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The Journal of Immunology, 2007, 179, 6184-6192
Copyright © 2007 by The American Association of Immunologists, Inc.
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Elastin Receptor (Spliced Galactosidase) Occupancy by Elastin Peptides Counteracts Proinflammatory Cytokine Expression in Lipopolysaccharide-Stimulated Human Monocytes through NF-{kappa}B Down-Regulation1

Thomas Baranek*, Romain Debret2,{dagger}, Frank Antonicelli{dagger}, Bouchaib Lamkhioued*, Azzaq Belaaouaj{ddagger}, William Hornebeck{dagger}, Philippe Bernard{dagger}, Moncef Guenounou* and Richard Le Naour3,*

* Laboratoire d’Immunologie et de Microbiologie, Immuno-Pharmacologie Cellulaire et Moléculaire, EA3796 Unité de Formation et de Recherche de Pharmacie, Reims, France; {dagger} Laboratoire de Biochimie et de Dermatologie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6198, Unité de Formation et de Recherche de Médecine, Reims, France; and {ddagger} Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche Santé 514, Centre Hospitalier Universitaire Maison Blanche, Reims, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-{gamma}) cell response in lymphocytes and to regulate IL-1beta expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-{alpha}, IL-1beta, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-beta secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-{kappa}B-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-{kappa}B-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The inflammatory process is tightly regulated involving both signals that initiate and maintain inflammation and signals that pull such process down. An imbalance between these two signaling pathways results in uncontrolled inflammation, leading to cellular and tissue damage. Monocytes are main players in the initiation, maintenance, and resolution of inflammation, notably through secretion of several biologically active molecules, among which cytokines play a pivotal function. During inflammation, monocytes produce proinflammatory cytokines such as IL-1beta, IL-6, and TNF-{alpha} (1, 2), which are followed by anti-inflammatory cytokine secretion as IL-10 and TGF-beta to keep the progression of inflammation under control (3, 4).

Such timely regulated process, corresponding to the initiation and resolution phases of inflammation, has been delineated in many human diseases (5). In pathologies involving elastin-rich tissues as artery, lung, and skin, inflammation is concomitant with important elastolysis, leading to elastin peptide (EP)4 generation (6, 7, 8). In those pathologies, a direct relationship between inflammation and EP levels has been established. Indeed, release by inflammatory cells of cytokines such as IL-1beta and TNF-{alpha}, which both activate the NF-{kappa}B pathway, enhances matrix metalloproteinase expression (9, 10, 11), some of them displaying potent elastinolytic activity (12, 13, 14). Besides, release of elastolytic proteases, i.e., elastase and proteinase-3, by polymorphonuclear neutrophils has been observed following Gram-negative bacteria infection (15). Recent investigation evidenced that these enzymes could liberate from hydrolysis of human elastin, VGVAPG-containing fragments (16). Such EPs further designated as elastokines trigger several biological activities.

EPs exhibit a wide range of properties, such as the following: 1) chemotactic activity for monocytes, fibroblasts, and tumor cells (17, 18, 19); 2) regulation of cell proliferation in normal and pathological conditions (20); and 3) control of vascular tone (21). These effects are largely mediated through interaction of EPs with a receptor complex that includes a 67-kDa elastin-binding protein (EBP) identified as an enzymatically inactive spliced variant of human beta-galactosidase designated as spliced galactosidase (S-gal) (22, 23, 24). Besides its elastin binding site, EBP also contains a beta-galactosugar binding site whose binding causes its shedding from the cell surface (25), and therefore, elastin receptor inactivation. Although this receptor is expressed at the surface of various cell types, including immune and inflammatory cells (26, 27), the observed effects following elastin/receptor interaction are dependent on cell type and therefore on the triggering of specific signaling cascade(s). For instance, activation of protein kinase G, cAMP, or cGMP by EPs was reported to induce chemotaxis of monocytes (28, 29, 30). Besides, activation of pertussis toxin-sensitive G proteins and MEK/ERK signaling cascade were involved in EP-mediated effects on cell proliferation as observed in arterial smooth muscle cells and fibroblasts (31, 32). In turn, S-gal occupancy by EPs led to the activation of both NF-{kappa}B and p38 in melanoma cells (33).

In previous studies, we showed the ability of EPs to orientate cytokine expression in human lymphocytes toward a Th1 profile (27), and to interfere on proinflammatory cytokine production in human melanoma cells (33). Based on these former results, we hypothesized that, subsequently to elastin fiber degradation in pathologies displaying an inflammatory reaction, EPs could interfere in the sustained inflammation through the regulation of pro- and/or anti-inflammatory cytokine expression. To that purpose, in this study we used LPS-activated human monocytes as a classical inflammatory model system, and showed that LPS-enhanced proinflammatory cytokine expression was down-regulated following EP interaction with its receptor. Inhibition of proinflammatory cytokine expression upon EP treatment was associated with inhibition of NF-{kappa}B pathway. Similarly, IL-1beta-mediated stimulation of NF-{kappa}B could be prevented by EPs in melanoma cells in culture.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

LPS from Escherichia coli 0111:B4, VGVAPG, brefeldin A, lactose, FITC-LPS from E. coli 0111:B4, and mouse monoclonal anti-human beta-actin (IgG1, clone AC-15, catalogue A5441) were purchased from Sigma-Aldrich. The scrambled VVGPGA peptide, not found in tropoelastin sequence, was synthesized by GENEPEP. RPMI 1640 medium, McCoy’s 5A medium, FCS, L-glutamine, penicillin, streptomycin, PBS, ethidium bromide, reverse transcriptase, and all primers and probes were obtained from Invitrogen Life Technologies. SYBR Green for real-time RT-PCR was purchased from Applied Biosystems. FACS lysing, FACS permeabilizing, mouse FITC-conjugated anti-human CD14 (IgG2a, clone M5E2, catalogue 555397), mouse PE-conjugated anti-human IL-1beta (IgG1, clone AS10, catalogue 340516), rat PE-conjugated anti-human IL-6 (IgG1, clone MQ2-13A5, catalogue 554545), mouse allophycocyanin-conjugated anti-human TNF-{alpha} (IgG1, clone Mab11, catalogue 340534), and corresponding isotype controls were from BD Pharmingen. Elastin-FITC was obtained from Elastin Products. Mouse PE-conjugated anti-human TLR4 (IgG1, clone HTA125, catalogue sc-13593), rabbit polyclonal Ab against human p50 NF-{kappa}B subunit (catalogue sc-114X), nonspecific IgG (catalogue sc-2027), and rabbit polyclonal Ab against human protein kinase C (PKC) {alpha} (catalogue sc-208) were purchased from Santa Cruz Biotechnology. Rabbit anti-human Thr180/Tyr182-phosphorylated p38 MAPK (catalogue 9211) and rabbit anti-human I{kappa}B-{alpha} (catalogue 9242) Abs were provided by Cell Signaling Technology. Rabbit polyclonal Abs against human PKCbetaII (catalogue 539605) and PKC{delta} (catalogue 539607) isoenzymes were purchased from Calbiochem.

Human peripheral blood monocyte isolation and culture

Human peripheral blood monocytes were obtained under sterile and endotoxin-free conditions by countercurrent centrifugal elutriation (service of hematology, Reims Hospital), followed by density-gradient centrifugation from heparinized venous blood of healthy consenting donors, after protocol approval by the Ethical Committee of Reims Hospital. Isolated human monocytes (1 x 106/ml) were cultured at 37°C in 5% CO2 under adherence-free conditions in Teflon wells and in RPMI 1640 medium containing L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and 10% heat-inactivated FCS (complete medium). For experiments, human monocytes were shown to be >98% by FACS analysis (data not shown), and cell viability was over 95%, as determined by trypan blue exclusion.

Treatment of human monocytes

Human monocytes cultured under experimental conditions above indicated were harvested, washed, and seeded onto Teflon wells at 1 x 106/ml in a serum-starved complete medium and incubated with LPS (1 µg/ml) in the presence or not of VGVAPG EP (10 µg/ml) or scrambled VVGPGA peptide (10 µg/ml). At 1 or 4 h following incubation at 37°C in 5% CO2, cells were either collected for intracellular cytokine staining, CD14 and TLR-4 surface staining, or total RNA extraction. Cell culture supernatants were also collected for quantification of cytokine protein level by ELISA. In Western blotting experiments, human monocytes were collected for protein extraction at 15, 30, and 60 min following incubation with LPS in the presence or not of VGVAPG. EMSA and chromatin immunoprecipitation (ChIP) assay analysis were performed at 60 min following incubation. Cell viability was >95% when cells or cell culture supernatants were collected. To detect intracytoplasmic cytokines, brefeldin A (5 µg/ml) was added to the culture medium (34). In some experiments, LPS or elastin binding on human monocytes was determined following 4-h incubation of cells with VGVAPG or LPS alone, respectively. Pretreatment of monocytes with 10 mM lactose (3 h) was used to appreciate the specificity of EP effects.

Melanoma cell line culture and treatment

Melanoma cell line M3Da was established from metastases of a patient with malignant melanoma and has been previously characterized. A total of 20,000 M3Da cells/cm2 was initially seeded in plastic plates and grown as monolayer cultures in McCoy’s 5A medium supplemented with 10% heat-inactivated FCS at 37°C in 5% CO2 until 80% confluence. Cells were then washed twice with serum-starved McCoy’s 5A medium prior treatment with IL-1beta (10 ng/ml) in the presence or not of VGVAPG (10 µg/ml). Four hours following incubation, cells were collected for total RNA and nuclear protein extractions. Cell viability was above 90% when cells were collected.

Intracellular staining and flow cytometric analysis

Nonpermeabilized monocytes (5 x 105) were incubated with FITC-conjugated anti-CD14 mAb at room temperature for 20 min in the dark to prevent fluorescence quenching; then cells were washed twice in PBS. Following two successive centrifugations (500 x g, 5 min), cell pellets were resuspended successively in FACS lysing and FACS permeabilizing solution for 10 min each. Cells were then stained with 1 µg/ml allophycocyanin-conjugated anti-TNF-{alpha}, PE-conjugated anti-IL-1beta, and PE-conjugated anti-IL-6 mAbs at room temperature for 20 min in the dark. In parallel, isotype-matched controls were used to determine nonspecific labeling. Finally, cells were washed and pellets were resuspended in 500 µl of PBS containing 1% paraformaldehyde, and stored in the dark at 4°C until analysis. A two-color flow cytometric analysis was performed with a FACSCalibur instrument (BD Biosciences). Human monocytes from healthy donors were initially gated on forward and side light-scattering properties, and thereafter on the presence of the surface marker CD14. Analysis was performed on a logarithmic scale using CellQuest software (BD Biosciences). Surface staining for TLR-4 was performed, as described above, using PE-conjugated anti-TLR-4 mAb. In some experiments, LPS or elastin binding on human monocytes was determined following 20-min incubation of cells with 1 µg/ml LPS-FITC or 10 µg/ml elastin-FITC, respectively. Cells were then washed twice and resuspended in 500 µl of PBS containing 1% paraformaldehyde, and stored in the dark at 4°C until analysis.

Confocal microscopy

Human monocytes (1 x 106) were incubated at 37°C for 20 min in the dark with 10 µg/ml elastin-FITC in PBS containing 1% BSA. After incubation, cells were washed twice in PBS to remove excess dye and then resuspended in PBS. In some experiments, monocytes were preincubated with 10 mM lactose for 3 h to remove EBP. Fluorescence emission was recorded over 520 nm for FITC using a laser scanning confocal microscope (Bio-Rad MRC-1024ES) coupled to an Olympus IX70 microscope.

RT-PCR analysis

Total RNA was extracted from 5 x 106 monocytes using NucleoSpin RNA II (Macherey-Nagel) in accordance with the Macherey-Nagel technical data sheets and analyzed on agarose gels stained with ethidium bromide. One microgram of total RNA was reverse transcripted into cDNA with a reverse transcriptase using SuperScript First-Strand Synthesis System in accordance with the Invitrogen Life Technologies technical data sheets. After reverse transcription, the cDNA product was amplified by PCR, as previously described (35). Primer sequences for growth-related oncogene (GRO)-{alpha}, IL-8, S-gal, and for the internal controls 18S and beta-actin were as follows: GRO-{alpha} sense, 5'-ACTGAACTGCGCTGCCAGTG-3'; GRO-{alpha} antisense, 5'-GGCATGTTGCAGGCTCCTCA-3'; IL-8 sense, 5'-ATTTCTGCAGCTCTGTGTGAA-3'; IL-8 antisense, 5'-TGAATTCTCAGCCCTCTTCAA-3'; S-gal sense, 5'-CCATCCAGACATTACCTGGCA-3'; S-gal antisense, 5'-GATGTTGCTGCCTGCACTGTT-3'; 18S sense, 5'-GCGAATTCCTGCCAGTAGCATATGCTTG-3'; 18S antisense, 5'-GGAGCTTAGAGGAGCGAGCGACCAAAGG-3'; beta-actin sense, 5'-TGCTATCCAGGCTGTGCTA-3'; beta-actin antisense, 5'-ATGGAGTTGAAGGTAGTTT-3'. PCR amplifications of cDNA were performed under the following conditions: initial DNA denaturation for 10 min at 94°C, primer annealing at 58°C (beta-actin) or 62°C (GRO-{alpha}, IL-8, S-gal) or 65°C (18S) for 1 min, and extension at 72°C for 1 min, for a total 40 cycles. The ethidium bromide-stained amplified cDNA obtained by RT-PCR were analyzed from 1.5% agarose gels under UV light (Gel Doc 2000; Bio-Rad). The PCR products for GRO-{alpha}, IL-8, and S-gal were analyzed comparatively to the amount of the beta-actin or 18S housekeeping gene detected in the same mRNA sample.

In some experiments, real-time RT-PCR analysis was used. Using this approach, the mRNA levels for TNF-{alpha}, IL-1beta, IL-6, and beta-actin were determined with the 7000 sequence detection system ABI Prism sequence detector (Applied Biosystems), using the double strand-specific SYBR Green (Applied Biosystems) dye system. All reactions were performed according to the following thermal profile: denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min (data collection was performed during the annealing/extension step). Primer sequences for TNF-{alpha}, IL-1beta, IL-6, and the internal control beta-actin were determined with Primer Express software (Applied Biosystems) and were as follows: TNF-{alpha} forward, 5'-GAGACCAGGGAGCCTTTGGT-3'; TNF-{alpha} reverse, 5'-TTGTGTCAATTTCTAGGTGAGGTCTT-3'; IL-1beta forward, 5'-TAAAGCCCGCCTGACAGAA-3'; IL-1beta reverse, 5'-ATAGGGAAGCGGTTGCTCATC-3'; IL-6 forward, 5'-ACTTAAGCCAGGGTTTCTCATATGTTA-3'; IL-6 reverse, 5'-CACCCGAGGCTTGCTAAGTC-3'; beta-actin forward, 5'-AAATGCTTCTAGGCGGACTATGA-3'; beta-actin reverse, 5'-TGTTTTCTGCGCAAGTTAGGTTT-3'. Data analysis was performed with the SDS software (Applied Biosystems). Results are presented as ratios between the target gene mRNA and the beta-actin mRNA.

Detection of cytokine concentration

Determination of TNF-{alpha}, IL-1beta, IL-6, and IL-10 in cell culture supernatants of human monocytes was performed in duplicate using commercially available high-sensitivity ELISA kit (Quantikine; R&D Systems), according to the manufacturer’s instructions. The sensitivity of each ELISA kit was 4.4, 1.5, 0.70, and 3.9 pg/ml for TNF-{alpha}, IL-1beta, IL-6, and IL-10, respectively.

ChIP

Human monocytes were fixed with 1% formaldehyde, neutralized with glycine for 5 min to a final concentration of 125 mM, rinsed twice with TBS (100 mM NaCl, 50 mM Tris-HCl (pH 8.1)), and then syringed with TBS containing 0.5% SDS. The cell pellet was resuspended in the same buffer completed with 2% Triton X-100. The cell pellet was sonicated to yield DNA fragments of ~400 bp in average, immunoprecipitated with 1 µg of Abs: rabbit anti-p50 NF-{kappa}B subunit polyclonal Ab and rabbit anti-IgG Ab as negative control, or no Ab as positive control (inputs), this last tube being conserved at 4°C until DNA isolation step. For the other tubes, after overnight incubation at 4°C with continuous mixing, aggregates were removed by centrifugation and protein A beads were added to supernatant. After 2 h at 4°C, immune complexes were collected by centrifugation at 4°C and washed three times with wash buffer containing 150 mM NaCl, 20 mM Tris (pH 8.1), 5 mM EDTA (pH 8), 6% sucrose, 0.2% NaN3, 1% Triton X-100, and 0.2% SDS; twice with solution containing 1 mM EDTA, 50 mM HEPES (pH 7.5), 500 mM NaCl, 1% Triton X-100, 0.2% NaN3, and 0.1% deoxycholic acid; and twice with LiCl detergent solution (10 mM Tris (pH 8), 1 mM EDTA (pH 8), 250 mM LiCl, 0.2% NaN3, 0.5% Nonidet P-40, and 0.5% deoxycholic acid; and finally in TE buffer. All washes were 5 min at 4°C with mixing. To eluate immune complexes from beads, an incubation for 30 min at 65°C in reversal buffer (1% SDS, 100 mM NaHCO3) was done. Eluate was then incubated overnight at 65°C in the same buffer to reverse cross-links. DNA was extracted with phenol-chloroform and precipitated with ethanol using glycogen as a carrier. The purified DNA isolated by immunoprecipitation was analyzed by PCR. The human TNF-{alpha} mRNA (GenBank accession NM_000594) was aligned against the overlapping genomic sequence (GenBank accession NC_000006.10), which maps to chromosome 6p21.3. The promoter sequence was identified from alignment and annotated relatively to the translation start site. Primers encompassing the three {kappa}B sites located at –1058, –822, and –793 were then designed, using Vector NTI v10 (Invitrogen Life Technologies). The sequences of the primers were as follows: 5'-GCAATGGGTAGGAGAATGTC-3' (sense) and 5'-CAAACACAGGCCTCAGGACT-3' (antisense). PCR amplifications of cDNA were performed under the following conditions: initial DNA denaturation for 10 min at 94°C, primer annealing at 62°C for 1 min, and extension at 72°C for 1 min, for a total of 40 cycles.

EMSA

Nuclear extracts were prepared from human monocytes or melanoma cells, as already described (36). Briefly, cells were 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 (13,000 x g, 20 s). 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 stirred for 20 min at 4°C, followed by centrifugation (10,000 x g, 5 min). Nuclear proteins (4 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 [{gamma}-32P]adenosine 5' triphosphate-labeled probes and submitted to electrophoresis on a 6% nondenaturing polyacrylamide gel. Gel-shift experiments were performed on both NF-{kappa}B consensus sequence and TNF-{alpha} promoter (GenBank accession NC_000006.10). DNA binding on NF-{kappa}B oligonucleotides was assessed by autoradiography. Sequences of probes have been synthesized as follows: NF-{kappa}B consensus oligonucleotides, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; NF-{kappa}B-specific probe for DNA binding on TNF-{alpha} DNA, 5'-GCTCATGGGTTTCTCCA-3'. In competition experiments, the nuclear extract was incubated with a 50-fold molar excess of the appropriate unlabeled specific and nonspecific competitor oligonucleotides. In supershift studies, a rabbit anti-p50 NF-{kappa}B subunit polyclonal Ab or a nonspecific IgG was preincubated with the crude nuclear extract for 1 h at room temperature before addition of the labeled probe.

Determination of I{kappa}B-{alpha} and phospho-p38 expression

Human monocytes were rinsed twice in ice-cold PBS and incubated on ice for 30 min with lysis buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM NaF, 0.2 mM Na3VO4, 1% Triton X-100, and 0.5% Nonidet P-40) containing a minicomplete protease inhibitor mixture tablet (Roche Diagnostic Systems). Extracts were clarified at 12,000 x g at 4°C for 5 min, and protein concentration was determined using the Bradford assay (Bio-Rad). With the use of one-dimensional SDS-PAGE, 30 µg of protein extracts was resolved and electrophoretically transferred to Trans-Blot Transfert Nitrocellulose membrane (0.45 µm) (Bio-Rad). Membranes were blocked using 5% nonfat dry milk in TBST (0.05% Tween 20, 10 mM TBS (pH 7.5)) for 1 h at room temperature and then washed twice for 10 min with TBST. Membranes were then incubated overnight at 4°C with Abs raised against I{kappa}B-{alpha} (1/1000e), Thr180/Tyr182-phosphorylated p38 MAPK (1/1000e), or beta-actin (1/1000e). Membranes were subsequently washed twice with TBST and incubated at room temperature for 1 h with the HRP-conjugated donkey anti-mouse (1/5000e) or goat anti-rabbit (1/5000e) IgG Abs used as secondary Abs. The bound secondary Abs were detected using the Luminol Chemiluminescence detection kit (Santa Cruz Biotechnology). The signal was visualized on hyperfilm ECL (Amersham Biosciences).

Determination of PKC isoenzymes

The cytosolic fractions were prepared by lysis of the human monocytes 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 collected by centrifugation (13,000 x g, 20 s). The remaining protein pellets were treated with the same lysis buffer supplemented with 1% Triton X-100 to obtain the membrane fractions. Protein concentrations were measured using the Bradford assay (Bio-Rad). Proteins were then acetone precipitated during 4 h at –20°C, boiled in Laemmli buffer for 10 min at 100°C, separated on 8% SDS-PAGE gel, and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were then blocked 1 h in PBS with 5% (w/v) dry milk, and probed at 4°C overnight with the polyclonal primary Abs specific to PKC{alpha}, PKCbetaII, and PKC{delta} isoenzymes (1/1000e), or beta-actin (1/1000e). Membranes were subsequently incubated for 1 h with the HRP-conjugated goat anti-rabbit Igs used as secondary Abs (1/5000e). The immunoreactive bands were visualized by a chemiluminescent detection using the Amersham ECL Western blotting system (Amersham).

Statistical analysis

Statistical analysis was performed using a Wilcoxon nonparametric test. Values of p < 0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
EPs decrease LPS-induced proinflammatory cytokine production in human monocytes

In keeping with the presence of S-gal on human monocytes (26, 37), we first investigated whether EPs, i.e., VGVAPG, could interfere with cytokine production by monocytes. To that purpose, human monocytes isolated by countercurrent centrifugal elutriation were incubated for 4 h with VGVAPG (10 µg/ml) and/or LPS (1 µg/ml), and then TNF-{alpha}, IL-1beta, and IL-6 intracellular expression was determined by flow cytometry. On untreated monocytes, VGVAPG alone had no effect on TNF-{alpha}, IL-1beta, and IL-6 intracellular expression (Fig. 1A, blue vs green curves). We then studied the ability of VGVAPG to modulate proinflammatory cytokines production in human monocytes stimulated by LPS. LPS treatment increased expression of the three proinflammatory cytokines tested (Fig. 1A, purple vs green curves) with a stronger effect for TNF-{alpha} as compared with IL-1beta and IL-6. When LPS-stimulated monocytes were cotreated with VGVAPG, expression of cytokines was decreased compared with LPS-treated cells (Fig. 1A, red vs purple curves), whereas cotreatment with scrambled peptide (VVGPGA) had no effect (Fig. 1A, black vs purple curves). Statistical significance of this result was confirmed using six different donors (Fig. 1B). Analysis of the VGVAPG inhibitory effects revealed that down-regulation of LPS-enhanced proinflammatory cytokine expression was either partial (TNF-{alpha}) or total (IL-1beta and IL-6) as compared with untreated human monocytes. Using similar experimental conditions, we then determined by ELISA the secretion of IL-10, an anti-inflammatory cytokine known to induce a negative feedback loop of the proinflammatory cytokine production in monocytes. Fig. 1C shows that following 4-h treatment with LPS, IL-10 basal expression was significantly increased; coincubation with VGVAPG did not modify its expression level.


Figure 1
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  		FIGURE 1. EPs decrease LPS-induced proinflammatory cytokine expression. A, Human monocytes were incubated with LPS (1 µg/ml) and/or the EP VGVAPG (10 µg/ml). The scrambled VVGPGA peptide (10 µg/ml), not found in tropoelastin sequence, was used as negative control. Four hours after incubation, TNF-{alpha}, IL-1beta, and IL-6 intracellular expression was determined by two-color flow cytometry analysis. Data are representative of six independent experiments. B, Statistical analysis of results obtained from the six independent experiments studying cytokine intracellular expression upon LPS/VGVAPG or LPS/VVGPGA coincubation was performed using a Wilcoxon nonparametric test. Data are expressed as mean fluorescence intensity. *, p < 0.05. C, Human monocytes were incubated with LPS (1 µg/ml) in the presence or not of VGVAPG (10 µg/ml). One, 2, and 4 h after incubation, IL-10 secretion in cell culture supernatants was determined by ELISA. Data are representative of six independent experiments. *, p < 0.05 using a Wilcoxon nonparametric test.

 
LPS receptor expression and binding of LPS to its receptor are not affected by coincubation with EPs

To further investigate the mechanisms involved in EP-mediated effect on reduced LPS-stimulated cytokine expression, we then analyzed whether cotreatment with VGVAPG displayed any influence on the expression of two components of LPS receptor, i.e., CD14 and TLR-4, at the monocyte cell surface (38). Flow cytometry analysis using CD14- and TRL-4-specific mAbs revealed that treatment with VGVAPG did not alter CD14 or TLR-4 expression (Fig. 2A, orange vs purple curves). Accordingly, LPS binding on monocyte surface was not modified by the presence of VGVAPG in the coincubation medium (Fig. 2B).


Figure 2
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  		FIGURE 2. CD14/TLR-4 expression and LPS binding on human monocytes are not affected by EPs. A, Human monocytes were incubated with LPS (1 µg/ml) in the presence or not of VGVAPG (10 µg/ml). One and 4 h after incubation, CD14 and TLR-4 expression levels were determined by flow cytometry analysis. Data are representative of three independent experiments. B, Human monocytes were incubated with LPS-FITC (1 µg/ml) in the presence or not of VGVAPG (10 µg/ml). Four hours after incubation, LPS binding on human monocytes was determined by flow cytometry analysis. Data are representative of three independent experiments.

 
Involvement of elastin receptor in LPS-induced proinflammatory cytokine down-regulation by EPs

The involvement of the elastin receptor (S-gal) activation by VGVAPG on the regulation of LPS-enhanced cytokine expression was further examined. To that end, we first studied the expression of S-gal and showed that human monocytes constitutively expressed S-gal mRNA, and that this expression was not affected by LPS treatment (Fig. 3A). Furthermore, comparative analysis by flow cytometry revealed that binding of elastin to its receptor was not modified by LPS coincubation (Fig. 3B). Specificity of elastin binding to its receptor was then demonstrated by confocal microscopy using elastin-FITC on monocytes pretreated or not with 10 mM lactose, a galactosugar known to induce the shedding of S-gal from the cell surface (25) (Fig. 3C). To determine whether VGVAPG down-regulate the production of TNF-{alpha}, IL-1beta, and IL-6 in LPS-activated human monocytes via interaction with its receptor, cells were pretreated with 10 mM lactose. Accordingly, the subsequent lactose-dependent elastin receptor desensitization abrogated the inhibitory effect of VGVAPG on LPS-induced proinflammatory cytokine production at both mRNA and protein levels (Fig. 3D).


Figure 3
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  		FIGURE 3. EP/S-gal interaction down-regulates LPS-induced proinflammatory cytokine expression. A, Human monocytes were treated or not with LPS (1 µg/ml) for 4 h, and then total RNA was extracted and S-gal and beta-actin mRNA expression was determined by RT-PCR analysis. Data are representative of three independent experiments. B, Human monocytes were incubated 4 h with elastin-FITC (10 µg/ml) in the presence or not of LPS (1 µg/ml). Elastin binding on human monocytes was determined by flow cytometry analysis. Data are representative of three independent experiments. C, Human monocytes were preincubated or not with lactose (10 mM) for 3 h, and then were treated 20 min with elastin-FITC (10 µg/ml) before analysis by phase-contrast microscopy (pictures 1 and 3) coupled to laser scanning confocal microscopy (pictures 2 and 4). Data are representative of three independent experiments. D, Human monocytes preincubated or not with lactose (10 mM) during 3 h were treated with LPS (1 µg/ml) in the presence or not of VGVAPG (10 µg/ml). Four hours after incubation, TNF-{alpha}, IL-1beta, and IL-6 mRNA expression and secretion were determined by real-time RT-PCR and ELISA, respectively. Data are representative of six independent experiments. *, p < 0.05, using a Wilcoxon nonparametric test.

 
EPs inhibit LPS-induced NF-{kappa}B DNA binding

NF-{kappa}B is one of the main transcription factors involved in proinflammatory cytokine production upon LPS stimulation (39, 40). Thus, the effect of VGVAPG on LPS-induced proinflammatory cytokine expression in monocytes was addressed at the NF-{kappa}B level. The activation of NF-{kappa}B from nuclear extracts was first investigated through its capacity to bind a consensus DNA sequence. Gel-shift analysis showed that NF-{kappa}B DNA binding was increased upon LPS monocyte stimulation (Fig. 4A, lane 2 vs lane 1). We first verify DNA-binding specificity by competition experiments (Fig. 4A, lanes 4 and 5) and characterization of the NF-{kappa}B complex by supershift experiments performed with an Ab against the p50 subunit protein (Fig. 4B, lane 2 vs lane 1). Then, we clearly demonstrated that VGVAPG decreased the NF-{kappa}B-DNA complex formation generated by monocytes treated with LPS alone (Fig. 4A, lane 3 vs lane 2). To establish the relationship between NF-{kappa}B and proinflammatory cytokine regulation, we then performed gel-shift experiments with a NF-{kappa}B-specific DNA-binding sequence delineated from the TNF-{alpha} promoter. Results obtained with the TNF-{alpha}-specific NF-{kappa}B probe revealed that the increase in NF-{kappa}B DNA binding induced by LPS (Fig. 4C, lane 2 vs lane 1) was also reduced when monocytes were coincubated with VGVAPG (Fig. 4C, lane 3 vs lane 2). To further demonstrate a direct involvement of NF-{kappa}B in TNF-{alpha} expression at the genomic level, ChIP was performed on monocytes stimulated by LPS in the presence or not of VGVAPG. Chromatin was immunoprecipitated with an Ab raised against the NF-{kappa}B p50 subunit, using the TNF-{alpha} promoter sequence containing the three NF-{kappa}B sites located at –1058, –822, and –793, respectively. This experiment showed that LPS increased the NF-{kappa}B p50 subunit DNA binding on the TNF-{alpha} promoter. This LPS effect was abolished by VGVAPG cotreatment. These variations were validated by using a nonspecific IgG as a negative control and no Ab immunoprecipitation (inputs) to show equal loading in all samples (Fig. 4D).


Figure 4
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  		FIGURE 4. EPs inhibit LPS-induced NF-{kappa}B DNA binding. Human monocytes were incubated for 1 h with LPS (1 µg/ml) in the presence or not of VGVAPG (10 µg/ml). A, Nuclear proteins were extracted, and NF-{kappa}B DNA-binding activity was assessed by EMSA using a consensus DNA sequence. Specificity of DNA binding was assessed by competitions using specific and nonspecific unlabeled probes. Data are representative of three independent experiments. B, Identification of NF-{kappa}B complex present in the nucleus of human monocytes was studied by supershift assays in which nuclear proteins were incubated or not with Abs specific for p50 NF-{kappa}B subunit. Data are representative of three independent experiments. C, Nuclear proteins were extracted, and NF-{kappa}B-specific DNA-binding activity was assessed by EMSA using a TNF-{alpha}-specific NF-{kappa}B probe. Data are representative of three independent experiments. D, Chromatin was fragmented, immunoprecipitated with an anti-NF-{kappa}B p50 Ab, and analyzed by PCR. Rabbit anti-IgG Ab was used as negative control, and no Ab as positive control (inputs). Data are representative of three independent experiments.

 
Analysis of NF-{kappa}B upstream pathways involved in EP down-regulation of LPS monocyte stimulation

We first investigated the canonical NF-{kappa}B signaling pathway. Kinetic analysis of I{kappa}B-{alpha} expression by Western blot showed a decrease of this protein after 30 min following LPS stimulation (Fig. 5A, lane 5). This down-regulation of I{kappa}B-{alpha}, characteristic of NF-{kappa}B activation, was not observed in monocytes costimulated with LPS and VGVAPG (Fig. 5A, lane 6). We then examined the p38 transduction pathway, also known to interfere on NF-{kappa}B regulation (41), and observed that although LPS highly increased the level of p38 phosphorylation after 30 min (Fig. 5A, lane 5), VGVAPG did not modify LPS effect (Fig. 5A, lane 6). Other kinases such as PKC may interact with NF-{kappa}B pathway and cytokine production (42, 43). We consequently investigated the activation of PKC{alpha}, betaII, and {delta} following LPS and VGVAPG treatments. The distribution of PKC isoforms among cytosolic and membrane protein fraction showed that LPS-treated monocytes induced cytosol-to-membrane translocation of PKC{alpha} and PKC{delta} isoenzymes (Fig. 5B, lanes 2 and 5), but not PKCbetaII. This transient effect, which was mainly observed after 15 min of incubation, was not significantly changed when cells were coincubated with VGVAPG (Fig. 5B, lanes 3 and 6).


Figure 5
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  		FIGURE 5. EPs target I{kappa}B-{alpha}, but not p38 and PKC activation in LPS-stimulated monocytes. Human monocytes were incubated for 15, 30, or 60 min with LPS (1 µg/ml) in the presence or not of VGVAPG (10 µg/ml). A, Cytosolic proteins were extracted, and I{kappa}B-{alpha} and phospho-p38 expression was determined by Western blot analysis using Abs raised against I{kappa}B-{alpha} and Thr180/Tyr182-phosphorylated p38 MAPK, respectively. Data are representative of three independent experiments. B, Cytosolic and membrane protein fractions were isolated and tested for the presence of PKC{alpha}, betaII, and {delta} isoenzymes by Western blot using specific Abs raised against human PKC{alpha}, betaII, and {delta} isoforms. Data are representative of three independent experiments.

 
EPs down-regulate NF-{kappa}B and associated proinflammatory cytokine expression in activated melanoma cells

To determine whether the EP effect on the regulation of LPS-mediated proinflammatory cytokine expression as shown in human monocytes could be extended to other cell type and system, we used IL-1beta-activated melanoma cells. The proinflammatory status of melanoma cells was addressed through expression of GRO-{alpha} and IL-8, two proinflammatory cytokines whose regulation is linked to NF-{kappa}B pathway. Fig. 6A shows that increase of GRO-{alpha} and IL-8 mRNA expression induced by IL-1beta (lane 2 vs lane 1) was nearly abolished by VGVAPG cotreatment (lane 3 vs lane 2). Effects of VGVAPG on cytokine expression were also linked to NF-{kappa}B pathway, as shown by gel-shift experiments. Indeed, increase of NF-{kappa}B DNA binding (Fig. 6B, lane 2 vs lane 1) observed in IL-1beta-stimulated melanoma cells was reduced by cotreatment with VGVAPG (Fig. 6B, lane 3 vs lane 2).


Figure 6
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  		FIGURE 6. EPs down-regulate proinflammatory cytokine expression and NF-{kappa}B in activated melanoma cells. Melanoma cells M3Da were incubated 4 h with IL-1beta (10 ng/ml) in the presence or not of VGVAPG (10 µg/ml). A, Total RNA was extracted, and GRO-{alpha} and IL-8 mRNA expression was determined by RT-PCR analysis. Data are representative of three independent experiments. B, Nuclear proteins were extracted, and NF-{kappa}B DNA-binding activity was assessed by EMSA using a consensus DNA sequence. Specificity of DNA binding was assessed by competitions using specific and nonspecific unlabeled probes (lanes 4 and 5). Data are representative of three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In keeping with the potentiality of elastokines to exert biological function in several cell types, we considered, so far, these matrix fragments as potent mediators acting on inflammation. At first, we thus analyzed their influence on cytokine expression by human monocytes. Although VGVAPG EP alone did not affect TNF-{alpha}, IL-1beta, and IL-6 production, we evidenced that VGVAPG down-regulated, both at mRNA and protein levels, the synthesis of these proinflammatory cytokines in LPS-activated human monocytes. Down-regulation of proinflammatory cytokine expression by VGVAPG in LPS-stimulated monocytes was not associated with a regulatory effect on the anti-inflammatory cytokine IL-10. We already showed a direct relationship between EP and cytokine expression and demonstrated the ability of VGVAPG to polarize a Th lymphocyte response by favoring Th1 cytokine production (IL-2, IFN-{gamma}) (27), and to up-regulate IL-1beta proinflammatory cytokine in melanoma cells (33). Taken together, our data suggest that EPs have the potential to interfere with the cascade of events leading to cytokine production, and therefore to orientate the inflammatory and/or immune cytokine balance on various cell types.

In LPS-stimulated monocytes, negative regulation of cytokine production could involve several mechanisms. Physiologically, the inflammatory process can be controlled by production of anti-inflammatory mediators such as IL-10 (44) or TGF-beta (45). Absence, upon VGVAPG, of IL-10 up-regulation in LPS-stimulated human monocytes eliminates the possibility of any participation of a negative feedback loop limiting the duration and intensity of the inflammatory process by this cytokine. We further speculated that VGVAPG could induce TGF-beta, although such factor is generally expressed at later stage of the inflammatory response (46). However, lack of VGVAPG effect on TGF-beta expression supported the absence of an anti-inflammatory feedback loop in our model (data not shown). Additional investigations also revealed that EP inhibitory effect on cytokine expression could not be attributed to a decrease of LPS interaction with its receptor because neither LPS receptor (CD14 and TLR-4) expression nor LPS binding was altered following EP treatment. Furthermore, a direct hydrophobic interaction between VGVAPG and LPS could be excluded because scrambled peptide with similar hydrophobicity had no effect. Alike, in monocytes costimulated with LPS, EPs still bound to S-gal, which presence had been clearly demonstrated by RT-PCR and confocal microscopy. Furthermore, abrogation by lactose of VGVAPG effect on LPS-enhanced proinflammatory cytokine expression underlined the major role of EP/S-gal interaction on LPS/receptor-enhanced cytokine production. It suggested that EPs could down-regulate LPS-mediated proinflammatory cytokines by a trans regulation mechanism. Such notion of receptor-mediated trans regulation of adjacent receptors has already been demonstrated in many systems (47, 48, 49), and recently extended to S-gal, where elastin/receptor interaction was found to increase platelet-derived growth factor/receptor-mediated arterial smooth muscle cell proliferation (32).

NF-{kappa}B is the main transcription factor involved in proinflammatory cytokine expression such as IL-1beta, IL-6, and TNF-{alpha} (50, 51, 52). We recently demonstrated the prominent role of NF-{kappa}B transcription pathway in the up-regulation of the proinflammatory IL-1beta expression following stimulation of melanoma cells by VGVAPG (33). In the present study, despite the lack of effect of VGVAPG on human resting monocytes both on cytokine expression and NF-{kappa}B transcription (data not shown), we found that increased proinflammatory cytokine expression following LPS stimulation was down-regulated by EP cotreatment via a decrease of NF-{kappa}B DNA binding. Implication of the canonical NF-{kappa}B signaling pathway in this regulation was demonstrated both upstream by analyzing the dissociation and degradation of I{kappa}B-{alpha} and downstream by showing the interaction of p50 on the TNF-{alpha} promoter through ChIP experiments. The transcription factor NF-{kappa}B can also be regulated independently of I{kappa}B degradation. For instance, it has been shown that p38 pathway increases trans activation capacity of NF-{kappa}B (53), leading to gene up-regulation. Furthermore, we previously evidenced p38 activation upon VGVAPG treatment of melanoma cells (33). However, any effect of VGVAPG in LPS-stimulated monocytes on the p38 pathway was detectable. Therefore, the p38 pathway is not linked to the regulatory mechanism induced upon VGVAPG costimulation to down-regulate proinflammatory cytokines. Because p38 pathway has also been associated to IL-10 regulation, the lack of effect observed on this signaling pathway is in setting with the absence of regulation observed on such anti-inflammatory cytokine expression. Another mechanism for the cell to end NF-{kappa}B activation and gene expression relies on the activation of PKC, which shuts down trans activation by exporting I{kappa}B-{alpha} associated to the heterodimer p50/p65 from the nucleus to cytosol (54). We found that LPS stimulation of human monocytes results in the translocation of PKC{alpha} and PKC{delta} from cytosol-to-nucleus, which is in keeping with previous data from the literature (43). Like for the p38 pathway, cytokine down-regulation by VGVAPG was not associated with variation in PKC translocation. Altogether, these data strengthen the pivotal function of NF-{kappa}B in EP effects on the down-regulation of LPS-induced proinflammatory cytokine.

To our knowledge, it is the first time that down-regulation of a signaling pathway, as demonstrated in this study for NF-{kappa}B, is observed upon cell treatment with EPs. Up to now, EPs were only involved in up-regulation of signaling cascade and gene expression (28, 55, 56). Thus, biological effects exerted by EPs should take into account the cell type used (37, 57), the signaling pathway analyzed (31, 56), and, as delineated in this study, the presence or not of coeffectors. As a matter of fact, most of the effects of EPs have been previously investigated using resting cells. The nature of the coeffector also seems to be essential because Mochizuki et al. (32) found a positive costimulatory effect of platelet-derived growth factor and EPs, whereas in this study we showed a negative effect of EPs on LPS-stimulated monocytes. Importantly, the inhibitory effect of EPs on cytokine expression is not specific to the LPS-stimulated monocyte model, because it could be extended to IL-1beta-stimulated melanoma cells in relation to proinflammatory cytokine IL-8 and GRO-{alpha} expression. Furthermore, in IL-1beta-activated melanoma cells, a model mimicking tumor progression associated with an inflammatory process, EP inhibitory effect was also observed at the NF-{kappa}B level similarly as in LPS-stimulated monocytes. As a whole, our data further illustrate the critical importance of elastolysis and EP generation in the control of inflammatory diseases.


   Acknowledgments
 
We thank Pr. Philippe Nguyen, Catherine Mace, and the Etablissement de Transfusion Sanguine Nord-Est (France) for providing heparinized venous blood of healthy donors. We are very grateful to Dr. Hervé Raoul (Cervi-Lyon, Institut National de la Santé et de la Recherche Médicale, France) for many helpful suggestions in the PKC isoenzyme analysis and advice throughout the course of these experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 T.B. is the recipient of a bursary from the French government (Ministère de l’Education Nationale et de la Recherche). Back

2 Current address: Institut de Biologie et de Chimie des Protéines, Lyon, France. Back

3 Address correspondence and reprint requests to Dr. Richard Le Naour, Laboratoire d’Immunologie et de Microbiologie, Immuno-Pharmacologie Cellulaire et Moléculaire, EA3796, IFR53, Unité de Formation et de Recherche de Pharmacie, 1 Rue du Maréchal Juin, 51096 Reims Cedex, France. E-mail address: richard.lenaour{at}univ-reims.fr Back

4 Abbreviations used in this paper: EP, elastin peptide; ChIP, chromatin immunoprecipitation; EBP, elastin-binding protein; GRO, growth-related oncogene; PKC, protein kinase C; S-gal, spliced galactosidase. Back

Received for publication April 25, 2007. Accepted for publication August 9, 2007.


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