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B-Dependent Inflammatory Responses of Human Endothelial Cells and Macrophages to Atherogenic Stimuli1
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* Rayne Laboratory, Medical Research Council Centre for Inflammation Research, Medical School, and
Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, United Kingdom;
Centre for Gene Therapeutics and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; and
University of Tromsø, Tromsø, Norway
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Abstract |
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were chosen as model stimuli of arterial wall inflammation and led to production of the chemokine IL-8 in endothelial cells. To counteract the activity of HNE, we have examined the effects of adenoviral gene delivery of the anti-elastases elafin, previously demonstrated within human atheroma, and murine secretory leukocyte protease inhibitor (SLPI), a related molecule, on the inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. We developed a technique of precomplexing adenovirus with cationic lipid to augment adenoviral infection efficiency in endothelial cells and to facilitate infection in macrophages. Elafin overexpression protected endothelial cells from HNE-induced IL-8 production and cytotoxicity. Elafin and murine SLPI also reduced endothelial IL-8 release in response to oxidized low density lipoprotein, LPS, and TNF- and macrophage TNF- production in response to LPS. This effect was associated with reduced activation of the inflammatory transcription factor NF-
B, through up-regulation of IB, in both cell types. Our work suggests a novel and extended anti-inflammatory role for these HNE inhibitors working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. Our findings indicate that elafin and SLPI may be gene therapy targets for the treatment of atheroma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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have an established role in plaque inflammation (2) and, more recently, bacterial components including LPS have been implicated as causative agents (3, 4, 5).
The neutrophil chemokine IL-8 is increased in atheroma (6, 7), and neutrophils from patients with multiple atherosclerotic plaques and widespread coronary inflammation demonstrate evidence of increased degranulation, becoming depleted of myeloperoxidase (8). Human neutrophil elastase (HNE) is contained in and released from the same azurophil granules as myeloperoxidase, and its damaging effects on endothelial monolayers have been described previously (9). Therefore, we have chosen to study HNE, in addition to oxidized LDL, TNF-, and LPS, as model stimuli of endothelial cell injury in atherosclerosis.
Elafin, a specific HNE inhibitor, has been identified within human coronary artery intima (10). Recent work has suggested extended anti-inflammatory roles for murine secretory leukocyte protease inhibitor (mSLPI), an inhibitor of HNE sharing homology with elafin (11). Murine SLPI has been shown to attenuate inflammatory cytokine production by macrophages in response to bacterial LPS (12, 13). Human SLPI has been demonstrated to prevent activation of the inflammatory transcription factor NF-B (14, 15). Therefore, we hypothesized that overexpression of elafin and mSLPI using adenoviral vectors may reduce the inflammatory responses of endothelial cells and macrophages to atherogenic stimuli.
The relative accessibility of vessels affected by atherosclerotic disease makes gene therapy attractive, and strategies directed at reducing inflammation within the vessel wall may slow plaque progression and prevent rupture and ensuing coronary thrombosis. We developed a technique to increase adenoviral gene delivery in human endothelial cells and to facilitate infection of macrophages, a cell type lacking natural tropism for adenovirus.
Our studies show for the first time that: 1) overexpression of elafin protects human endothelial cells from HNE-induced damage and 2) both elafin and mSLPI have broad-ranging anti-inflammatoryactivity, reducing endothelial cell IL-8 production in response to TNF-, LPS, and oxidized LDL and TNF- production by human macrophages in response to LPS. It is noteworthy that both of these anti-inflammatory actions were associated with reduced activation of the transcription factor NF-B and concomitant increase in IB protein. Our findings extend the observed potential of elafin augmentation in models of arterial wall inflammation, including vein graft degeneration (16) and transplant arteriosclerosis (17). They indicate that gene augmentation of both elafin and mSLPI may have therapeutic potential in the treatment of atherosclerosis.
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Materials and Methods |
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HNE was obtained from Elastin Products (Owensville, MO). LPS was from Escherichia coli serotype 0127:B8 (Difco Laboratories, Detroit, MI). Endothelial basal medium-2 (EBM2) and growth supplements were obtained from Clonetics (BioWhittaker, Wokingham, U.K.). Penicillin G, streptomycin sulfate, DMEM, and IMDM were obtained from Life Technologies (Paisley, U.K.). Falcon tissue culture material was from A. & J. Beveridge (Edinburgh, U.K.). ELISAs for IL-8, TNF-, and murine eotaxin (m-eotaxin) were obtained from R&D Systems Europe (Abingdon, U.K.). All other chemicals were purchased from Sigma-Aldrich (Poole, U.K.).
LDL: isolation and oxidation
Human plasma was obtained from the Department of Transfusion Medicine, Royal Infirmary of Edinburgh (U.K.). LDL was isolated by sequential ultracentrifugation, followed by dialysis against PBS and 0.2 g/L chelex (pH 7.4). The LDL was oxidized against CuCl2 and the degree of oxidation was monitored by the formation of conjugated dienes at 234 nm (18). At maximum absorbance (usually 60–90 min after initiation), oxidation was terminated by the addition of 10% excess EDTA. Both native and oxidized LDL were concentrated in an ultrafiltration unit (Amicon 52; Millipore, Watford, U.K.) using polyethersulfone membranes (diameter, 44.5 mm; nominal molecular weight limit, 30,000; Millipore) to yield 5 ml of concentrated material. Traces of Cu2+ were then removed by gel filtration chromatography over a Sephadex G25 column (PD-10; Amersham Pharmacia, Uppsala, Sweden). Protein concentrations were measured by the Lowry method (19). The endotoxin concentration of native and oxidized LDL at working dilutions was measured by the Limulus amebocyte lysate assay (Chromogenix, Charleston, SC) and was found to be <100 pg/ml or 0.01 endotoxin U/ml.
Murine SLPI oligonucleotides
Murine SLPI oligonucleotides were obtained from MWG Biotech (Milton Keynes, U.K.). A forward oligonucleotide (5'-GCTCTAGAGCTTCACCATGAAGTCCTGCGG-3') was engineered to contain an XbaI site (5'-GCTCTAGAG-3') and oligonucleotides 6–26 from the mSLPI cDNA sequence containing the endogenous Kozak’s sequence and coding for the first 4 aa (20). The reverse oligonucleotide (5'-GGAATTCCTTTGCATAGAGAAATGAATGCG-3') was designed to contain an EcoRI site (5'-GGAATTCC-3') and oligonucleotides 634–655 from the cDNA sequence. The two oligonucleotides were designed to span introns and to amplify a 667-bp product (containing a poly(A) signal and 265 bp of 3' untranslated region).
RNA preparation and cloning of mSLPI
A fresh lung from a murine C57BL6/CBA hybrid was obtained and snap-frozen in liquid nitrogen, and total RNA was prepared using TRIzol reagent (21). RNA was quantified in the final solution (0.29 µg/µl) and used as a template for reverse transcription (RT) and PCR (all reagents from Promega, Southampton, U.K.). Briefly, 1 µg of total RNA was used in the RT reaction (37°C for 45 min) with 5 ng/µl reverse primer, 0.5 mM dNTPs, 10 U/µl reverse transcriptase (Moloney murine leukemia virus), and 2 U/µl RNasin in PCR buffer containing 3 mM MgCl2. After a 5-min incubation at 95°C, the RT mixture was added for PCR (final volume, 100 µl of PCR buffer: 1 ng/µl reverse and forward primers, 0.03 U/µl Taq polymerase, and 0.5 mM MgCl2). Thirty cycles of amplification were conducted in a thermal cycler (DNA Engine; MJ Research, Cambridge, MA) using the following conditions for each cycle: 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. Amplified products were then analyzed on 0.8% agarose gels, cut from the gel, purified, and ligated by conventional methods into pGEM T-easy (Promega). An EcoRI mSLPI cDNA digest was then subcloned into pDK6. The correct orientation was assessed and sequencing was performed to rule out any unwanted mutagenesis. Further directional cloning was performed by subcloning the XbaI-EcoRV mSLPI cDNA fragment into NheI-EcoRV digested pIRESGFP2 (gift from F. Graham and R. Marr, Department of Biology, McMaster University, Hamilton, Ontario, Canada) to generate pIRESmSLPI-GFP2.
RNA preparation and cloning of m-eotaxin
Murine eotaxin cDNA was amplified by RT-PCR from total RNA extracted from female BALB/c murine lung that had been subjected to induced allergic airway eosinophilia (22). RT was conducted with random hexamers and PCR was performed (as above) using a primer pair containing restriction sites EcoRI and BamHI on forward and reverse primers, respectively (forward, 5'-CGGAATTCCGGTAACTTCCATCTGTCTCC-3'; reverse, 5'-CGGGATCCCGGTCCCTGTTTCAA ACAAGC-3'). An 800-bp EcoRI/BamHI fragment of full-length m-eotaxin was isolated from a 1% agarose gel and ligated into the multicloning site of the shuttle vector pDK6 to generate pDK6-m-eotaxin (23).
Adenovirus (Ad) constructs
E1, E3-deleted Ad encoding mSLPI cDNA (Ad-mSLPI).
pIRES-mSLPI-GFP2 and pBHGlox
E1,3Cre were used to cotransfect 293 cells. Cre-mediated, site-specific recombination (24) resulted in the generation of Ad-mSLPI. Ad-mSLPI was purified and titered by conventional methods (23). The integrity of mSLPI and its activity in HUVECs were assessed by dot blot analysis (using anti-mSLPI polyclonal Abs) and anti-HNE activity, respectively (data not shown).
Ad-m-eotaxin. The pDK6m-eotaxin plasmid was cotransfected into 293 cells along with the rescuing vector pBHG10. Homologous recombination resulted in the generation of Ad-m-eotaxin. HUVECs were infected with Ad-m-eotaxin, and m-eotaxin production was verified by ELISA (data not shown). The Ad-m-eotaxin virus was purified, titered, and stored as above (23).
Ad-IB.
Construction of the vector expressing a mutant form of IB protein (in which serines 32 and 36 are replaced by alanine residues, therefore preventing inducible IB phosphorylation) has been described before (25).
Ad-elafin. The Ad vector encoding for the 95-aa elafin molecule has been described before (26). E1, E3-deleted empty adenoviral vector (Ad-dl703) coding for no transgene was described before (27).
Ad expressing green fluorescent protein (Ad-GFP) was a gift from F. Graham and R. Marr.
Cell isolation and culture
HUVECs. Ethical approval was obtained from Lothian Research and Ethics Committee (2001/R/UO/01) for the procurement of human umbilical veins from healthy term pregnancies during elective cesarean sections. Human umbilical cord was digested using collagenase type IV according to published protocols (28), and HUVECs were grown in EBM2 culture medium containing growth supplements at 37°C, 5% CO2. Cells were not grown beyond six passages.
PBMC-derived macrophages. Mononuclear cells were isolated from peripheral blood as described (29). Freshly citrated blood was centrifuged at 400 x g for 20 min, and the platelet-rich plasma supernatant was used to prepare autologous serum by addition of CaCl2 (10 mM final concentration). Leukocytes were isolated after removal of erythrocytes by sedimentation using 6% (w/v) dextran T500 in saline by fractionation on a discontinuous gradient of isotonic Percoll solutions made in Ca2+/Mg2+-free PBS. Percoll concentrations of 55, 68, and 79% were used, and the leukocytes were centrifuged at 700 x g for 20 min. Mononuclear cells were aspirated from the 55/68% interface and were washed three times in Ca2+/Mg2+-free PBS before culture. Monocytes were enriched from the mononuclear fraction by selectively attaching them to 48- or 12-well plates for 40 min at 37°C. Nonadherent lymphocytes were removed and adherent monocytes were washed twice in PBS. Monocytes were then cultured in IMDM containing 10% autologous serum, penicillin G (final concentration 100 U/ml), and streptomycin sulfate (final concentration 100 µg/ml) at 37°C in a 5% CO2 atmosphere. Maturation into macrophages with this culture protocol has previously been demonstrated using myeloid-specific markers, including CD16 and CD51/CD61 (29).
A549 lung epithelial cells. A549 lung epithelial cells (30) were incubated in DMEM containing 10% FCS, penicillin G (final concentration 100 U/ml), and streptomycin sulfate (final concentration 100 µg/ml) and were grown to confluence in 48-well plates 37°C in a 5% CO2 atmosphere.
Incubations with HNE, TNF-, LPS, and oxidized LDL
For HNE-mediated injury experiments, HUVECs were washed with PBS to remove serum (because of the presence of HNE inhibitors in serum) and were incubated in serum-free EBM2 containing HNE for 8 h, after which photomicrographs were taken. For HNE pretreatment studies, HUVECs were washed in PBS and preincubated with HNE for 1 h before replenishment with serum-containing EBM2 containing the second LPS or oxidized LDL stimulus. All incubations with TNF- and oxidized LDL were for 8 h. When LPS was the sole stimulus, it was incubated with HUVECs and A549s for 8 h and with macrophages for 3 h.
Ad infection protocols
To augment adenoviral gene transfer in HUVECs, we adapted a protocol involving precomplexing adenoviral vectors with the cationic liposome lipofectamine (31). We also tested this protocol for gene delivery on human monocyte-derived macrophages, a notoriously difficult cell type to transfect (32). HUVECs and A549 cells were grown to confluency and one well was used for cell count determination. Ad was dosed for each experiment at 100 PFU/cell. Ad vectors were preincubated for 15 min at room temperature in OptiMEM I reduced serum medium (Invitrogen, Paisley, U.K.) with lipofectamine (Invitrogen) at a ratio of 5 x 104 lipofectamine molecules to each adenovirus particle. Ad particle concentration was determined from absorbance at 260 nm for each Ad construct, according to published protocols (33). HUVECs, peripheral blood-derived macrophages, and A549s were incubated in the virus-containing medium for 2 h before replenishment with their respective normal growth media.
Preparation of nuclear extracts and EMSAs
Nuclear extracts were prepared after 1-h treatments from HUVECs and macrophages according to the method of Staal et al. (34). Briefly, cells were scraped and lysed in 400 µl of buffer A (10 mM HEPES, 10 mM KCl, 2 mM MgCl2 1 mM DTT, 0.1 mM EDTA, 0.4 mM PMSF, 0.2 mM NaF, 0.2 mM Na3VO4, and 1 µg/ml leupeptin), to which was added 25 µl of buffer B (10% Nonidet P-40), and the nuclei were collected by centrifugation (10,000 x g for 2 min). Nuclei were resuspended in 50 µl of buffer C (50 mM HEPES, 50 mM KCl, 10% glycerol, 0.2 mM NaF, and 0.2 mM Na3VO4) and agitated for 20 min at 4°C, followed by centrifugation (10,000 x g for 5 min). Nuclear proteins (7 µg) were incubated with 5x binding buffer (50 mM Tris-HCl, 0.5 M KCl, 5 mM EDTA, 2.5 mM MgCl2, 40% glycerol) and [
-32P]-labeled NF-B consensus oligonucleotide and were electrophoresed on a 6% nondenaturing polyacrylamide gel. The NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') oligonucleotide was obtained from Promega. DNA binding was assessed by autoradiography. Quantitative analysis was performed with a Storm 860 PhosphorImager using ImageQuant software (Molecular Dynamics, Buckinghamshire, U.K.). A mutant NF-B oligonucleotide (NF-B) with a "G" 
"C" substitution in the NF-B/Rel DNA binding motif (Santa Cruz Biotechnology, Santa Cruz, CA) was used to establish the specificity of the sample nuclear protein binding to NF-B.
Western blot analysis for IB protein content in HUVECs and macrophages
Total cell (HUVECs and macrophages) extracts were prepared with cell lysate buffer (10% glycerol, 2% SDS, 62.5 mM Tris-Cl, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM PMSF) after 10 min of LPS (100 ng/ml) or TNF- (100 pg/ml) stimulation. Cell extracts were analyzed for IB protein content by SDS-PAGE. Briefly, equal volumes were loaded onto a 12% Bis-Tris/MES polyacrylamide gel (Invitrogen) and electrophoresis was performed. Electroblotting was then conducted on nitrocellulose (Amersham Pharmacia, Buckingham, U.K.), and membranes were treated with rabbit anti-IB Ab, 1/1000 dilution (Cell Signaling Technology, New England Biolabs, Hitchin, U.K.), for either 16 h at 4°C or 2 h at room temperature and subsequently with goat anti-rabbit, 1/2000 dilution (DAKO, Ely, U.K.) for 1.5 h at room temperature. The Western blots were then developed by ECL (Western Lightning Chemiluminescence Reagent Plus; Perkin-Elmer Life Sciences, Cambridge, U.K.) according to the manufacturer’s instructions.
Statistics
Results are presented as mean ± SD, and differences between treatments were tested using the Student t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and HNE on IL-8 production by HUVECs
To validate our in vitro model, HNE, LPS, oxidized LDL, and TNF- were chosen as stimuli relevant to endothelial injury occurring during the development of atherosclerosis. HUVECs produced the chemotactic cytokine IL-8 (pg/ml) as follows: 92.7 ± 7.05 (untreated), 126 ± 9.65 (post 100 µg/ml native LDL), 317 ± 38.2 (post 100 µg/ml oxidized LDL), 608 ± 144 (post 100 ng/ml LPS), 5144 ± 721 (post 1 ng/ml TNF-), 126 ± 8.45 (post 2.5 x 10-8 M HNE), 145 ± 14.4 (post 5.0 x 10-8 M HNE). Nonoxidized, native LDL was included as a control and in agreement with the literature (35) produced a small increase in IL-8 production over untreated cells. This increase was considered negligible compared with the 3-fold increase over basal levels observed with oxidized LDL (36). Results pertaining to native LDL therefore are not shown in the rest of the manuscript. Addition of low concentrations of HNE for 1 h followed by replacement of the HNE-containing medium with serum-containing medium led to a small increase in IL-8 production. In addition, a 1-h HNE pretreament led to increased IL-8 response after subsequent stimulation with both LPS and oxidized LDL. The data, expressed as a percentage of non-HNE-pretreated cells (Table I), show a dose-dependent effect for HNE pretreatment. Our results indicate that a brief exposure to HNE (in which no morphological damage was observed, as evidenced by absence of lifting and rounding of the HUVEC monolayer; data not shown) not only induced IL-8 production on its own but also primed cells to further stimulation by LPS and oxidized LDL.
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As a marker for our Ad-lipofectamine infection method, we chose to measure levels of the HNE inhibitor elafin. Endogenous elafin could not be detected in the supernatants from uninfected HUVECs or macrophages in the presence or absence of stimuli (data not shown). In contrast, elafin levels in the supernatants from HUVECs and macrophages as measured by ELISA (37) were dramatically increased after infection with Ad-elafin that had been precomplexed with lipofectamine compared with infection with Ad-elafin alone. Indeed, HUVEC elafin production, after infection with Ad-elafin at an multiplicity of infection of 100 in the absence and presence of lipofectamine, was 61.7 (±6.82) ng/ml and 782 (±27.5) ng/ml, respectively. Lipofectamine also had a facilitatory effect on Ad infection of macrophages, increasing production from 17.1 (±3.71) ng/ml to 250 (±18.3) ng/ml. A549 elafin production was 571 (±23.1) ng/nl, increasing to 893 (±34.1) ng/ml with lipofectamine (all values are means and SDs derived from one representative experiment performed in quadruplicate).
Ad-elafin reduced HNE induced injury and cytokine production by HUVECs
Having demonstrated the inflammatory effects of oxidized LDL, LPS, and HNE on HUVECs and having established an efficient Ad-infection method (see above), we hypothesized that elafin may modulate the responses to these stimuli by both inhibiting HNE activity and interfering with LPS signaling in a similar fashion to mSLPI (13). Ad-mediated overexpression of elafin reduced the rounding and lifting observed in HUVEC monolayers after an 8-h incubation with HNE (2.5 x 10-8 M) (Fig. 1a). Ad-dl703 and Ad-GFP were included to demonstrate the specificity of effect for Ad-elafin. Ad-GFP was used in this experiment specifically to visualize both infection efficiency and cell damage after HNE injury (Fig. 1a) and was not used further in our study. Ad-IB was included as a positive control of cytokine inhibition and reduced HUVEC IL-8 production in response to HNE, HNE and LPS in combination, and LPS alone (Fig. 1b). Ad-elafin also inhibited IL-8 production after a brief exposure to HNE alone (5 x 10-8 M for 1 h), after HNE pretreatment followed by LPS, and, most notably, after LPS stimulation in isolation (Fig. 1b). An extended anti-inflammatory role for elafin was suggested by its disruptive effect on LPS signaling, and the remainder of the study focused on investigation of this property.
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The anti-inflammatory activities of elafin were further studied on HUVECs, macrophages, and lung alveolar epithelial cells (A549 cells). Epithelial cells are not relevant to atherosclerosis and were included to test whether the inhibitory effect demonstrated cellular specificity. The effects of both Ad-elafin and Ad-mSLPI, a closely related antiprotease, were examined on responses to LPS, TNF-, and oxidized LDL. In addition to the empty Ad construct Ad-dl703, we included a further control Ad expressing m-eotaxin (Ad-m-eotaxin), a secreted chemokine of similar molecular mass to elafin and mSLPI (38), with no known anti-inflammatory activity in the cell types studied. In HUVECs, Ad-elafin and Ad-mSLPI produced small but significant reductions in basal IL-8 production, an effect not observed in Ad-IB-infected cells (Fig. 2b). Ad-elafin and Ad-mSLPI had comparable inhibitory effects to Ad-IB after stimulation of HUVECs with LPS (Fig. 2d, same data as Fig. 1b) and oxidized LDL (Fig. 2e) when compared with control vectors. TNF- stimulation led to a large increase in IL-8 production that was significantly attenuated by Ad-IB, Ad-mSLPI, and Ad-elafin (Fig. 2c). TNF- was chosen as a marker for the ability of macrophages to respond to LPS, and stimulation of macrophages with LPS produced a response in both uninfected and virally infected cells (Fig. 3a). In contrast with IL-8 production in HUVECs (Fig. 1b), there was no significant difference in the basal level of TNF- production among the five adenovirus constructs (Fig. 3b). Ad-mSLPI and Ad-elafin significantly reduced the TNF- response to LPS compared with Ad-dl703 and Ad-m-eotaxin (Fig. 3c).
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B retained an inhibitory effect, significantly reducing basal IL-8 production and attenuating the response to LPS in A549 cells.
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B activation in HUVECs and macrophages by reducing IB degradation
In HUVECs and macrophages, the level of NF-B activity in unstimulated cells was very low. There was no difference between virally infected and uninfected cells (data not shown). In HUVECs, stimulation with LPS led to an increase in NF-B activity that was significantly reduced by Ad-elafin and Ad-mSLPI, but only at the lowest concentration of LPS used (100 ng/ml) (Fig. 5a). Inhibitory effects disappeared at higher LPS concentrations (1–5 µg/ml) by comparison with the inhibitory effect of Ad-IB, which remained constant (Fig. 5a).
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B was most inhibitory, but both Ad-elafin and Ad-mSLPI attenuated NF-B activation significantly compared with control vectors. Similarly, the rise in NF-B activity after stimulation with TNF- (100 pg/ml) was reduced in Ad-elafin- and Ad-mSLPI-infected cells. In parallel with their inhibitory effects on LPS signaling (Fig. 5a), this effect disappeared with a higher concentration (1 ng/ml) of TNF- (Fig. 6b). In contrast, the inhibitory effect of Ad-IB was retained at the higher TNF- concentration.
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B activity after LPS stimulation when compared with control vectors, although Ad-IB was the most effective (Fig. 7).
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B inhibition by Ad-elafin and Ad-mSLPI was further studied by stimulating Ad-infected HUVECs and macrophages with TNF- and LPS, respectively. Total cell lysates were then analyzed for IB content by Western blot analysis.
Fig. 8 shows that Ad-mSLPI and Ad-elafin significantly protected HUVECs and macrophages from TNF-- and LPS-induced IB degradation, respectively. Ad-IB produced overexpression of mutated IB (as evidenced by its higher m.w.), but in accordance with other studies it did not completely prevent endogenous IB degradation.
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Discussion |
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At the molecular level, endothelial cells demonstrate increased adhesion molecule and chemokine expression during plaque development (7, 41), and macrophages are the predominant cell of the mononuclear cell infiltrate. Murine models of atheroma have exemplified the importance of chemokine expression in atherogenesis. Indeed, atherosclerosis-susceptible, LDL receptor-deficient mice that were irradiated and repopulated with marrow cells lacking the neutrophil chemokine receptor CXCR-2 had reduced atheroma (42). This array of data prompted us to examine endothelial cell chemokine production in response to oxidized LDL, TNF-, and LPS. We demonstrate here that all three stimuli increased production of the neutrophil chemokine IL-8 (Table I and Fig. 2a) in endothelial cells. We also show for the first time that brief exposure to the neutrophil enzyme HNE increases endothelial cell IL-8 production (see Results) in a similar fashion to its previously published effects on lung epithelial cells (43, 44) and that HNE pretreatment potentiated the IL-8 response to subsequent stimulation by LPS and oxidized LDL (Table I). In accordance with previously published work (9), we found that a more prolonged incubation of HNE with endothelial cells was cytotoxic (Fig. 1a). Although neutrophils are not present in large quantities within atherosclerotic plaques, myeloperoxidase, a major neutrophil protein, has been identified as a key mediator of plaque development (45, 46). The observation that defensin, a further neutrophil granule-derived protein, is present within the coronary artery intima (47) and may interact with LDLs (48), supports our hypothesis that neutrophil-derived HNE augments endothelial cell inflammatory responses to oxidized LDL and LPS.
We chose, therefore, to investigate whether Ad-mediated overexpression of HNE inhibitors would modulate the inflammatory responses of endothelial cells observed in the first part of our study. Elafin and mSLPI are cationic low-m.w. protease inhibitors with spectrums of antiprotease activity that include HNE, proteinase 3, and porcine pancreatic elastase for elafin and HNE, trypsin, and chymotrypsin for mSLPI (11). In humans, these protease inhibitors show 40% protein sequence homology and have been characterized from a variety of epithelial lining fluids (49). SLPI and elafin are present in monocytes, alveolar macrophages, and neutrophils, albeit to varying degrees (11, 50). The N-terminal of elafin provides a substrate for transglutamination and binding to interstitial molecules, potentially accounting for the colocalization of elafin with tissue transglutaminase in human coronary arteries (10).
Using a novel technique involving precomplexing of adenovirus with cationic liposomes, we were able to significantly augment Ad gene delivery in HUVECs. Other workers have also demonstrated that precomplexing cationic compounds, including calcium phosphate precipitates and liposomes with Ad, augment gene delivery both in vivo and in vitro (31, 51, 52, 53). Using our methodology, Ad-mediated elafin overexpression in HUVECs prevented direct HNE damage (Fig. 1a) and inhibited HNE-induced IL-8 production (Fig. 1b). Remarkably, the elafin inhibitory effect was comparable to that of the NF-B inhibitory subunit IB, suggesting that elafin and IB may share a common HNE inhibitory pathway. Walsh et al. (44) have recently shown that HNE signals through the MyD88-IL-1R-associated kinase-TNFR-associated factor-6-NF-B pathway, and a recent report has demonstrated Toll-like receptor 4-mediated IL-8 induction by HNE (54). Our new findings extend in endothelial cells the above-mentioned observations in epithelial cells and support promoter mutation studies indicating that NF-B is the predominant cis-acting element in IL-8 gene expression (55).
Importantly, Ad-elafin has modulatory effects even in the absence of HNE (Fig. 1b), suggesting that elafin may be exerting additional anti-inflammatory effects beyond neutralization ofHNE. This possibility was tested further in a range of cell types by comparing the anti-inflammatory actions of elafin, its homologue mSLPI (previously shown to inhibit LPS) (12, 13), and IB. In HUVECs, Ad-elafin and Ad-mSLPI significantly reduced IL-8 production in basal conditions and in response to TNF-, LPS, and oxidized LDL (Fig. 2, a–d). Because human macrophages are relatively refractory to Ad infection expressing low levels of adenoviral receptors (56), we used our Ad-lipofectamine technique on these cells and showed that elafin expression was increased vastly (see Results). Overexpression of elafin, mSLPI, and IB significantly reduced macrophage TNF- production in response to LPS stimulation (Fig. 3c). These results confirm the previously known anti-LPS activity of mSLPI (12, 13) using a different approach and establish for the first time the broad-ranging antagonism of elafin and mSLPI against different stimuli, suggesting a general role in dampening inflammation.
Interestingly, Ad-elafin and Ad-mSLPI had no inhibitory effect on basal or LPS-induced IL-8 production by lung epithelial cells; however, Ad-IB was inhibitory (Fig. 4, a and b), possibly reflecting a cellular or organ-specific action for these antiproteases. Indeed, recent work from our own group suggests that overexpression of elafin is not anti-inflammatory and may prime murine lung innate immune responses after an LPS challenge (57, 58).
Because both IL-8 and TNF- production are dependent on NF-B-regulated transcription (59), we investigated whether elafin could also interfere with the NF-B pathway. NF-B activity is increased within the intimal cells of human plaques and is a possible therapeutic target regulating the expression of many proatherogenic genes (60). We performed EMSAs to examine the effects of the Ad vectors on NF-B activation in HUVECs and macrophages. Oxidized LDL increases endothelial cell production of reactive oxygen species and activates NF-B through interaction with the lectin-like oxidized LDL receptor 1 scavenger receptor (61), and oxidized LDL, LPS, and TNF- stimulation were associated with increased activity of the proinflammatory transcription factor in HUVECs (Figs. 5 and 6) (62, 63). After Ad-elafin and Ad-mSLPI infection, NF-B activity was reduced in LPS- (Fig. 5), oxidized LDL- (Fig. 6a), and TNF--stimulated HUVECs (Fig. 6b). This provides evidence for an inhibitory effect before or at the level of gene transcription for these stimuli. Ad-elafin and Ad-mSLPI also reduced LPS-induced macrophage NF-B activation in accordance with their inhibitory action on production of the NF-B-regulated proinflammatory cytokine TNF- (Fig. 7). This was further studied by Western blot analysis, which showed that Ad-elafin and Ad-mSLPI inhibited NF-B activity by reducing TNF-- and LPS-induced IB degradation (Fig. 8). Interestingly, and as demonstrated by others (64), Ad-IB did not prevent endogenous IB degradation, confirming that overexpression of IB can saturate the endogenous IB degrading cellular machinery. IB protein content was highest in the Ad-IB-infected cells (Fig. 8) mirroring the EMSA results, which showed that IB was most efficient at down-regulating NF-B activity (Figs. 6 and 7). Consistent with its inhibitory activity on NF-B in the present work, SLPI-/- murine macrophages demonstrate sustained increases in NF-B activation after LPS stimulation (65). Accordingly, Lentsch et al. (14) demonstrated that inhibition of NF-B activity in a rat immune complex lung injury model, after administration of human SLPI, was associated with increased levels of the IB
inhibitory subunit. In that model, mutated human SLPI sequences coding for proteins that lack trypsin inhibitory activity failed to inhibit NF-B activation (66). Although SLPI has been shown to bind physically to LPS (67), Jin et al. (12) as well as J. McMichael (unpublished observations) in our group have reported that SLPI-containing medium was unable to transfer LPS-inhibiting activity to fresh cells, suggesting that extracellular SLPI may not be able to interfere with LPS signaling. Two reports have supported an intracellular mode of action for this secreted protein. Zhu et al. (13) showed that a SLPI construct lacking a leader sequence and hence coding for a nonsecreted protein was still able to exert its anti-LPS effect and subsequent NF-B activity. More recently (68), SLPI’s anti-inflammatory activity against LPS was shown to be inhibited by proteasome inhibitors. In addition to SLPI and elafin, other serine protease inhibitors have been shown to block NF-B activation through inhibition of proteosomal proteases (69, 70).
Our results do not exclude elafin and mSLPI interference with parallel proinflammatory signaling pathways activated by TNF-, LPS, and oxidized LDL because inhibition of cytokine production was more striking than was the sole suppression of NF-B activation (particularly for TNF-) supporting this possibility.
In conclusion, our experiments have shown for the first time that overexpression of HNE inhibitors is protective against HNE-mediated endothelial cell damage and proinflammatory signaling in both endothelial cells and macrophages and can, through NF-B inhibition, attenuate inflammatory responses to LPS, TNF-, and oxidized LDL. Overall, our data support the concept of endogenous defense molecules such as elafin and SLPI working as effectors of innate immunity to protect tissues against maladaptive inflammatory responses. We believe that the properties of these molecules make them attractive anti-inflammatory agents in a range of diseases, including arterial wall inflammation occurring during atherosclerosis.
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Acknowledgments |
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B and Ad-dl703 and A. Harris and T. Sheldrake for excellent technical assistance. We also thank many colleagues for helpful advice in preparation of this work, particularly Drs. A. J. Simpson, C. Ward, and S. Fujihara. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Jean-Michel Sallenave, Rayne Laboratory, Medical School, University of Edinburgh, Edinburgh EH8 9AG, U.K. E-mail address: j.sallenave{at}ed.ac.uk
3 Abbreviations used in this paper: LDL, low density lipoprotein; HNE, human neutrophil elastase; mSLPI, murine secretory leukocyte protease inhibitor; EBM2, endothelial basal medium-2; m-eotaxin, murine eotaxin; RT, reverse transcription; Ad, adenovirus; Ad-mSLPI, E1, E3-deleted Ad encoding mSLPI cDNA; Ad-GFP, Ad expressing green fluorescent protein; Ad-dl703, E1, E3-deleted empty adenoviral vector.
Received for publication January 24, 2003. Accepted for publication January 27, 2004.
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B and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 87:9943. and oxidized phospholipids. Arterioscler. Thromb. Vasc. Biol. 21:1585.-defensins stimulate proteoglycan-dependent catabolism of low-density lipoprotein by vascular cells: a new class of inflammatory apolipoprotein and a possible contributor to atherogenesis. Blood 96:1393.v3 and v5 on human monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery. J. Virol. 69:2257.[Abstract]
B reveals that macrophage tumor necrosis factor production in rheumatoid arthritis is NF-B dependent. Proc. Natl. Acad. Sci. USA 95:8211.B in atherogenesis. Exp. Physiol. 82:297.[Abstract]
B through an increased production of intracellular reactive oxygen species. J. Biol. Chem. 275:12633.B kinase complex is an intracellular target for endotoxic lipopolysaccharide in human monocytic cells. Blood 94:1711.B activation in human endothelial cells involves degradation of IB but not IB. Exp. Cell Res. 243:425.[Medline]
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