Interleukin (IL)-1β is produced primarily by activated mononuclear phagocytic cells in the lung airway and functions as a potent proinflammatory cytokine. Release of IL-1β in the airway microenvironment induces the production of proinflammatory factors from parenchymal airway cells, including IL-8. To study the regulation of lung epithelial cell responsiveness to IL-1β, the human type II–like airway epithelial cell line A549 and primary normal human bronchial epithelial (NHBE) cells were assayed for IL-1–specific response modifiers. Specifically, the IL-1 type I receptor (IL-1RI), IL-1 type II receptor (IL-1RII), IL-1 receptor accessory protein (IL-1RAcP), and IL-1 receptor antagonist (IL-1Ra) were analyzed. Constitutive expression of IL-1RI, IL-1RAcP, and IL-1Ra was detected in both immortalized and primary human airway epithelial cells. Interestingly, a complete absence of IL-1RII expression was demonstrated under all study conditions in both A549 and NHBE cells. Both cell types were responsive to IL-1β at concentrations as low as 50 to 500 pg/ml when measured by IL-8 release into cell supernatants. IL-1β–induced chemokine production and release were inhibited by a 10- to 1,000-fold molar excess of recombinant IL-1RII or IL-1Ra, whereas IL-1RI was a less effective inhibitor. On the basis of our results, we propose that human lung epithelial cells lack the ability to downregulate IL-1β activity extracellularly because of an inability to express IL-1RII. Release of extracellular IL-1 inhibitors, including soluble IL-1Ra and soluble IL-1RII, by other inflammatory cells present in the airway may be critical for regulation of IL-1β activity in the airway microenvironment.
Interleukin (IL)-1β is a highly potent cytokine that induces many proinflammatory effects throughout the body and in the lung airway (1). The mononuclear phagocyte is the primary source of IL-1β in the lung airway (2). Release of IL-1β from the activated alveolar macrophages stimulates the surrounding parenchyma, which includes lung epithelial cells. Release of second messengers by epithelial cells enhances the recruitment and retention of additional inflammatory cells, including neutrophils, lymphocytes, and monocytes (3-10). In humans, IL-1β–induced activation of the lung epithelium has been implicated as a key inflammatory pathway in lung diseases, including interstitial pulmonary fibrosis, cystic fibrosis, and asthma (5).
Growing evidence suggests that IL-1β activity is tightly regulated outside of the cell in tissue compartments. In support of this, three isoforms of the IL-1 receptor antagonist (IL-1Ra) exist: secreted IL-1Ra and two distinct intracellular forms, termed type I and type II (11). The type I intracellular form of IL-1Ra (type I icIL-1Ra) is expressed mainly by epithelial cells, lacks a signal peptide, and is generally retained inside the cell (12). However, recent evidence has demonstrated release of this molecule from human lung epithelial cells exposed to IL-4, IL-13, and interferon (IFN)-γ, supporting the notion that IL-1 is tightly regulated in the inflammatory lung microenvironment (13).
In addition to the pure antagonist IL-1Ra, there are two known receptors for IL-1, termed the IL-1 type I receptor (IL-1RI) and the IL-1 type II receptor (IL-1RII). Both receptors exist in membrane-bound and soluble, extracellular forms (14, 15). The 80-kD type I receptor is found on the surface of all IL-1–responsive cells and, in conjunction with the IL-1 receptor accessory protein (IL-1RAcP), is responsible for IL-1β–mediated signal transduction (1, 16-18). The membrane-associated IL-1RII has a molecular weight of 60 kD, does not induce a signal cascade, and can undergo post-translational cleavage by metalloproteases to yield the soluble form of the inhibitor protein with a molecular weight ranging from 45 to 50 kD, depending on the cell source (19-21). In addition, a splice variant of the soluble IL-1RII has been demonstrated (15). Although different IL-1RII species exist, all forms preferentially bind IL-1β with higher affinity when compared with IL-1α and IL-1Ra, which suggests an essential anti- inflammatory role for IL-1RII (22-24). Taken together, regulation of IL-1RII and IL-1Ra expression in the lung microenvironment, and in particular the ability to downmodulate IL-1β activity, is essential in the host inflammatory response.
Cells that express IL-1RII and participate in lung airway inflammation include neutrophils, B lymphocytes, and monocyte/macrophages (1). Interestingly, elevated levels of soluble IL-1RII have been measured in serum taken from patients with sepsis, in synovial fluid taken from patients with inflammatory joint disorders, and in bronchoalveolar lavage fluid (BALF) taken from patients with acute eosinophilic pneumonia (25-27). The studies consistently identified inflammatory cells as the source of soluble IL-1RII and support a role for IL-1RII shedding as a mechanism for regulating IL-1 activity in inflammatory disease processes. Regulation of IL-1β bioactivity within the airway microenvironment and, in particular, lung epithelial cells is likely quite complex. Previous work has shown that primary human airway epithelial cells and relevant human lung cell lines respond to very low concentrations of IL-1β (1, 5). Although not yet proven, this would imply that IL-1RI and IL-1RAcP act in concert on the lung epithelial cell surface to amplify IL-1–induced cell activation (28, 29). It has been shown that lung epithelial cells constitutively produce only the type I intracellular form of IL-1Ra which, under native conditions, remains cell associated (12, 13). Furthermore, previous investigations have identified human bronchial epithelial cells as a source of IL-1β (30, 31). To our knowledge, with the exception of IL-1Ra, critical evaluation of IL-1–specific response modifiers in human airway epithelial cells has not been conducted. Furthermore, conflicting data has demonstrated expression of both IL-1RI and IL-1RII in primary human epithelial keratinocytes, whereas primary rat intestinal epithelial cells expressed only IL-1RI (32, 33). These results suggest that IL-1 receptor expression in epithelial cells may be a consequence of the tissue environment.
Activation of the airway epithelium by IL-1β and, in particular, the regulation of IL-1β by IL-1 receptors in the lung epithelium are poorly understood. The present investigation attempts to define the relationship between IL-1β, activation of lung epithelial cells, the expression of IL-1 response modifiers, and the net effect on cell activation and release of IL-8. We propose that human lung epithelial cells lack the ability to express IL-1RII and hence are limited in their ability to downregulate IL-1β activity extracellularly. As such, release of extracellular IL-1 inhibitors, including soluble IL-1RII and soluble IL-1Ra, by other inflammatory cells present in the airway may be critical for regulation of IL-1β activity in the airway microenvironment.
A549 cells, a type II–like human lung epithelial cell line (34), were purchased from the American Type Culture Collection (Rockville, MD) and grown in 75-cm2 tissue-culture flasks. Cultures were maintained in F-12 nutrient mixture (GIBCO BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO BRL) (complete growth medium) at 37°C with 5% CO2. A549 cells were passaged at 80 to 100% confluency using 0.25% trypsin and 1 mM ethylenediaminetetraacetic acid (EDTA)⋅4Na (GIBCO BRL).
Normal human bronchial epithelial (NHBE) cells were purchased from Clonetics Corp. (San Diego, CA). Cells were propagated according to the manufacturer's instructions in bronchial epithelial cell growth medium (BEGM) (modified LHC-9) with complete supplementation. NHBE cells were passaged at 80% confluency per manufacturer's recommendations. All experiments with NHBE cells were performed at the second or third passage. For studies performed with IL-1β and soluble IL-1 inhibitors, hydrocortisone was removed from the cell culture 24 h before treatment and during the time of the study, unless otherwise stated. Removal of hydrocortisone from cell culture medium was performed to allow optimal release of IL-8.
Peripheral blood mononuclear cells (PBMC) were purified from normal healthy volunteers. Heparinized (heparin sodium 15 U/ml; Elkins–Sinn, Inc., Cherry Hill, NJ) blood was obtained (60 ml), and PBMC were purified using polysucrose/sodium diatrizoate (Histopaque; Sigma Diagnostics, St. Louis, MO) density gradient centrifugation. The PBMC (typically 20% monocytes, 80% lymphocytes) were counted, washed, and resuspended at a concentration of 5 × 106/ml in RPMI 1640 (BioWhitaker, Walkersville, MD)/5% FBS (Hyclone) with 10 μg/ml of polymyxin B (Rorer Pharmaceuticals, New York, NY) to neutralize any contaminating endotoxin. Cells were incubated overnight after the addition of dexamethasone at 1 × 10−7 M (American Reagent Laboratories, Shirley, NY) and lipopolysaccharide (LPS) (LPS W, Escherichia coli 0127:B8; Difco Laboratories, Detroit, MI) at 1 μg/ml. HepG2 cells were purchased from the American Type Culture Collection and grown in 75-cm2 tissue-culture flasks. Cultures were maintained in minimum essential medium (MEM) with nonessential amino acids and sodium pyruvate with Earle's balanced salt solution (GIBCO BRL) supplemented with 10% FBS (Hyclone), 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO BRL) at 37°C with 5% CO2.
For experiments involving dose and time response with IL-1β, A549 cells were passaged and plated in six-well polystyrene plates (Falcon/Fisher, Pittsburgh, PA) at a density of 3 × 105 cells/well in complete growth medium to 80% confluency. Because cell passage alone can induce IL-8 release in A549 cells (personal observation), cells were incubated for 2 d after passage before further manipulation to allow cells to reach a native resting state. At this point, the medium was removed and replaced with complete growth medium that contained increasing concentrations of recombinant human IL-1β (rhIL-1β) (0 to 5 ng/ml; Biological Response Modifiers Program, National Cancer Institute, Frederick, MD) and incubated for 24 h. The following day, cell-free supernatants were collected and IL-8 release was measured by enzyme-linked immunosorbent assay (ELISA). An IL-1β dose of 50 pg/ml was then used to determine the time-dependent release of IL-8 by A549 cells into the cell culture medium. Cell-free supernatants were collected and measured for IL-8 by ELISA at 0, 1, 2, 4, 8, and 24 h after addition of IL-1β to the culture medium. Similar studies were performed using NHBE cells with an IL-1β dosage range of 0 to 50 ng/ml using BEGM without hydrocortisone. Based on dose-response studies, doses of 50 and 500 pg/ml of IL-1β were used in all subsequent studies involving A549 and NHBE cells, respectively. These concentrations of IL-1β led to a statistically significant increase in IL-8 release, as determined previously.
For experiments involving the analysis of IL-1RII and IL-1Ra protein expression, A549 and NHBE cells were grown for 24 h in standard culture conditions. The following day, cell-free supernatants were obtained as well as cell lysates after treatment with lysis buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, 0.2 mM methoxysuccinyl-Ala-Ala-Pro-Val chloromethylketone (Enzyme System Products, Livermore, CA), 10 μg/ml leupeptin (Sigma Diagnostics), and 10 μg/ml pepstatin (Sigma Diagnostics). Supernatants and cell lysates were stored at −80°C until further use.
For inhibition experiments, A549 cells were passaged and plated in either 96-well plates or six-well plates at a cell density of either 2 × 104 or 3 × 105 cells/well, respectively, in complete growth medium. Two days after passage, culture medium was removed and the cells were rinsed with serum-free medium and then incubated for 24 h with rhIL-1β (50 pg/ml) alone or in combination with one or more of the following soluble inhibitors: a 10- to 1,000-fold molar excess hIL-1RI (a kind gift of Dr. J. E. Sims, Immunex Corp., Seattle, WA), a 10- to 1,000-fold molar excess hIL-1RII (also a gift of Dr. J. E. Sims), or a 10- to 1,000-fold molar excess of IL-1Ra (a kind gift from Dr. Daniel Tracey, Pharmacia-UpJohn, Kalamazoo, MI). Before the addition to A549 cells, IL-1β and soluble IL-1 inhibitors were mixed and preincubated in complete growth medium at 37°C, 5% CO2 for 15 min. At the end of the experiment, cell-free supernatants were collected and assayed for IL-8 by ELISA.
Similar experiments were performed with NHBE cells with the following modifications: 24 h before the experiment was carried out, BEGM was replaced with BEGM without hydrocortisone as previously described, NHBE cells were stimulated with 500 pg/ml IL-1β instead of 50 pg/ml, and the dose of IL-1 inhibitors was adjusted on the basis of IL-1β concentration. As before, following incubation for 24 h, the cell-free supernatants were assayed for IL-8 by ELISA.
A549 and NHBE cell lines were also stimulated with 0 to 1 μg/ml of LPS. Stimulation was carried out in a fashion similar to that described above. Briefly, cells were plated in appropriate medium and incubated until they reached 80% confluence. A549 cells were maintained in complete growth medium, whereas NHBE cells were placed in BEGM without hydrocortisone 24 h before stimulation and throughout the incubation period with LPS. Cells were incubated for 24 h in the presence of LPS, and on the following day cell-free supernatants were harvested and assayed for IL-8 release by ELISA. In addition, A549 cells were stimulated with IL-4 (10, 20, and 30 ng/ml), IFN-γ (250 and 500 U/ml), and dexamethasone (1 × 10−7 M) for 24 h as described previously. Cell-free supernatants were collected and assayed for IL-1RII and IL-1Ra release by ELISA.
IL-8 release was measured by a sandwich ELISA as previously described (35). Briefly, a mouse monoclonal antihuman IL-8 antibody (R&D Systems, Minneapolis, MN) was used as the capture antibody, and a rabbit antihuman IL-8 polyclonal antibody (Endogen, Inc., Boston, MA) was used to complex the antigen. This complex was detected colorimetrically by the enzymatic reaction between a goat antirabbit immunoglobulin (Ig)G conjugated to horseradish peroxidase (HRP) (Bio-Rad Laboratories, Hercules, CA) and 3,3′,5,5′-tetramethylbenzidine (TMB) (Moss, Inc., Pasadena, MD) as substrate. Samples were read by a Dynatech MRX plate reader at 450 nm and compared with rhIL-8 (R&D) using Revelation software (Dynatech Laboratories, Chantilly, VA).
The amount of IL-1RII was measured using a sandwich ELISA (36). Briefly, a monoclonal rat antihuman IL-1RII (0.25 μg/well) (Genzyme Diagnostics, Cambridge, MA) was used as the capture antibody and a polyclonal rabbit antihuman IL-1RII antibody was used to complex the antigen (provided by Dr. Mark Wewers). The complex was detected as described above, and samples were compared with human recombinant IL-1RII. Plates were read using Revelation software (Dynatech).
The amount of IL-1Ra was measured using a sandwich ELISA as previously described (37). Briefly, a rabbit polyclonal antihuman IL-1Ra against IL-1Ra was generated in Dr. Wewers's laboratory. This antibody was used as the capture antibody and a goat antihuman IL-1Ra (R&D) was used as the detecting antibody for the ELISA. Bound goat antibody was detected using a goat antirabbit IgG conjugated to HRP (Sigma Diagnostics) and developed with TMB (Moss, Inc.). Unknown samples were measured by comparison with rhIL-1Ra.
Total RNA for Northern blot analysis and reverse transcriptase–polymerase chain reaction (RT–PCR) was purified by a modification of the method of Chomczynski and Sacchi (38). Approximately 1 × 106 A549 or NHBE cells were resuspended in 1 ml Trizol Reagent (GIBCO BRL). Cells were lysed by repetitive pipetting, and RNA was extracted with chloroform and precipitated with isopropanol. The RNA pellet was washed twice in 75% ethanol, vacuum-dried, and resuspended in 20 μl of ribonuclease-free water. The total RNA concentration was measured by absorbance at 260/280 nm (GeneQuant II; Pharmacia BioTech, Piscataway, NJ). Purified RNA samples were maintained at −80°C until further use.
Ten or thirty micrograms per sample of total RNA taken from A549 or NHBE cells, respectively, was resuspended in RNA sample loading buffer at a 5:1 ratio (Sigma, St. Louis, MO). The RNA mixture was denatured at 65°C for 15 min, chilled on ice, and immediately loaded onto a 1% agarose/2.2 M formaldehyde gel. Following resolution, the ethidium bromide–stained samples were photographed and then blotted to nylon membranes (Nytran; Schleicher & Schuell, Keene, NH) by capillary transfer with 20× saline sodium phosphate EDTA (SSPE). The RNA was then immobilized by ultraviolet cross-linking (GS Gene-Linker; Bio-Rad). Northern blots were probed sequentially with α32P-labeled complementary DNA (cDNA) probes for human IL-8 (a generous gift of Genentech, Inc., South San Francisco, CA), human IL-1RII (a generous gift from Dr. Ueli Gubler, Hoffman LaRoche, Nutley, NJ), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion, Austin, TX) . Briefly, restriction fragments were generated from amplified plasmids encoding IL-8 (480 base pairs [bp]), IL-1RII (100 bp), and GAPDH (328 bp), resolved on a 1% agarose gel, isolated, and labeled by random priming (Strip-EZ DNA probe synthesis and removal kit; Ambion) with α-32P deoxyadenosine triphosphate (Easytides; NEN, Boston, MA). Hybridization was performed overnight at 42°C in 50% formamide, 5× SSPE, 0.1% sodium dodecyl sulfate (SDS), 2× Denhardt's solution, 100 μg/ml denatured salmon sperm DNA, and 1 to 5 × 106 cpm of labeled probe per milliliter of hybridization buffer. After hybridization, blots were washed twice at room temperature for 15 min each in 2× SSPE and 0.5% SDS, and once at 55°C for 1 h in 0.1× SSPE and 0.5% SDS. Autoradiography was performed overnight and quantitatively analyzed the following day using a Phospho-Imager (Molecular Dynamics, Sunnyvale, CA).
Total cellular RNA was isolated from both A549 and NHBE cell lines as described above. Starting with 2 μg of total RNA per sample, contaminating DNA was removed using deoxyribonuclease I (amplification grade; GIBCO BRL) at 2 U/20 μl reaction for 15 min at room temperature. This was followed by the addition of 2 μl of 25 mM EDTA and 2 μl random primers (50 ng/μl; GIBCO BRL) and heat inactivation (70°C for 10 min). The reaction was divided in half and 5× first-strand buffer (GIBCO BRL), 2 μl of 100 mM dithiothreitol, and 2 μl of 10 mM deoxynucleotide triphosphate (dNTP) mix were added to each. Superscript II (100 U; GIBCO BRL) was added to one of the two tubes and an equal volume of water was added to the other, for a final volume of 20 μl for each reaction. Reverse transcription was performed at 37°C for 1 h followed by heat deactivation at 70°C for 10 min. For PCR amplification, Taq Gold polymerase (0.5 U/reaction) (Perkin– Elmer, Foster City, CA), 10× reaction buffer (5 μl/reaction), 25 mM MgCl2 (3.2 μl/reaction), and 0.8 μl of 10 mM dNTP mix were combined with 10 μl of each RT reaction. Each reaction also contained primer pairs (2.5 × 10−3 μmol of each primer/50 μl reaction) for one of the following: primers D722-01 and D722-02 (5′-ATGCATCCTACACATACTTGG-3′ and 5′-CATCTGAAGCTTTTATTGGG-3′, respectively) were used to amplify a 555-bp fragment of IL-1RI; primers B912-01 and B912-02 (5′-CTCTGGAAGTTGTCAGGAGC-3′ and 5′-TGAGGCCATAGCACAGTCAG-3′, respectively) were used to amplify a 1,318-bp fragment of IL-1RII; and primers IL-1RAcP-5F and IL-1RAcP-6R (5′-GTGCCAGCTCCAAGATACACAGT-3′ and 5′-ATGAGCAGGGCCTCTCGTATTCAT-3′, respectively) were used to amplify a 665-bp fragment of IL-1RAcP. Amplification of β-actin message RNA with primers β-actin (+) and β-actin (−) (5′-GTGACGAGGCCCAGAGCAAGAG-3′ and 5′-AGGGGCCGGACTCATCGTACTC-3′, respectively) were used to amplify a 950-bp fragment as a positive control for each sample. For all amplifications, PCR was performed for 35 cycles (94°C × 1 min, 57°C × 1 min, and 72°C × 1 min) in a total volume of 50 μl. Identity of amplification products was confirmed by asymmetric restriction enzyme digestion. The IL-1RI RT–PCR products were digested with BamHI to yield the predicted 423- and 123-bp fragments and the IL-1RAcP RT–PCR product was digested with SstI to yield the predicted 393- and 272-bp fragments.
The full-length, transmembrane-coding, human IL-1RII cDNA was provided in the pEF-BOS mammalian expression vector by Dr. Ueli Gubler (Hoffman LaRoche). Using PCR, the full-length, membrane-associated IL-1RII (14) and a truncated, soluble form of the IL-1RII (amino acids 1–296) (15) were amplified. The PCR sample composition was as outlined above using the following primers: primers mIL1RII-F (5′-GGCAAGCTTCCACCATGTTGCGCTTGTACGTGTTG-3′) and mIL1RII-R (5′-GGCTCTAGAATCATCACTTGGGATAGGATTG-3′) were used for amplifying the membrane-bound IL-1RII construct. For the soluble IL-1RII construct, primers mIL1RII-F and sIL1RII-R (5′-GGCTCTAGAATCATTACTGGCGTGGCCCCTC-3′) were used. The primers contained a kozac consensus sequence (as underlined above) and unique 5′-HindIII and 3′-XbaI flanking sites. Following restriction digestion with HindIII and XbaI, amplified fragments were directionally subcloned into the pRc/CMV mammalian expression vector (Invitrogen, Carlsbad, CA). The membrane-associated IL-1RII–expressing construct (pRcCMV-mIIR) and the soluble IL-1RII–expressing construct (pRcCMV-sIIR) were transformed into E. coli (TOP 10F′; Invitrogen) by electroporation and amplified in LB medium (Difco Laboratories) containing ampicillin at a concentration of 100 μg/ml. After positive identification by restriction analysis, the plasmids were purified using the Endofree Giga Plasmid Prep buffer set (Qiagen, Santa Clarita, CA) according to the manufacturer's instructions.
The pRcCMV-mIIR, pRcCMV-sIIR, and pRcCMV control plasmids were then used to transiently transfect A549 cells using the transfection reagent TransIT-LT2 (PanVera Corp., Madison, WI). Transfections were carried out according to the manufacturer's instructions using previously optimized transfection conditions. Briefly, 3 × 105 A549 cells were plated per six-well plate in complete growth medium. The following day, 2 μg of pRcCMV-mIIR, pRcCMV-sIIR, or pRcCMV vector alone was mixed with 25 μl of the TransIT-LT2 reagent in Opti-MEM I serum-free medium (GIBCO BRL) and allowed to complex for 30 min at room temperature. During this time, the cells were rinsed twice with Opti-MEM I medium and then covered with 2 ml of serum-free medium. The complexed DNA was layered on top of the cells and incubated for 4 h at 37°C, 5% CO2. At this point, the medium was aspirated and replaced with 2 ml of complete growth medium. Cells were incubated an additional 72 h before the addition of IL-1β at a concentration of 50 pg/ml. Immediately before the addition of IL-1β, an aliquot of medium was retrieved for analysis of IL-1RII concentration by ELISA. Finally, transfected and control cells were incubated an additional 12 h in the presence of IL-1β before cell-free supernatants were removed and measured for IL-8 by ELISA.
All data are expressed as means ± SEM. Analysis of variance (ANOVA) with Tukey's honestly significant difference (HSD) testing was used to compare the IL-1β dose response in A549 and NHBE cells (Systat, Evanston, IL). Statistical significance was defined as P ⩽ 0.05. The independent sample t test was used to compare IL-1Ra concentrations in NHBE cells. Statistical significance was defined as P ⩽ 0.001.
Under standard culture conditions, A549 and NHBE cells were incubated for 24 h with increasing concentrations of IL-1β to determine their responsiveness with respect to IL-8 release. The amount of IL-8 in the resulting cell-free supernatants was measured by ELISA. Both A549 cells and NHBE cells were found to respond to IL-1β in the picomolar range (approximately 3 and 30 pmol, respectively; Figures 1a and 1b). In particular, A549 cells demonstrated a rapid and marked increase in IL-8 release when stimulated with as little as 50 pg/ml of IL-1β, whereas NHBE cells demonstrated a significant increase in IL-8 release at a dose of 500 pg/ml of IL-1β. When IL-1β–induced IL-8 release was measured as a function of time, A549 cells released an appreciable amount of IL-8 (3.81 ± 0.47 ng/ml) in as little as 4 h after the addition of IL-1β (50 pg/ml) (Figure 1c). The amount of IL-8 production continued to increase throughout the experiment. Time-response experiments were not performed with NHBE cells.
To ensure that endotoxin was not a source of contamination in our studies, similar experiments were performed in which A549 and NHBE cells were incubated with increasing doses of LPS (0 to 1 μg/ml). Neither cell type demonstrated an appreciable increase in IL-8 production over the background level, regardless of LPS dose (data not shown). Therefore, under the conditions tested, A549 and NHBE IL-8 release was not influenced by trace contamination of endotoxin present in culture medium or, in particular, plasmid DNA vectors used in subsequent studies.
Initially, A549 and NHBE cells were grown under standard culture conditions and studied for release of soluble IL-1RII into cell supernatants. NHBE cells were cultured using BEGM with and without hydrocortisone because previous studies demonstrated upregulation of soluble IL-1RII release in monocytes and neutrophils after treatment with dexamethasone (20, 39). Cells were also treated overnight with IL-1β as previously described, and were monitored for upregulation of IL-1RII protein expression. After overnight culture, cell-free supernatants and cell lysates were harvested and analyzed for IL-1RII protein by ELISA. Under all conditions studied, IL-1RII could not be detected in A549 or NHBE cells. As a positive control, IL-1RII release was measured from freshly isolated human PBMC treated overnight with LPS and dexamethasone as previously described (40). Soluble IL-1RII was detectable in the cell-free supernatants (Table 1). In addition, A549 cells were stimulated with IL-4, IFN-γ, and dexamethasone, and measured for IL-1RII release, because previous studies have shown release of IL-1RII by these factors (13, 16, 19-21). In each case, IL-1RII could not be detected in the cell-free supernatants (data not shown).
IL-1β | − | + | − | + | ||||||
---|---|---|---|---|---|---|---|---|---|---|
HC | − | − | + | + | ||||||
A549 | S | 0 | 0 | n.d. | n.d. | |||||
L | 0 | 0 | n.d. | n.d. | ||||||
NHBE | S | 0 | 0 | 0 | 0 | |||||
L | 0 | 0 | 0 | 0 | ||||||
PBMC | S | 0 | n.d. | 303 ± 26* | n.d. |
Expression of the type I icIL-1Ra has been demonstrated in NHBE and related lung cell lines as a cytosol-retained protein. However, recent evidence has demonstrated release of this molecule from human airway epithelial cells (13). We performed studies to determine the amount of type I icIL-1Ra retained in the cell and, in particular, the amount of IL-1Ra released into the extracellular compartment under our study conditions. To accomplish this, both A549 cells and NHBE cells were grown under standard culture conditions. A549 cells were stimulated with IL-1β (50 pg/ml), IL-4 (10 to 30 ng/ml), IFN-γ (250 and 500 U/ ml), and dexamethasone (1 × 10−7 M), whereas NHBE cells were stimulated with IL-1β alone (500 pg/ml). On the following day, cell-free supernatants and/or cell lysates were measured for IL-1Ra content by ELISA. In A549 cells stimulated with IL-1β, all of the IL-1Ra was detected in the cell lysate fraction of the sample (115 ± 5.4 pg/ml; Figure 2, upper panel), which was not influenced by IL-1β treatment (121 ± 19.3 pg/ml). On the basis of previous work by others, and because ELISA cannot differentiate between different isoforms, we presume this is the type I icIL-1Ra (12, 13). IL-1Ra was not detected in the supernatants of A549 cells stimulated with IL-4, IFN-γ, or dexamethasone (data not shown).
IL-1Ra was detected in supernatants from both unstimulated and IL-1β–stimulated NHBE cells (393 ± 26.4 and 591 ± 30.2 pg/ml, respectively); however, approximately 10 times the amount of IL-1Ra was detected in the cell lysates when compared with cell supernatants (6.2 ± 0.9 and 5.9 ± 0.4 ng/ml, respectively; Figure 2, lower panel). Similar to the results obtained from A549 cells, stimulation with IL-1β did not significantly alter the total IL-1Ra found in NHBE cells. However, IL-1β did induce a statistically significant increase in IL-1Ra release (P ⩽ 0.001). In comparison with A549 cells, NHBE cells produced significantly more IL-1Ra. Our results indicate that under our conditions, IL-1Ra remains largely cell-associated and is not released in high concentration in lung epithelial cells under native conditions or when stimulated with IL-1β.
Next, experiments were performed to assess the relative efficiency of known IL-1 inhibitors to prevent IL-1β–induced IL-8 release in airway epithelial cells. In our experiments, A549 cells were stimulated with IL-1β (50 pg/ml) that had been incubated previously with varying individual molar-excess doses and combinations of soluble IL-1 inhibitors, including IL-1RI, IL-1RII, and IL-1Ra. Following 24-h incubation, cell-free supernatants were collected and assayed for IL-8 release. Blocking with a 10- to 1,000-fold molar excess of soluble IL-1RI did not affect IL-1β–induced IL-8 production when compared with stimulation with IL-1β alone (10.6 ± 0.4 to 8.6 ± 0.3 with IL-1RI versus 9.8 ± 0.2 control; Figure 3a). Significant inhibition of IL-8 production was achieved with either a 1,000-fold molar excess of soluble IL-1RII (1.66 ± 0.32 ng/ml) or a 100-fold molar excess of IL-1Ra (1.66 ± 0.97 ng/ml). Our results demonstrate that a large molar excess of individual IL-1 inhibitors is necessary to inhibit IL-8 release. Furthermore, IL-1Ra is a more effective inhibitor of IL-1β–induced IL-8 release than is IL-1RII. Although IL-1RII and IL-1Ra treatment alone effectively inhibited IL-8 release, inhibition of chemokine release was not significantly enhanced by the combination of a 1,000-fold molar excess of soluble IL-1RII and a 100-fold molar excess of IL-1Ra (Figure 3c). In corroboration with previous results, when IL-1Ra and IL-1RI were combined, the ability of IL-1Ra to reduce the production of IL-8 was antagonized.
Similar results were obtained with studies involving NHBE cells; however, similar molar-excess doses of the soluble IL-1 inhibitors were more effective in preventing IL-8 release from NHBE cells than from the A549 cell line (Figure 3b). This may be because of the fact that cell lines generally have a greater number of cell-surface IL-1RI than do primary cells (1). Also, in contrast to the results obtained from A549 cells, inhibition of IL-8 production was seen when molar-excess doses of the soluble IL-1RI were added. In fact, 1,000-fold IL-1RI brought the level of IL-8 down nearly to that of the untreated control (0.37 ± 0.07 ng/ml versus 0.30 ± 0.17 ng/ml). Soluble IL-1RII and IL-1Ra once again were effective inhibitors. However, significant inhibition of IL-8 production was achieved with even less IL-1RII and IL-1Ra (100- and 10-fold, respectively) than needed with the A549 cell line. Similar to A549 cells, the addition of IL-1RI in combination with IL-1RII did not augment the ability to block release of IL-8 (data not shown). The addition of excess IL-1RI to IL-1Ra did, however, interfere with the ability of the receptor antagonist to block IL-1β signaling as the measured amount of IL-8 more than doubled when compared with a 100-fold molar excess of IL-1Ra alone (Figure 3d). As shown, a 10-to 100-fold molar excess of IL-1Ra was needed to inhibit IL-1β–induced IL-8 release; therefore, endogenous IL-1Ra release by NHBE cells (as shown in Figure 2b) was probably insufficient to inhibit IL-8 release. Together, these results indicate that local production of the IL-1 type II receptor and IL-1Ra, at a relative molar excess, inhibit the response of lung epithelial cells to IL-1β as measured by IL-8 release. The soluble IL-1RI, however, appears to contribute to a much lesser extent and, in fact, may interfere with IL-1β inhibition by antagonizing soluble IL-1Ra from binding to IL-1RI at the cell surface.
We postulated that decreased IL-8 release by A549 and NHBE cells treated with soluble IL-1 inhibitors resulted from direct competition with the native IL-1RI signal transducing receptor. As such, we anticipated that decreased IL-8 release was a direct consequence of decreased IL-1– mediated signal transduction and therefore IL-8 gene expression. To test this, Northern blot analysis was performed with total RNA isolated from A549 and NHBE cells. The cells had been treated overnight with IL-1β in the absence or presence of IL-1 inhibitors, as previously described. As expected, in both A549 and NHBE cells, an increase in IL-8 messenger RNA (mRNA) was detected in samples that had been treated with IL-1β compared with those that were not (Figure 4).
In both cell types, samples that had been stimulated with IL-1β plus soluble IL-1 inhibitors were also examined. In accordance with the results obtained from ELISA, there was not a significant decrease in IL-1β–induced IL-8 mRNA expression in A549 cells treated with a 100- to 1,000-fold molar excess of soluble IL-1RI (Figure 4). Incubation of A549 cells in the presence of IL-1β plus either a 1,000-fold molar excess of IL-1RII or 100-fold molar excess of IL-1Ra resulted in a significant reduction in IL-8 message expression when compared with IL-1β treatment alone. Similar results were obtained with NHBE cells that had been treated with IL-1RII or IL-1Ra (Figure 4). The results from Northern blot analysis correlate well with our previous studies showing a decrease in IL-8 protein release, and indicate that decreased IL-8 release by the addition of IL-1RII or IL-1Ra is due to inhibition of IL-1 signal activation and active gene transcription.
In addition, the same samples taken from A549 and NHBE cells were analyzed with a partial cDNA probe coding for the human IL-1RII (data not shown). Consistent with the lack of IL-1RII protein expression, mRNA for IL-1RII was not detected in any of the samples, whereas hybridization with a human GAPDH probe demonstrated a strong signal with similar intensity in all samples.
Preliminary analysis of A549 and NHBE cells revealed the absence of measurable IL-1RII. To confirm the absence of IL-1RII expression, RT–PCR analysis of the IL-1RII transcript was performed. RNA isolated from HepG2 cells, a cell line known to express the transmembrane form of the IL-1RII, was used as a positive control (41). Transcripts for IL-1RII could not be detected in either A549 or NHBE cells, regardless of culture conditions. Treatment with IL-1β or hydrocortisone produced similar results, suggesting that neither factor induced expression of IL-1RII (Figure 5). Primers for human β-actin served as a positive control for the RT reactions of each sample to demonstrate the integrity of the RNA. On the basis of our results, we conclude that both A549 and NHBE cells are defective in their ability to express the IL-1RII that contributes to their sensitive response to IL-1.
To elucidate further the role of IL-1β signaling in lung epithelium with respect to related IL-1 receptor family members, the same RNA samples were tested further by RT–PCR for the presence of both IL-1RI and IL-1RAcP. As expected, on the basis of previous experiments demonstrating exquisite sensitivity of A549 and NHBE cells to IL-1β, our results show that A549 and NHBE cells constitutively express mRNA for IL-1RI, as demonstrated by amplification of a DNA fragment at the correct size whose identity was confirmed by restriction enzyme digestion (Figure 5). In addition, an amplified DNA fragment corresponding to IL-1RAcP was observed in A549 and NHBE cells under both unstimulated and IL-1β–stimulated conditions. Our results confirm that A549 and NHBE cells respond to IL-1β at relatively low concentrations, which is partly because of constitutive expression of IL-1RI and IL-1RAcP.
Previous work has demonstrated that the glycosylation of inhibitory, soluble IL-1 receptors, along with the receptor location (extracellular versus membrane-bound), may influence their effectiveness in IL-1 blockade (42, 43). Therefore, despite limited transient transfection in the A549 cell line, we postulated that overexpression of the transmembrane and/or soluble IL-1RII protein by A549 cells may enhance the ability to downregulate IL-1β–mediated IL-8 release. To accomplish this task, mammalian expression vectors coding for the membrane-bound and soluble IL-1RII proteins designated pRcCMV-mIIR and pRcCMV-sIIR, respectively, were constructed in our laboratory and used to overexpress both receptor proteins transiently in A549 cells. In our preliminary experiments, cell-free supernatants and cell lysates taken from native and vector-only transfected A549 cells demonstrated the absence of any detectable IL-1RII protein by ELISA. The cell lysates and culture supernatants obtained from A549 cells transfected with pRcCMV-mIIR or pRcCMV-sIIR demonstrated a marked amount of IL-1RII within 24 h after transfection (Figure 6, upper panel).
Next, we performed the same transfection experiments, allowed the cells to remain in static culture for 3 d after transfection, and then incubated the cells and conditioned medium with IL-1β (50 pg/ml) for 12 h. Despite production of immunodetectable IL-1RII with both the pRcCMV-mIIR and pRcCMV-sIIR constructs, IL-1β–induced release of IL-8 was not inhibited (Figure 6b). Furthermore, transfection had a positive effect on IL-8 release in addition to that induced by IL-1, as demonstrated by the vector control (Figure 6, lower panel). In comparison with experiments already described, in which soluble IL-1RII was exogenously added to A549 cells to block IL-1β, native production of IL-1RII in transiently transfected A549 cells produced only a 3- to 5-fold molar excess of IL-1RII. This is well below the dose of recombinant IL-1RII needed to inhibit IL-8 release. Together, our results suggest that native production of the IL-1RII protein in a soluble or membrane-bound form does not significantly enhance the potency for IL-1 blockade.
IL-1β has been implicated as a central mediator of inflammation in a variety of lung disorders. Excessive production and release of IL-1β by the alveolar macrophage results in activation of surrounding parenchymal airway cells which, in turn, release potent chemokines, such as IL-8 and monocyte chemotactic protein-1, that are capable of recruiting inflammatory cells (1-5, 7). In addition to enhancing the recruitment of inflammatory cells to the airway, IL-1β can induce lung epithelial cells to release granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). Lung epithelial cell– derived GM-CSF and M-CSF can inhibit apoptosis of inflammatory cells present in the airway and enhance cell retention and therefore contribute to the progression of chronic lung disease (6-9). Furthermore, activation of epithelial cells by IL-1 can lead to secretion of growth factors that cause fibroblast proliferation, collagen production, and remodeling of the lower airway, all of which contribute to progressive lung disease (1, 22, 23). Our results confirm the exquisite sensitivity of both A549 and human primary bronchial epithelial cells to IL-1β. Furthermore, our results are the first to demonstrate that IL-1RI and IL-1RAcP, which are necessary for the amplification of the IL-1 signal, are expressed constitutively in lung epithelial cells. Coexpression of both factors results in amplification of the IL-1 signal in lung epithelial cells and a pronounced increase in IL-8 release. It is interesting to note that preliminary results obtained with NHBE cells demonstrate induction of IL-1RAcP expression with IL-1β treatment (data not shown), suggesting that IL-1β may enhance its own signaling in lung epithelium in a paracrine fashion. This work further supports IL-1β activation of lung epithelial cells as a critical pathway in the recruitment and retention of immune cells from the vasculature to the lung airway. However, it also identifies a relative deficiency in our understanding of extracellular IL-1β regulation in the airway microenvironment.
Currently, there are three known endogenous inhibitors of IL-1β that likely regulate IL-1 bioactivity in the airway. These factors include soluble IL-1RI, membrane-bound and soluble IL-1RII, and the IL-1Ra family of molecules. The soluble IL-1RI and both IL-1RII receptors bind IL-1β, IL-1α, and IL-1Ra but with distinctly different affinities, suggesting a distinct role for each molecule in IL-1 modulation (1). Soluble IL-1RI binds IL-1Ra with greatest affinity, followed by IL-1α and then IL-1β. In contrast, the type II receptor binds IL-1β with greatest affinity, followed by IL-1α and then IL-1Ra (1). Importantly, all studies with the IL-1RII protein isoforms have established its role as an inhibitory decoy receptor for IL-1 (1, 16, 19, 21, 22). Finally, IL-1Ra exists as three different functional species that can compete directly with IL-1 for binding to the IL-1 receptors (11). Most relevant to our studies is the type I icIL-1Ra, which has previously been reported to be expressed by lung epithelial cells (12) and can be released in response to various cytokines, therefore attenuating extracellular IL-1 activity (13). Our results demonstrate that each individual inhibitor, in a soluble recombinant form, can prevent IL-1β–induced IL-8 release in a consistent manner with both A549 and NHBE cells. IL-1Ra was a superior inhibitor of IL-1β activity, followed by soluble IL-1RII and then soluble IL-1RI, which had significantly less inhibitory activity. When paired combinations of the three inhibitors were studied in the presence of IL-1β, a lack of synergistic activity (particularly with IL-1Ra and IL-1RII) was noted. Furthermore, a reversal of IL-1Ra inhibitory activity occurred when IL-1RI was present. The latter finding is not surprising, and is consistent with previous investigations showing that soluble IL-1RI binds IL-1Ra, allowing unimpeded binding between IL-1β and the membrane-bound, signal-transducing IL-1RI protein (44, 45).
The ability of lung epithelial cells to express IL-1RII and, therefore, provide a mechanism to self-regulate IL-1β–induced activity was carefully examined. Our results consistently demonstrate a lack of IL-1RII expression by lung epithelial cells under all study conditions. This finding may further explain why lung epithelial cells are so responsive to IL-1β activation. Lung epithelial cells are defective in their ability to downmodulate the response to IL-1β extracellularly or, in particular, at the cell surface because of absent expression of IL-1RII. Previous work has demonstrated that other relevant factors, including endotoxin, tumor necrosis factor (TNF)-α, IL-4, IL-13, and glucocorticoids, induce shedding of soluble IL-1RII from neutrophils and mononuclear phagocytes (16, 19-21). It is important to note that constitutive expression of IL-1RII was detected in monocyte/macrophages and neutrophils before TNF-α, IL-4, and IL-13 treatment. With the exception of IL-1, dexamethasone, IL-4, IFN-γ, and endotoxin, we cannot exclude the possibility that other factors may induce IL-1RII expression in lung epithelial cells. However, constitutive expression of IL-1RII was clearly not present in lung epithelial cells. Our studies were performed in a cell line representative of type II alveolar epithelial cells (A549) and epithelial cells representing the upper airway space (NHBE); therefore, we also cannot exclude the possibility of IL-1RII expression by other lung parenchymal cells. However, analysis of BALFs obtained from healthy adult volunteers has consistently demonstrated a lack of IL-1RII protein as measured by ELISA (data not shown). In the context of the airway microenvironment, our results suggest that lung epithelial cells must rely on soluble IL-1RII shed from other sources in the lung airway to regulate their response to IL-1β.
Previous investigations have shown that lung epithelial cells produce an intracellular form of IL-1Ra. In our studies with A549 cells, IL-1Ra could be detected only in cell lysates, and production was not influenced by treatment with IL-1β. In NHBE cells, IL-1Ra was largely detected in the cell lysates; however, a small amount was found in the supernatants that did increase following treatment with IL-1β. As with A549 cells, the cumulative amount of IL-1Ra measured in the supernatants and cell lysates of NHBE cells was not increased following treatment with IL-1β. Furthermore, the amount of IL-1Ra detected in NHBE supernatants was significantly less than the concentration(s) used to inhibit IL-1 activity; therefore, native IL-1Ra had little to no effect on inhibiting IL-1β in the extracellular environment in our studies. Similar to previously described studies with IL-1RII, other factors present in the airway, including IL-4, IL-13, and IFN-γ, cause increased release of the type I icIL-1Ra from lung epithelial cells, thus providing a mechanism to regulate externally the lung epithelial cell response to IL-1β (13). However, in these studies the concentration of type I icIL-1Ra increased only 2- to 3-fold above baseline, characteristic of an acute-phase response. In contrast, our studies with a different but related cell line (A549) did not show an increase in type I icIL-1Ra release in cells treated with IL-4, IFN-γ, or dexamethasone. Therefore, release of type I icIL-1Ra by lung epithelial cells alone may not account for the substantial excess needed to prevent IL-1 signaling. Similar to soluble IL-1RII, increased IL-1Ra expression and release in the inflammatory airway may be dependent on other cellular sources, including neutrophils and monocytes, to regulate IL-1 activity in the lung (37, 46).
In summary, our results indicate that lung epithelial cells are defective in their ability to downmodulate the response to IL-1β because of a lack of IL-1RII production. Clearly, IL-1β is an important proinflammatory mediator of lung disease. In particular, the pleiotropic effects of this molecule on lung epithelial cells have been well documented. Therefore, IL-1 bioactivity must be tightly regulated in the airway microenvironment. The ability of lung epithelial cells to regulate their response to IL-1 in vivo may, in large part, be determined by the availability of soluble inhibitors—namely, IL-1Ra and IL-1RII—released by immune regulatory cells present in the airway, which include neutrophils, lymphocytes, and monocyte/macrophages. Furthermore, our results suggest that a relative abundance of each inhibitor is required to inhibit effectively IL-1β engagement of the signal-transducing receptor complex. With regard to extracellular IL-1 bioactivity in the lung microenvironment, the relative abundance of each IL-1 response modifier and the local concentration of IL-1β determine the overall pro- or anti-inflammatory effect.
This research was supported by a grant from the American Lung Association of Ohio (D.L.K.), and National Institutes of Health grants HL56336 (D.K.W.) and HL40871 (M.D.W.). One author (K.R.C.) is supported by an R35 award from the NHLBI. The authors give special thanks to Alissa Winnard for assistance with construction of primers used in PCR experiments.
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Abbreviations: bronchial epithelial growth medium, BEGM; base pairs, bp; complementary DNA, cDNA; ethylenediaminetetraacetic acid, EDTA; enzyme-linked immunosorbent assay, ELISA; fetal bovine serum, FBS; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; interferon, IFN; interleukin, IL; IL-1 receptor antagonist, IL-1Ra; IL-1 receptor accessory protein, IL-1RAcP; IL-1 type I receptor, IL-1RI; IL-1 type II receptor, IL-1RII; lipopolysaccharide, LPS; messenger RNA, mRNA; normal human bronchial epithelial, NHBE; peripheral blood mononuclear cells, PBMC; recombinant human, rh; reverse transcriptase–polymerase chain reaction, RT–PCR; sodium dodecyl sulfate, SDS; saline sodium phosphate EDTA, SSPE; type I intracellular IL-1Ra, type I icIL-1Ra.