Clara cell secretory protein (CCSP) is an inhibitor of secretory phospholipase A2. It is produced by airway epithelial cells and is present in airway secretions. Because interferon (IFN)-γ can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis. A human bronchial epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was detected by ribonuclease protection assay. IFN-γ was found to increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-γ (300 U/ml) treatment for 8–36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-γ induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on, CCSP reporter gene activity assay, and CCSP mRNA half-life assay demonstrated that IFN-γ-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The present study demonstrates that IFN-γ can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.
- airway inflammation
- airway secretion
clara cell secretory protein (CCSP) (10), also designated Clara cell 10-kDa protein (CC10) (37), Clara cell 16-kDa protein (3), Clara cell 17-kDa protein (41), or polychlorinated biphenyl (PCB)-binding protein (20), was first named from the apparent molecular mass of this protein in nonreducing SDS-PAGE (37). Recent evidence (4) indicates it is also identical to human urinary protein-1. CCSP protein consists of two identical subunits of 70 amino acids joined by two disulfide bonds (2, 24, 41). The cDNAs and the derived amino acid sequences of human CCSP show striking homology with the rat and murine CC10 and rabbit uteroglobin (1, 11, 30, 34, 35, 39). The CCSP in rat and human lungs is the counterpart of rabbit uteroglobin in these species (22). CCSP is expressed in many nonrespiratory organs (27, 43) and also is highly expressed in airway epithelial cells (10,12, 33, 36, 37). Although the CCSP gene (12, 43), amino acid sequence (14, 26, 35), and distribution (27, 37) of this protein are known, its physiological function is still unclear. Because a markedly similar protein, uteroglobin, is reported to have immunosuppressive, anti-inflammatory, antiproteinase, anti-phospholipase A2(PLA2), and progesterone-binding activities (6, 22, 34), it is postulated that the CCSP gene product might also be an immunomodulatory or protective protein in the airway. Mice homozygous for deletion of the uteroglobin gene have increased serum PLA2 activity and develop glomerulonephritis (45). In addition, CCSP can bind methylsulfonyl PCBs (20, 26) to inhibit secretory PLA2activity.
Epithelia provide a protective barrier for many organs exposed to the external environment. It is now recognized that the pulmonary epithelium actively participates in the inflammatory response and is capable of releasing various mediators, which have the potential to modulate local inflammatory reactions (40). The airway epithelium may play an active role in initiating and modulating airway inflammation (31). CCSP expression is not limited to Clara cells of the airway; instead, CCSP may be expressed by cells along the conducting airway. Therefore, we were interested in studying CCSP gene expression and translation in human bronchial epithelial cells.
Interferons (IFN-α, IFN-β, and IFN-γ) are a family of potent multifunctional cytokines produced in the course of viral infections or during the inflammatory response. In addition to their antiviral properties, IFNs are also known to induce a variety of other physiological responses including antiprotozoal, antiproliferation, and immunoregulatory activities (17, 28, 32). IFN-γ exerts its biological functions via a distinct cell-surface receptor (42). Human airway epithelial cells express IFN-γ receptors and respond to IFN stimulation with increased expression of intercellular adhesion molecule-1, and, similarly, BEAS-2B cells, an immortalized human airway epithelial cell line, respond to IFN-γ stimulation with increased intercellular adhesion molecule-1 expression (19). IFN-γ also induces PLA2 activity, PLA2 gene expression, and protein synthesis in a variety of cell lines (29, 44). An IFN-γ response element has been identified in the 5′ promoter region of the murine CCSP gene (21). Because IFN-γ can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis.
Cell culture. BEAS-2B cells, a human bronchial epithelial cell line transformed by an adenovirus 12-SV40 virus hybrid, were a gift from Dr. J. E. Lecher and C. Harris (National Cancer Institute, Bethesda, MD). The cells were maintained in serum-free, hormonally defined culture medium (LHC-8, Biofluids, Rockville, MD) and grown in 175-cm2 tissue culture flasks (Nalge Nunc, Naperville, IL) that were precoated with a thin layer of rat tail type I collagen (Collaborative Research, Bedford, MA). Experiments were performed when the cells reached 80% confluence (∼3 million cells/flask).
Ribonuclease protection assay. The cells were treated with IFN-γ (10, 100, 300, and 1,000 U/ml; Boehringer Mannheim, Indianapolis, IN) for 4–36 h. Total cellular RNA was extracted from 175-cm2culture flasks by the single-step guanidinium thiocyanate-phenol-chloroform extraction method (Tri-Reagent, Molecular Research, Cincinnati, OH). The RNA pellet was precipitated with isopropanol and washed with 70% ethanol, then redissolved in diethyl pyrocarbonate water. To construct the probe for CCSP mRNA, a 367-bp product of CCSP cDNA was amplified by PCR with the following sets of sense and antisense primers: 5′ primer, 5′-CTCCACCATGAAACTCGCTG-3′ (bases 1–20) and 3′primer, 5′-GAAGAGAGCAAGGCTGGTGG-3′ (bases 367–348) (Bio-Synthesis, Lewisville, TX). The product of the CCSP gene (bases 1–367) was cloned into the TA cloning vector (Invitrogen, San Diego, CA). Orientation of the insert was determinated by DNA sequencing with the dideoxynucleotide chain termination method. The CCSP and glyceradehyde-3-phosphate dehydrogenase (GAPDH; Ambion, Austin, TX) RNA probes were prepared by in vitro transcription using T7 polymerase with [α-32P]CTP. A ribonuclease protection assay (RPA) kit (RPAII, Ambion) was used for the following procedures. Hybridization was performed at 45°C for 16 h and with 10 (for GAPDH) or 50 (for CCSP) μg of total RNA and 104 (for GAPDH) and 2 × 104 (for CCSP) counts/min of the32P-labeled RNA probe. After hybridization, the unhybridized RNA was digested by the addition of diluted (1:150) RNase A-T1 mix at 37°C for 60 min. Digestion was terminated by the addition of an RNase inactivation and precipitation mixture. The protected RNA fragment was analyzed by autoradiography after separation on 6% polyacrylamide-8 M urea gels.
Immunoblot of CCSP protein. The BEAS-2B cells were grown in 175-cm2 flasks and treated with IFN-γ (300 U/ml; Boehringer Mannheim); the cell culture media of control and treated BEAS-2B cells were collected after IFN-γ treatment for 8 and 18 h. After the culture media were dialyzed with a 3,500-molecular-weight membrane (Baxter, McGaw Park, IL) against distilled water, 100 ml of the supernatant were concentrated to 0.5 ml by lyophilization. Total protein was assayed with bicinchoninic acid (BCA Reagent, Pierce, Rockford, IN). Samples containing 10 μg of total protein were separated on 18% polyacrylamide gels (Novex, San Diego, CA) under reducing conditions for 5 h. Another experiment was done to verify the specific effect of IFN-γ by treating the BEAS-2B cells with IFN-γ (300 U/ml; Boehringer Mannheim), interleukin (IL)-4 (40 ng/ml), and IL-13 (10 ng/ml; R&D Systems, Minneapolis, MN); the cell culture media of control and treated BEAS-2B cells were collected after treatment with the different cytokines for 18 h. After dialysis with a 3,500-molecular-weight membrane (Baxter) against distilled water, 25 ml of the culture media were concentrated to 1 ml by lyophilization. Total protein was assayed with the BCA Reagent (Pierce). Samples containing 25 μg of total protein were separated on 18% polyacrylamide gels (Novex) under reducing conditions for 5 h. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH), then blocked with 5% nonfat dry milk overnight. CCSP protein expression was detected by using a 1:500 dilution of a rabbit anti-human CC10 polyclonal antibody (37) (a gift from Dr. Gurmukh Singh, Veterans Affairs Medical Center, Pittsburgh, PA) followed by a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG as the second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and developed with the enhanced chemiluminescence Western blotting detection system (Amersham, Arlington Heights, IL).
Nuclear run-on assay. Nuclear run-on assay was performed with a modification of previously described methods (9, 15). The cells were stimulated with IFN-γ (300 U/ml) for 0, 0.5, 2, and 4 h. The cells were harvested after digestion with 0.1% collagenase in Hanks’ balanced salt solution without Ca2+or Mg2+ [HBSS(−)] for 5–10 min. After being washed with cold HBSS(−), the cell pellet was resuspended with 4 ml of lysis buffer (10 mM Tris-buffered saline, 0.5% Nonidet P-40, 100 μg/ml of leupeptin, 50 μg/ml of aprotinin, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonyl fluoride), incubated on ice for 5 min, and centrifuged at 500g for 5 min. The nuclear pellet was resuspended in 1 ml of lysis buffer and spun at 500g for 5 min. The nuclei were resuspended with 200 μl of reaction buffer [10 mM Tris ⋅ HCl, pH 8.0; 5 mM MgCl2; 300 mM KCl; 1 mM dithiothreitol; 0.5 mM each ATP, CTP, and GTP; and 200 μCi of [α-32P]UTP (3,000 Ci/mmol; Amersham)] and then reacted at 30°C for 1 h. RNA was extracted by the single-step guanidinium thiocyanate-phenol-chloroform extraction method with the addition of 100 μg of yeast transfer RNA. The samples were resuspended to equal counts per minute per milliliter (5–6 × 106 dpm/ml) in hybridization buffer (50 mM PIPES, pH 6.8, 10 mM EDTA, 600 mM NaCl, and 0.2% SDS). The samples were allowed to hybridize to denatured DNA targets (10 μg) that were slot blotted on nitrocellulose filters at 65°C for 40 h after prehybridization at 70°C for 2 h in hybridization buffer containing 1% SDS. The DNA targets included the linearized plasmid PCR II containing human cytosolic PLA2(cPLA2) cDNA as a positive control, CCSP cDNA and GAPDH as an internal control, or the plasmid PCR II as a negative control. At the end of the hybridization period, the filters were washed in 2× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0)-0.2% SDS, 1× SSC-0.2% SDS, and 0.5× SSC-0.2% SDS, respectively, at 65°C for 1 h and evaluated by autoradiography.
Plasmid construction for reporter plasmids. Analysis of the nucleotide subsequences of the 5′ portion of the CCSP gene was performed in MacVector Version 5.0.2. The 5′-flanking sequences of the CCSP gene were generated by PCR from a human genomic DNA library with primers derived from a published sequence (EMBL accession no. X59875). A common 24-mer 3′ primer was synthesized for all the constructs, which corresponds from bases +31 to +13 of the sequence of human CCSP gene and contains a Bgl II restriction site at its 5′-end (5′-GAAGATCTTCTCTGGTTCCGTTCTCTG-3′). Individual 5′ primers were constructed to generate various deletion constructs. Different 24-mer 5′ primers were synthesized, which contain a 5′Kpn I site and started at base −801 (5′-GGGGTACCAGAATAAACATCTAAAGA-3′) and base −168 (5′-GGGGTACCTGGGGACAGAAACTGGGT-3′), respectively. The different truncated promoters (bases −801 to +31 and −168 to +31) were cloned upstream from the firefly luciferase coding region in the pGL3-basic vector (Promega, Madison, WI).
Transient transfection of reporter plasmids. BEAS-2B cells were maintained at 37°C under 5% CO2 in LHC-8 medium (Biofluids). For each transfection experiment, the cells were seeded in six-well plates and transfected with 2 μg of reporter plasmid DNA with Lipofectamine Reagent (Life Technologies, Gaithersburg, MD) when the cells reached 80% confluence. As a control for transfection efficiency, 0.2 μg of a secreted placental alkaline phosphatase (SEAP) plasmid (pCMV/SEAP Vector; Tropix, Bedford, MA) was added. After a 2-h transfection period, the cells were placed in culture medium and incubated for 16 h. The cells were then treated with IFN-γ (300 U/ml) for 8–18 h, the transfected cells were harvested and lysed, and the extracts were evaluated with luciferase assay reagent (Promega) in a luminometer (model 2010, Analytical Luminescence Laboratories, Ann Arbor, MI). SEAP activity was assayed in culture media with a SEAP assay kit (Tropix).
Measurement of CCSP mRNA half-life in BEAS-2B cells. BEAS-2B cells were grown in 175-cm2 flasks for the determination of CCSP mRNA half-life in control cells or in cells after IFN-γ stimulation. Control cells or cells stimulated with IFN-γ (300 U/ml) for 18 h before the addition of actinomycin D (50 μg/ml; Calbiochem, San Diego, CA) were harvested for RNA at 0, 8, 18, and 24 h after the addition of actinomycin D. Total RNA was prepared, and RPAs were performed as described in Ribonuclease protection assay. The protected fragments were quantitated with a densitometer (Molecular Dynamics, Sunnyvale, CA). The quantity of CCSP mRNA was normalized to the amount of GAPDH by calculating a CCSP-to-GAPDH ratio for each sample. All time points were performed in triplicate.
Statistical analysis of the mRNA half-life was performed by ANOVA in Microsoft Excel, Version 5.0.
BEAS-2B cells produce CCSP mRNA and IFN-γ increases CCSP steady-state mRNA levels. To determine whether BEAS-2B cells produce CCSP mRNA, steady-state levels of CCSP mRNA were measured by RPA of total cellular RNA extracted from cells that were incubated without or with IFN-γ (300 U/ml) for 4–36 h. As shown in Fig. 1, these cells produce CCSP mRNA, and the steady-state level of CCSP mRNA is significantly induced by IFN-γ treatment over 8–36 h.
To determine a dose-related effect of IFN-γ on the changes in steady-state CCSP mRNA, cells were incubated with 10–1,000 U/ml of IFN-γ for 24 h. Total cellular RNA was extracted, and an RPA for CCSP mRNA was performed. IFN-γ in concentrations of 100–1,000 U/ml induced a dose-related change in CCSP mRNA (Fig.2).
IFN-γ induces CCSP protein production and release in BEAS-2B cells. To determine the effect of IFN-γ treatment on the level of CCSP synthesized and released into the cell culture medium, BEAS-2B cells were treated with IFN-γ (300 U/ml) for 8 and 18 h, the time points at which CCSP mRNA levels significantly increased after IFN-γ stimulation. The culture media were concentrated and subjected to polyacrylamide gel electrophoresis followed by Western blotting. IFN-γ treatment increased the release of CCSP immunoreactive material into the supernatant at both 8 and 18 h (Fig. 3). This material had an apparent molecular mass of ∼7 kDa on polyacrylamide gel electrophoresis.
Additional experiments were performed to determine whether IFN-γ has a specific effect on CCSP synthesis and release. BEAS-2B cells were treated with IFN-γ (300 U/ml) or with the TH2 cytokine IL-4 (40 ng/ml) or IL-13 (10 ng/ml) for 18 h, the time point at which CCSP mRNA levels significantly increased after IFN-γ stimulation. The culture media were concentrated and subjected to polyacrylamide gel electrophoresis followed by Western blotting. IFN-γ treatment increased the release of CCSP immunoreactive material into the cell culture medium at 18 h; neither IL-4 nor IL-13 treatment had an effect (Fig. 4).
IFN-γ regulates CCSP gene expression at posttranscriptional level. To further understand the mechanism involved in CCSP gene regulation by IFN-γ, three studies were performed. The investigation of CCSP promoter activation by IFN-γ was done by transfecting two different constructs that contained the upstream sequence of the CCSP promoter into BEAS-2B cells, followed by stimulation with IFN-γ. Although both reporter genes exhibited promoter activity, IFN-γ treatment of cells for 8 or 18 h resulted in no increase in luciferase activity above baseline (Fig. 5). Second, control of CCSP steady-state mRNA levels was studied with a transcriptional assay. An in vitro transcriptional assay was performed after IFN-γ treatment for 0.5–4 h. Although the cPLA2 gene transcriptional rate was increased after treatment for 0.5–4 h, no increase was observed in the nuclear transcription rate of the CCSP gene over the period studied (Fig. 6). Therefore, both assays did not demonstrate changes in CCSP mRNA transcription. Because these two assays for transcription did not suggest transcriptional control of the observed changes in CCSP mRNA, the half-life of CCSP mRNA was studied. Total RNA was extracted from control and IFN-γ-stimulated cells treated with the inhibitor of RNA synthesis, actinomycin D, as described inmethods. CCSP mRNA levels were analyzed by RPA. As shown in Fig. 7, IFN-γ-stimulated cells displayed a prolonged stability of CCSP mRNA compared with the unstimulated control cells. While the calculated half-life of CCSP mRNA in control cells was ∼15 h, the calculated half-life of CCSP mRNA in the IFN-treated cells was prolonged to ∼40 h (P < 0.01). Therefore, in airway epithelial cells, IFN-γ increased the steady-state level of CCSP mRNA, at least in part, at a posttranscriptional level.
The expression of CCSP protein in the airway epithelium has been previously reported (10, 11, 27, 34, 37). Human CCSP protein has striking structural and sequence similarities with the rat and murine CCSPs and the uteroglobin gene product in the rabbit. The physiological role(s) of CCSP is still unclear. The human CCSP protein has been found to have several biological properties including inhibition of PLA2 activity (22, 23, 34), binding of PCBs, and reduction of foreign protein antigenicity by forming a complex with transglutaminase (22). It has been reported that CCSP has a clear inhibitory effect on PLA2 activity in lung fibroblasts in vitro (16). CCSP was able to inhibit fibroblast chemotaxis in vitro by mechanisms that may be related to the inhibition of PLA2 activity. Significantly higher levels of CCSP have been observed in bronchoalveolar lavage fluid from patients with acute lung injury (13). The inhibition of PLA2 by CCSP may be important in controlling extracellular inflammatory activity in the lung. Inhibition of PLA2 activity might result in a reduction in the metabolism of arachidonic acid and decrease the production of lipid mediators of inflammation.
The results of our experiments indicate that human bronchial epithelial cells in culture contain detectable levels of CCSP mRNA and can synthesize and secrete CCSP. The secretion of CCSP protein is induced by IFN-γ but not by the TH2 cytokines IL-4 and IL-13. For Western blot studies, we have used an antibody utilized in a previous study of human CCSP (37). In our studies, the secreted CCSP migrated on SDS-PAGE under reducing conditions with an apparent molecular mass of ∼7 kDa. This is in agreement with the Clara cell protein size reported by Bernard and colleagues (3, 4). They analyzed CCSP by electron spray-mass spectrometry to determine the extact size of this protein. Their results suggested that the CCSP has a 15.8-kDa molecular mass and is 7.8 kDa after reduction of the protein.
IFN treatment may alter the rates of synthesis and the steady-state level of a variety of cellular mRNAs and the corresponding proteins (32). Although IFNs can increase transcription rates within minutes after exposure of the cells to IFN and the steady-state level of IFN-inducible mRNAs may be primarily regulated at the transcriptional level, posttranscriptional regulation may also be involved (8, 38). Examples of posttranscriptional regulation of mRNA have been well documented, particularly at the level of mRNA stability (18, 25, 28). Friedman et al. (8) found that the mRNA of one of the genes they cloned from IFN-γ-induced T98G neuroblastoma cells (designated mRNA 1–8) continued to accumulate after 8–12 h of IFN-γ stimulation, whereas the transcription rate began to fall after 4–6 h. Our study showed that IFN-γ may alter the rate of synthesis of CCSP protein at a posttranscriptional level. We observed that an increase in steady-state CCSP mRNA paralleled the increased protein level in human bronchial epithelial cells after IFN-γ stimulation. Although there are two areas in the 5′ promoter region of the CCSP gene that might be putative [IFN-γ response element consensus sequence sites (5) at base −758 (10 of 14 nucleotides) and at base −148 (10 of 14 nucleotides)], the reporter gene constructs (bases −801 to +31 and −168 to +31) contained both of these sites, and the luciferase activity measured in the different reporter gene constructs did not increase after IFN-γ treatment. Our data also showed an increase in CCSP mRNA after 8–36 h of treatment with IFN-γ despite no change in the transcription rate after IFN-γ stimulation. This suggests that the stimulatory effects of IFN-γ on human CCSP gene expression in BEAS-2B cells is controlled, at least in part, at the posttranscriptional level.
Dierynck et al. (7) found that CCSP potentially inhibits IFN-γ production and biological activity in peripheral blood mononuclear cells. We observed that recombinant human IFN-γ can stimulate CCSP gene expression and protein synthesis in cultured human bronchial epithelial cells. Therefore, IFN-γ may modulate CCSP production, and CCSP may feed back to modulate IFN-γ production by peripheral blood lymphocytes. This loop may represent another example of cytokine modulation of epithelial cell function and epithelial cell modulation of the inflammatory response in the lung.
Airway epithelial cells are increasingly recognized for their ability to participate in the regulation of local inflammatory and immune responses. Our present study demonstrates that IFN-γ can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.
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