in resting monocytes from ZZ individuals compared with normal monocytes. Monocytes were isolated from MM (n = 7) and ZZ (n = 7) individuals and UPR gene expression analyzed by real-time RT-PCR. Data was normalized for b-actin for each sample. pp , 0.05, one-way ANOVA compared with MM monocytes. Downloaded from retrotranslocation of misfolded proteins from the ER to the cyto- and liver disease (51). In addition, it has been established that the plasm for proteasome-mediated degradation (40). secretion of AAT is decreased in monocytes from ZZ individuals Lastly, our finding that phosphorylation of the serine/threonine due to the intracellular accumulation of Z AAT (52). More re- kinase Akt is increased and that IkBa is degraded in monocytes cently, Z AAT has been shown to associate in vitro with an array of chaperones, including grp78 and grp94 in several different cell from ZZ individuals suggests a mechanism linking exaggerated http://www.jimmunol.org/ cytokine production to UPR activation and also provides a possi- lines (53), but conversely, work from the same group has shown ble explanation for the continued survival of the ZZ monocyte. that Z AAT does not cause UPR activation in mouse liver cells Prolonged ER stress can activate apoptosis by both mitochondria- (54). However, work from our group indicates that the UPR is dependent and -independent pathways (41, 42). The activation of activated by Z AAT in human lung epithelial and liver cell lines, Akt is a critical prosurvival response to ER stress as it promotes suggesting species- and cell-specific differences may exist. We the expression of inhibitor of apoptosis protein family members have demonstrated that Z AAT induces proinflammatory IL-6 and (43) and inhibits several proapoptotic proteins by phosphorylation IL-8 gene expression and activates specific ER stress responses (44). Interestingly, XBP-1-mediated PI3K/Akt pathway activation (55). Using a novel in vitro model of Z AAT ER accumulation, we has been shown to be responsible for the marked resistance of established that both the ER overload response and the UPR are by guest on September 24, 2021 melanoma cells to chemotherapeutic drugs (45). The activation of activated by Z AAT, and not wild-type M AAT. The scenario of NF-kB leads to a well-characterized prosurvival transcription UPR activation in Z AAT-producing monocytes shown in this study adds another layer of complexity to the pathogenesis of program of cellular proliferation and the expression of genes en- AATD, and suggests a role for the monocyte in AATD-associated coding cytokines, chemokines, and growth factors (46), and NF- emphysema. kB can be activated downstream of IRE1 in response to ER stress The model of UPR-induced inflammatory gene expression in in vitro (47). These antiapoptotic but proinflammatory signals may monocytes producing Z AAT described here raises several in- facilitate the survival of the Z AAT-producing monocyte, pre- triguing scenarios with profound clinical and biological implica- venting ER stress-induced apoptosis, whereas rendering the tions. First, previous studies have demonstrated that LPS, IL-6, IL- monocyte intrinsically proinflammatory. Further work will be re- 1b, neutrophil elastase, and TNF-a upregulate AAT production in quired to tease out the exact mechanism of ER stress-induced monocytes and macrophages (56–58). The central role of the cytokine production, and determine the involvement of the MAPK macrophage as a mediator of neutrophil accumulation is long cascades that are known to be activated in cells undergoing ER established (59). If inflammatory stimuli, such as LPS, upregulate stress (48–50). local AAT production in monocytes from AATD individuals, the The concept of UPR involvement in the pathogenesis of AATD is subsequent increase in Z AAT production could cause activation not new. The synthesis of stress proteins Hsp70 and Hsp90 was of the UPR. Therefore, in monocytes producing Z AAT, UPR- shown to be increased in individuals with homozygous ZZ AATD induced cytokine production could represent a self-perpetuating cycle of amplified inflammatory gene expression and immune cell recruitment. As cytokine and chemokine levels become elevated in the local environment, increased numbers of neutrophils and monocytes would be attracted and contribute to the inflammatory milieu. In support of this theory, a 1991 study showed that alveolar macrophages from ZZ individuals spontaneously released 3-fold more chemotactic activity than normal macrophages (60). Second, there is the possibility that moderate UPR activation in FIGURE 8. Akt phosphorylation is increased and IkBa is degraded in ZZ monocytes compared with MM monocytes. Monocytes were isolated MZ individuals could confer an unexpected survival advantage. A from MM and ZZ individuals and probed for phospho-Akt (Ser473), total recent study investigating monocytes from CF homozygotes and Akt, IkBa and b-actin. MM monocytes treated with 100 nM TG for 1 h heterozygotes showed that a single allelic CFTR mutation was were also investigated. The immunoblots are representative of three in- sufficient to augment IL-8 secretion in response to LPS. The authors dependent experiments on different MM and ZZ individuals. postulated that the enhanced response to LPS in monocytes from CF The Journal of Immunology 4545 heterozygotes could be advantageous in priming the innate immune 11. Mahadeva, R., and D. A. Lomas. 1998. Genetics and respiratory disease. 2. Alpha 1-antitrypsin deficiency, cirrhosis and emphysema. Thorax 53: 501–505. system, leading to the rapid eradication of bacterial invaders (25). In 12. Greene, C. M., S. D. Miller, T. Carroll, C. McLean, M. O’Mahony, M. W. Lawless, a similar manner this advantage could apply to individuals het- S. J. O’Neill, C. C. Taggart, and N. G. McElvaney. 2008. Alpha-1 antitrypsin de- erozygous for the Z mutation, and provide an explanation as to why ficiency: a conformational disease associated with lung and liver manifestations. J. Inherit. Metab. Dis. 31: 21–34. the Z mutation has persisted for centuries. In support of this theory, 13. Brantly, M. L., L. D. Paul, B. H. Miller, R. T. Falk, M. Wu, and R. G. Crystal. a study of nonsmoking asymptomatic MZ subjects without airflow 1988. Clinical features and history of the destructive lung disease associated with obstruction demonstrated IL-8–related neutrophilic inflammation alpha-1-antitrypsin deficiency of adults with pulmonary symptoms. Am. Rev. Respir. Dis. 138: 327–336. in the airways (61). However, it is clear that many questions remain 14. Carrell, R. W. 1986. alpha 1-Antitrypsin: molecular pathology, leukocytes, and to be answered. What other UPR components are activated in tissue damage. J. Clin. Invest. 78: 1427–1431. 15. Lomas, D. A., D. L. Evans, J. T. Finch, and R. W. Carrell. 1992. The mechanism monocytes from ZZ individuals? Is the UPR activated in airway of Z alpha 1-antitrypsin accumulation in the liver. Nature 357: 605–607. epithelial cells that produce Z AAT? What precisely is the mech- 16. Iwakoshi, N. N., A. H. Lee, P. Vallabhajosyula, K. L. Otipoby, K. Rajewsky, and anism of UPR-regulated cytokine production? L. H. Glimcher. 2003. Plasma cell differentiation and the unfolded protein re- sponse intersect at the transcription factor XBP-1. Nat. Immunol. 4: 321–329. Our findings change the current paradigm regarding in- 17. Smith, J. A., M. J. Turner, M. L. DeLay, E. I. Klenk, D. P. Sowders, and flammation in the lung disease associated with AATD from R. A. Colbert. 2008. Endoplasmic reticulum stress and the unfolded protein a protease/antiprotease-driven hypothesis to one where a gain of response are linked to synergistic IFN-beta induction via X-box binding protein 1. Eur. J. Immunol. 38: 1194–1203. function from accumulated mutant AAT protein leads to ER stress 18. Li, Y., R. F. Schwabe, T. DeVries-Seimon, P. M. Yao, M. C. Gerbod-Giannone, and an exaggerated inflammatory phenotype. Previously, this group A. R. Tall, R. J. Davis, R. Flavell, D. A. Brenner, and I. Tabas. 2005. Free and others have demonstrated that polymerized Z AAT, in addition cholesterol-loaded macrophages are an abundant source of tumor necrosis factor- alpha and interleukin-6: model of NF-kappaB- and map kinase-dependent in- to being ineffective as an antiprotease, is a potent neutrophil flammation in advanced atherosclerosis. J. Biol. Chem. 280: 21763–21772. Downloaded from chemoattractant on the respiratory epithelial surface (62, 63). In 19. Mornex, J. F., A. Chytil-Weir, Y. Martinet, M. Courtney, J. P. LeCocq, and R. G. Crystal. 1986. Expression of the alpha-1-antitrypsin gene in mononuclear this study, we present compelling data supporting a novel mech- phagocytes of normal and alpha-1-antitrypsin-deficient individuals. J. Clin. In- anism where Z AAT accumulation in the ER of monocytes acti- vest. 77: 1952–1961. vates the UPR and induces an exaggerated immune response. This 20. Perlmutter, D. H., J. D. Daniels, H. S. Auerbach, K. De Schryver-Kecskemeti, H. S. Winter, and D. H. Alpers. 1989. The alpha 1-antitrypsin gene is expressed model of UPR activation in ZZ monocytes may lead to renewed in a human intestinal epithelial cell line. J. Biol. Chem. 264: 9485–9490. interest in pharmacological agents, such as 4-phenylbutyric acid, 21. Hu, C., and D. H. Perlmutter. 2002. Cell-specific involvement of HNF-1beta in http://www.jimmunol.org/ as interventions that enhance the secretion of Z AAT or accelerate alpha(1)-antitrypsin gene expression in human respiratory epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 282: L757–L765. the ERAD-mediated degradation of misfolded Z AAT may prove 22. Perlmutter, D. H., R. M. Kay, F. S. Cole, T. H. Rossing, D. Van Thiel, and effective in reducing the inflammatory consequences of the ac- H. R. Colten. 1985. The cellular defect in alpha 1-proteinase inhibitor (alpha 1- PI) deficiency is expressed in human monocytes and in Xenopus oocytes injected cumulated Z AAT protein. Therapies that target elements of the with human liver mRNA. Proc. Natl. Acad. Sci. USA 82: 6918–6921. UPR could represent novel strategies in the treatment of the lung 23. Barnes, P. J. 2000. Chronic obstructive pulmonary disease. N. Engl. J. Med. 343: disease associated with AATD. 269–280. 24. Morrison, H. M., J. A. Kramps, D. Burnett, and R. A. Stockley. 1987. Lung lavage fluid from patients with alpha 1-proteinase inhibitor deficiency or chronic Acknowledgments obstructive bronchitis: anti-elastase function and cell profile. Clin. Sci. (Lond.) 72: 373–381. by guest on September 24, 2021 We thank Grace Mullins, Ma´ire Stack, and Charlotte Hall for help with 25. Zaman, M. M., A. Gelrud, O. Junaidi, M. M. Regan, M. Warny, J. C. Shea, C. Kelly, patient recruitment and phlebotomy and Warren Thomas for help with B. P. O’Sullivan, and S. D. Freedman. 2004. Interleukin 8 secretion from mono- confocal imaging. cytes of subjects heterozygous for the deltaF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered. Clin. Diagn. Lab. Immunol. 11: 819–824. Disclosures 26. Vij, N., S. Fang, and P. L. Zeitlin. 2006. Selective inhibition of endoplasmic The authors have no financial conflicts of interest. reticulum-associated degradation rescues DeltaF508-cystic fibrosis trans- membrane regulator and suppresses interleukin-8 levels: therapeutic implications. J. Biol. Chem. 281: 17369–17378. 27. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression References data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. 1. Kaufman, R. J. 1999. Stress signaling from the lumen of the endoplasmic re- Methods 25: 402–408. ticulum: coordination of gene transcriptional and translational controls. Genes 28. Calfon, M., H. Zeng, F. Urano, J. H. Till, S. R. Hubbard, H. P. Harding, S. G. Clark, Dev. 13: 1211–1233. and D. Ron. 2002. IRE1 couples endoplasmic reticulum load to secretory capacity 2. Mori, K. 2000. Tripartite management of unfolded proteins in the endoplasmic by processing the XBP-1 mRNA. Nature 415: 92–96. reticulum. Cell 101: 451–454. 29. Guha, M., and N. Mackman. 2001. LPS induction of gene expression in human 3. Malhotra, J. D., and R. J. Kaufman. 2007. The endoplasmic reticulum and the monocytes. Cell. Signal. 13: 85–94. unfolded protein response. Semin. Cell Dev. Biol. 18: 716–731. 30. Treiman, M., C. Caspersen, and S. B. Christensen. 1998. A tool coming of age: 4. Harding, H. P., Y. Zhang, and D. Ron. 1999. Protein translation and folding are thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca(2+)-ATPases. coupled by an endoplasmic-reticulum-resident kinase. Nature 397: 271–274. Trends Pharmacol. Sci. 19: 131–135. 5. Harding, H. P., Y. Zhang, H. Zeng, I. Novoa, P. D. Lu, M. Calfon, N. Sadri, 31. Rosen, O. M., G. H. Chia, C. Fung, and C. S. Rubin. 1979. Tunicamycin-mediated C. Yun, B. Popko, R. Paules, et al. 2003. An integrated stress response regulates depletion of insulin receptors in 3T3-L1 adipocytes. J. Cell. Physiol. 99: 37–42. amino acid metabolism and resistance to oxidative stress. Mol. Cell 11: 619–633. 32. Misra, U. K., R. Deedwania, and S. V. Pizzo. 2006. Activation and cross-talk 6. Tirasophon, W., A. A. Welihinda, and R. J. Kaufman. 1998. A stress response between Akt, NF-kappaB, and unfolded protein response signaling in 1-LN pathway from the endoplasmic reticulum to the nucleus requires a novel bi- prostate cancer cells consequent to ligation of cell surface-associated GRP78. functional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes J. Biol. Chem. 281: 13694–13707. Dev. 12: 1812–1824. 33. Hu, M. C., H. Y. Gong, G. H. Lin, S. Y. Hu, M. H. Chen, S. J. Huang, C. F. Liao, 7. Yoshida, H., T. Matsui, A. Yamamoto, T. Okada, and K. Mori. 2001. XBP1 and J. L. Wu. 2007. XBP-1, a key regulator of unfolded protein response, ac- mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to tivates transcription of IGF1 and Akt phosphorylation in zebrafish embryonic produce a highly active transcription factor. Cell 107: 881–891. cell line. Biochem. Biophys. Res. Commun. 359: 778–783. 8. Yoshida, H., K. Haze, H. Yanagi, T. Yura, and K. Mori. 1998. Identification of 34. Kim, V., T. J. Rogers, and G. J. Criner. 2008. New concepts in the pathobiology the cis-acting endoplasmic reticulum stress response element responsible for of chronic obstructive pulmonary disease. Proc. Am. Thorac. Soc. 5: 478–485. transcriptional induction of mammalian glucose-regulated proteins. Involvement 35. Marciniak, S. J., C. Y. Yun, S. Oyadomari, I. Novoa, Y. Zhang, R. Jungreis, of basic leucine zipper transcription factors. J. Biol. Chem. 273: 33741–33749. K. Nagata, H. P. Harding, and D. Ron. 2004. CHOP induces death by promoting 9. Travers, K. J., C. K. Patil, L. Wodicka, D. J. Lockhart, J. S. Weissman, and protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes P. Walter. 2000. Functional and genomic analyses reveal an essential co- Dev. 18: 3066–3077. ordination between the unfolded protein response and ER-associated degrada- 36. Lee, A. S. 1981. The accumulation of three specific proteins related to glucose- tion. Cell 101: 249–258. regulated proteins in a temperature-sensitive hamster mutant cell line K12. 10. Laurell, C. B., and S. E. Eriksson. 1963. The electrophoretic alpha- J. Cell. Physiol. 106: 119–125. pattern of serum in alpha1-antitrypsin deficiency. Scand. J. Clin. Lab. Invest. 15: 37. Ni, M., and A. S. Lee. 2007. ER chaperones in mammalian development and 132–140. human diseases. FEBS Lett. 581: 3641–3651. 4546 UPR ACTIVATION IN a-1 ANTITRYPSIN DEFICIENCY

38. Frickel, E. M., P. Frei, M. Bouvier, W. F. Stafford, A. Helenius, R. Glockshuber, hibitors of glycosylation and of oligosaccharide processing on secretion and and L. Ellgaard. 2004. ERp57 is a multifunctional thiol-disulfide oxidoreductase. function. Biol. Chem. Hoppe Seyler 371: 231–238. J. Biol. Chem. 279: 18277–18287. 53. Schmidt, B. Z., and D. H. Perlmutter. 2005. Grp78, Grp94, and Grp170 interact 39. Argon, Y., and B. B. Simen. 1999. GRP94, an ER chaperone with protein and with alpha1-antitrypsin mutants that are retained in the endoplasmic reticulum. peptide binding properties. Semin. Cell Dev. Biol. 10: 495–505. Am. J. Physiol. Gastrointest. Liver Physiol. 289: G444–G455. 40. Lilley, B. N., and H. L. Ploegh. 2005. Multiprotein complexes that link dislo- 54. Hidvegi, T., B. Z. Schmidt, P. Hale, and D. H. Perlmutter. 2005. Accumulation of cation, ubiquitination, and extraction of misfolded proteins from the endoplas- mutant alpha1-antitrypsin Z in the endoplasmic reticulum activates caspases-4 mic reticulum membrane. Proc. Natl. Acad. Sci. USA 102: 14296–14301. and -12, NFkappaB, and BAP31 but not the unfolded protein response. J. Biol. 41. Schro¨der, M., and R. J. Kaufman. 2005. The mammalian unfolded protein re- Chem. 280: 39002–39015. sponse. Annu. Rev. Biochem. 74: 739–789. 55. Lawless, M. W., C. M. Greene, A. Mulgrew, C. C. Taggart, S. J. O’Neill, and 42. Ma, Y., and L. M. Hendershot. 2004. The role of the unfolded protein response in N. G. McElvaney. 2004. Activation of endoplasmic reticulum-specific stress tumour development: friend or foe? Nat. Rev. Cancer 4: 966–977. responses associated with the conformational disease Z alpha 1-antitrypsin de- 43. Hu, P., Z. Han, A. D. Couvillon, and J. H. Exton. 2004. Critical role of en- ficiency. J. Immunol. 172: 5722–5726. dogenous Akt/IAPs and MEK1/ERK pathways in counteracting endoplasmic 56. Perlmutter, D. H., L. T. May, and P. B. Sehgal. 1989. Interferon beta 2/in- reticulum stress-induced cell death. J. Biol. Chem. 279: 49420–49429. terleukin 6 modulates synthesis of alpha 1-antitrypsin in human mononuclear 44. Datta, S. R., A. Brunet, and M. E. Greenberg. 1999. Cellular survival: a play in phagocytes and in human hepatoma cells. J. Clin. Invest. 84: 138–144. three Akts. Genes Dev. 13: 2905–2927. 57. Perlmutter, D. H., J. Travis, and P. I. Punsal. 1988. Elastase regulates the syn- 45. Jiang, C. C., F. Yang, R. F. Thorne, B. K. Zhu, P. Hersey, and X. D. Zhang. 2009. thesis of its inhibitor, alpha 1-proteinase inhibitor, and exaggerates the defect in Human melanoma cells under endoplasmic reticulum stress acquire resistance to homozygous PiZZ alpha 1 PI deficiency. J. Clin. Invest. 81: 1774–1780. microtubule-targeting drugs through XBP-1-mediated activation of Akt. Neo- 58. Knoell, D. L., D. R. Ralston, K. R. Coulter, and M. D. Wewers. 1998. Alpha 1- plasia 11: 436–447. antitrypsin and protease complexation is induced by lipopolysaccharide, in- 46. Karin, M. 2009. NF-kappaB as a critical link between inflammation and cancer. terleukin-1beta, and tumor necrosis factor-alpha in monocytes. Am. J. Respir. Cold Spring Harbor Perspect Biol 1: a000141. Crit. Care Med. 157: 246–255. 47. Kaneko, M., Y.Niinuma, and Y.Nomura. 2003. Activation signal of nuclear factor- 59. Takemura, R., and Z. Werb. 1984. Secretory products of macrophages and their kappa B in response to endoplasmic reticulum stress is transduced via IRE1 and physiological functions. Am. J. Physiol. 246: C1–C9. tumor necrosis factor receptor-associated factor 2. Biol. Pharm. Bull. 26: 931–935. 60. Hubbard, R. C., G. Fells, J. Gadek, S. Pacholok, J. Humes, and R. G. Crystal. Downloaded from 48. Urano, F., X. Wang, A. Bertolotti, Y. Zhang, P. Chung, H. P. Harding, and 1991. Neutrophil accumulation in the lung in alpha 1-antitrypsin deficiency. D. Ron. 2000. Coupling of stress in the ER to activation of JNK protein kinases Spontaneous release of leukotriene B4 by alveolar macrophages. J. Clin. Invest. by transmembrane protein kinase IRE1. Science 287: 664–666. 88: 891–897. 49. Harper, S. J., and P. LoGrasso. 2001. Signalling for survival and death in neurones: 61. Malerba, M., F. Ricciardolo, A. Radaeli, C. Torregiani, L. Ceriani, E. Mori, the role of stress-activated kinases, JNK and p38. Cell. Signal. 13: 299–310. M. Bontempelli, C. Tantucci, and V. Grassi. 2006. Neutrophilic inflammation 50. Papa, S., F. Zazzeroni, C. G. Pham, C. Bubici, and G. Franzoso. 2004. Linking and IL-8 levels in induced sputum of alpha-1-antitrypsin PiMZ subjects. Thorax JNK signaling to NF-kappaB: a key to survival. J. Cell Sci. 117: 5197–5208. 61: 129–133.

51. Perlmutter, D. H., M. J. Schlesinger, J. A. Pierce, P. I. Punsal, and 62. Parmar, J. S., R. Mahadeva, B. J. Reed, N. Farahi, K. A. Cadwallader, M. T. Keogan, http://www.jimmunol.org/ A. L. Schwartz. 1989. Synthesis of stress proteins is increased in individuals with D. Bilton, E. R. Chilvers, and D. A. Lomas. 2002. Polymers of alpha(1)-antitrypsin homozygous PiZZ alpha 1-antitrypsin deficiency and liver disease. J. Clin. In- are chemotactic for human neutrophils: a new paradigm for the pathogenesis of vest. 84: 1555–1561. emphysema. Am. J. Respir. Cell Mol. Biol. 26: 723–730. 52. Gross, V., D. vom Berg, J. Kreuzkamp, U. Ganter, J. Bauer, G. Wu¨rtemberger, 63. Mulgrew, A. T., C. C. Taggart, M. W. Lawless, C. M. Greene, M. L. Brantly, C. Schulz-Huotari, H. Beeser, and W. Gerok. 1990. Biosynthesis and secretion of S. J. O’Neill, and N. G. McElvaney. 2004. Z alpha1-antitrypsin polymerizes in M- and Z-type alpha 1-proteinase inhibitor by human monocytes. Effect of in- the lung and acts as a neutrophil chemoattractant. Chest 125: 1952–1957. by guest on September 24, 2021