Long pentraxin 3 (PTX3), an acute-phase protein, is a newly clarified mediator for innate immunity and inflammation. As a soluble pattern recognition receptor, it has a nonredundant role in antifungal infection. Overexpression of PTX3 worsens acute lung injury. The lung epithelium is a critical factor in defense against pulmonary pathogens; it is also involved in acute inflammatory responses related to tissue injury. However, very little is known about how PTX3 is regulated in the lung epithelium. In this study, we found that i.v. injection of LPS induced PTX3 expression in rat lung alveolar epithelium. Using human lung cell lines and primary epithelial cells, we found that PTX3 expression was significantly up-regulated by TNF-α in a time- and dose-dependent manner, but not by LPS. Pretreatment with either actinomycin D or cycloheximide abolished TNF-α-induced PTX3 expression, indicating the requirement for both transcriptional and translational regulation. The TNF-α-induced PTX3 expression was blocked by SP600125, a JNK-specific inhibitor, but not by the inhibitors against NF-κB, ERKs, or p38 MAPK. Knockdown of either JNK1 or JNK2 with small interfering RNA also significantly reduced the regulated PTX3 expression. Thus, lung epithelial cells appear to be a major local source for PTX3 production, which could be induced in vivo from these cells by LPS or other inflammatory stimuli, and may be an important mediator for host defense and tissue damage. The importance of the JNK pathway for the regulated PTX3 expression may be a potential target for its regulation in the lung.

With breathing, the respiratory tract is frequently contacted with potentially infective microorganisms and noxious substances in the air. An effective defense system is crucial for protecting the lung from pathogen invasion (1). Located at the boundary between the environment and internal tissues, lung epithelial cells are an important component of the host defense. Both airway and alveolar epithelial cells function not only as a physical barrier but also as biological sensors for invading microorganisms and their products, by producing cytokines, chemokines and other inflammatory mediators (2, 3). These soluble factors, together with infiltrated immune cells, may initiate local inflammatory reactions, to eliminate the invaded pathogens, dilute the pathogen products, limit the damages, and eventually lead to recovery (4). However, excess of inflammation may induce persistent tissue injury and cause morbidity and mortality (4). Acute respiratory distress syndrome, for example, is a major cause of death for critically ill patients and acute inflammatory responses are a major underlying mechanism (5).

Evidence has accumulated that lung epithelial cells can produce a host of inflammatory mediators, such as MCP-1, IL-8, TNF-α, and IL-6, in response to a variety of inflammatory stimuli (6). Further identifying these factors and their regulatory mechanisms will help us understand the role of lung epithelial cells in host defense and acute inflammatory responses. Using microarray, we have recently reported a TNF-α-induced acute inflammatory response pattern in human alveolar epithelial A549 cells, including up-regulation of multiple genes encoding chemokines, proinflammatory mediators, and membrane-associated proteins (7). One of these genes is pentraxin 3 (PTX3)2 (also called TNF-α-stimulated gene 14), which encodes the first long pentraxin identified.

Pentraxins are a superfamily of conserved proteins, characterized by a cyclic multimeric structure and a conserved C-terminal domain. Classic pentraxins, such as C-reactive protein and serum amyloid P, are acute-phase proteins that are rapidly activated in responses to inflammation and play a role in immunity by regulating innate resistance to microbes and scavenging of cellular debris and components of extracellular matrix (8). PTX3 shares similarity with the classic pentraxins in the C-terminal domain but has an unrelated N-terminal sequence (9, 10). Unlike classic pentraxins made in the liver, PTX3 is produced by macrophages and a variety of tissue cells upon exposure to primary inflammation signals, such as TNF-α, IL-1β, and LPS (11). PTX3 levels are very low in serum and tissues of normal subjects but are rapidly increased in response to inflammatory stimulation with a wide range of diseases, including infectious, autoimmune, and degenerative disorders (12, 13, 14). The circulating level of PTX3 was elevated in critically ill patients, with an increasing level from systemic inflammatory response syndrome to septic shock (15). Clinical tests suggest that the elevation of PTX3 level could be a sensitive marker for early diagnosis and prognosis of some severe illnesses, such as acute myocardial infarction (16, 17).

PTX3 plays a nonredundant role in antifungal immune responses; PTX3-deficient mice were more susceptible to invasive pulmonary aspergillosis (14). Dias et al. (18) generated transgenic mice that carried multiple copies of PTX3 gene under the control of its own promoter. Exposing these transgenic mice to LPS induced PTX3 overexpression, which significantly improved survival (18). In contrast, when these animals were exposed to intestinal ischemia-reperfusion injury, overexpression of PTX3 increased mortality and inflammatory responses in the intestine and the lungs (19). Furthermore, PTX3 up-regulated tissue factor expression in human endothelial cells and activated monocytes (20). PTX3 also inhibited phagocytosis of apoptotic neutrophils by dendritic cells (21) and macrophages (22), suggesting its importance for innate immunity and inflammatory responses.

Because PTX3 may play an important role in the host defense and overexpression of PTX3 may contribute to acute lung injury, we have studied the mechanisms by which an early proinflammatory cytokine, TNF-α, induces PTX3 production from lung epithelial cells.

Recombinant human TNF-α and IL-1β were purchased from BioSource. NF-κB inhibitors pyrolidine dithiocarbamate (PDTC) and caffeic acid phenylethyl ester (CAPE), JNK inhibitor SP600125, MEK-1 inhibitor PD98059, and p38 MAP kinase inhibitor SB203580 were purchased from Calbiochem. Escherichia coli-derived LPS (026:B6), actinomycin D, and cycloheximide were from Sigma-Aldrich. Abs against NF-κB p65 subunit, IκBα, and PTX3 were from Santa Cruz Biotechnology. Abs against phosphorylated p42/44 ERKs, p38 MAP kinase, and JNK were purchased from Cell Signaling Technology. Polyclonal Ab against GAPDH was from Trevigen. The small interfering RNA (siRNA) was designed using a siRNA Target Finder program (Ambion), with the sense sequences of GCAAGCGUGACAACAAUUUdtdt against JNK1, and CCUUCACUGUCCUAAAACGdtdt against JNK2, respectively. The siRNAs and the noneffect control dsRNA (GAAUCCGCUGAUAAGUGACdtdt) were synthesized by Dharmacon.

Under anesthesia (80 mg/kg ketamine and 8 mg/kg xylazine, i.p. injection), male Sprague-Dawley rats (300–350 g body weight; four animals per group; Charles River Laboratories) were subjected to a tracheotomy and catheterized (14 gauge). The animals were subjected to mechanical ventilation with tidal volume of 6 ml/kg, positive end-expiratory pressure of 5-cm H2O, 50–60 breaths/min, and FiO2 (35%) (Flexi Vent; SCIREQ). The right carotid artery was cannulated (24 gauge) for arterial blood pressure measuring, blood sampling, and resuscitation. The tail vein was catheterized (22 gauge) for LPS injection and maintaining anesthesia (ketamine, 20 mg/kg/h; xylazine, 4 mg/kg/h; and pancronium, 0.3 mg/kg/h). After basal line measurements of arterial blood pressure and PaO2, the endotoxic shock was introduced by injection of LPS (5 mg/kg) into the tail vein. One hour later, animals were resuscitated with Ringer’s solution (mean ± SD, 18.2 ± 1.3 ml) for 30 min. The sham group of animals underwent the same surgical procedure and ventilation without LPS challenge and resuscitation. The mean arterial blood pressure, PaO2, peak airway pressure, and lung elastance were recorded every 30 min (23). At the end of experimentation, the left lung tissues were fixed with 10% Formalin for histology and immunohistochemistry staining. Frozen tissues from the right lung were used for RNA extraction and gene expression assay. The animal care and experimental protocol was approved by the Animal Care Committee of the Toronto General Research Institute.

Human alveolar type II-like epithelial (A549) cells (American Type Culture Collection (ATCC)) were grown in DMEM with 10% FBS (13). Human bronchial epithelial BEAS-2B cells (ATCC) were cultured in a 50/50 mixture of DMEM and F-12 nutrient mixture (DMEM/F-12; Invitrogen Life Technologies) supplemented with 10% FBS, 2 mM l-glutamine, and 1 mM HEPES (24). Primary human alveolar epithelial type II cells were isolated as previously described (25). The isolated human alveolar type II epithelial cells were cultured in DMEM/F-12 medium with 10% FBS and 2 mM l-glutamine (Invitrogen Life Technologies). Primary human small airway epithelial cells were purchased from Cambrex and were cultured in Small Airway Epithelial Cell Growth Medium supplemented (in 500 ml) with bovine pituitary extract (2 ml), hydrocortisone (0.5 ml), recombinant human epithelial growth factor (0.5 ml), epinephrine (0.5 ml), transferrin (0.5 ml), insulin (0.5 ml), retinoic acid (0.5 ml), triiodothyronine (0.5 ml), gentamicin (0.5 ml), and (fatty acid free) BSA (5 ml) (Cambrex). Human U937 monocytes (ATCC) were incubated in RPMI 1640 medium (Invitrogen Life Technologies) with 10% FBS and human microvascular endothelial cells (HMEC-1; Centers for Disease Control) were cultured in MCBD 131 medium supplement with 10% FBS and epidermal growth factor (10 ng/ml).

All cells were seeded in 6-well culture plates and cultured at 37°C, 5% CO2, in a humid atmosphere. The confluent cells were then cultured in serum-free medium overnight, rinsed once before treatment with TNF-α, IL-1β, or LPS at indicated concentrations for designated time. For inhibition studies, cells were preincubated with designated inhibitor for 30 min, and then treated with TNF-α or LPS in the presence of the inhibitor.

For siRNA studies, A549 cells were seeded at low density (105 cells/well) overnight in DMEM with 10% FBS. The cells at 30–50% confluence were transfected with siRNA against JNK1, JNK2, or control RNA at 25 nM using Oligofectamine following the manufacturer’s instruction (Invitrogen). The transfected cells were further grown for 48 h, and then challenged with TNF-α.

Once the treatment was completed, the supernatants of the culture media were collected from each well for measuring PTX3 protein. The cells were lysed at 4°C, either in TRIzol reagent (Invitrogen Life Technologies) for RNA extraction (1 ml/well), or in a lysis buffer (Tris-HCl (50 mM) (pH 7.5), NaCl (150 mM), EGTA (2 mM), EDTA (2 mM), 1% Triton X-100) containing aprotinin (10 μg/ml), leupeptin (10 μg/ml), PMSF (1 mM), Na3VO4 (1 mM), and NaF (10 mM) for collecting the protein (300 μl/well). All collected samples were stored at −80°C before further processing.

Quantitative real-time PCR was performed as described previously (7). In brief, total RNA was extracted from cells or lung tissues lysed in TRIzol reagent (Invitrogen Life Technologies), using an RNeasy Mini kit (Qiagen). High-quality RNA extracted (5 μg/sample) was used for synthesis of single-strand cDNA with Superscript II (Invitrogen Life Technologies). Real-time PCR was conducted with 45 ng of cDNA using an ABI Prism 7900HT PCR machine (Applied Biosystems). Primers were designed using the Primer Express 1.5 software (Applied Biosystems) and synthesized by ACGT Corp. The sequences of primers were as follows: PTX3 forward primer, 5′-GGGACAAGCTCTTCATCATGCT-3′; reverse primer, 5′-GTCGTCCGTGGCTTGCA-3′; and hydroxymethylbilane synthase (HMBS), a housekeeping gene, forward primer, 5′-TGAGCAAAGGAGCCAAAAACA-3′; reverse primer, 5′-AACCAGTTAATGGGCATCGTTAAG-3′.

The PTX3 released into the culture medium was measured using an ELISA kit from Alexis, following the manufacturer’s instructions with minor modification. In brief, the ELISA plates (Nunc) were coated with a monoclonal anti-human PTX3 Ab (700 ng/ml) in coating buffer (15 mM carbonate-bicarbonate buffer (pH 9.6)) overnight at 4°C. Then, at room temperature, the plates were blocked with 5% dry milk in washing buffer (0.05% Tween 20 in PBS (pH 7.2)) for 2 h, and incubated with either recombinant human PTX3 standards or the samples collected in duplicate (50 μl/well) for another 2 h. The plates were then incubated with a polyclonal anti-human PTX3 Ab conjugated with biotin (25 ng/ml) for 1 h, and freshly diluted (1/4000) streptavidin-HRP (Amersham Biosciences) for 30 min subsequently. After each step, the plates were washed four times with the washing buffer. A chromogen substrate tetramethylbenzidine (Pierce) was added (100 μl/well) and incubated for 15 min in dark. The reaction was stopped by adding of 2 M H2SO4 (100 μl/well), and the plates were read at 450 nm with an automatic ELISA reader (ThermoLabsystems).

Immunoblotting was performed as previously described (26). Briefly, cells lysates containing equal amounts of total protein were boiled with SDS sample buffer (0.06 M Tris (pH 8.0), 10% (v/v) glycerol, 2% (w/v) SDS, 5% (v/v) 2-ME, 0.0025% (w/v) bromphenol blue), and subjected to SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with 5% dry milk, and probed with the specific Ab indicated. The blots were visualized with an ECL detection kit from Amersham Biosciences. The membrane was stripped and reprobed with another Ab if desired.

Immunofluorescent staining was done as described previously (26). Briefly, the cells after treatment were fixed in 3.7% formaldehyde for 10 min, permeabilized with 0.25% Triton X-100 for 5 min, and then stained with NF-κB p65 Ab (0.5 μg) for 60 min and the FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch) at 1/1000 dilution (v/v) for 30 min in dark. Slides were examined under a fluorescent microscope (Nikon). Immunohistochemical staining of PTX3 in rat lung tissues was performed with a PTX3 pAb (1/50 dilution) and a Vectastain ABC (avidin-biotin complex)-AP kit (Vector Laboratories), following the protocol provided. Positive staining was visualized with Vector red. Slides were then counterstained with 1% methyl green for 10 s. The specificity of staining was determined by replacing the primary Abs with nonimmunized IgG isoforms (Sigma-Aldrich), respectively.

The in situ hybridization was conducted by a staff member of the core facility in our institute. Briefly, total RNA was isolated from LPS-treated rat lung tissue using TRIzol reagent (Invitrogen). RT-PCR was performed using a forward primer containing T7 promoter sequence (5′-TAATACGACTCACTATAGGGCTGAGACCTCCT-3′) and a reverse primer containing SP6 promoter sequence (5′-ATTTAGGTGACACTATAGATGGGAAGAAAATCG-3′). A 511-bp PCR product was purified, confirmed by DNA sequencing, and used as a template for in vitro transcription with a DIG RNA labeling system (Roche Diagnostics). The antisense RNA was made with SP6 polymerase and the sense RNA was generated with T7 polymerase. The frozen rat lung tissue was fixed in 4% paraformaldehyde. Frozen sections (7 μm) were permeabilized with protease K (5 μg/ml), and incubated with digoxigenin-labeled RNA probes (250 ng/ml) at 55°C overnight. The signal was amplified with alkaline phosphatase-conjugated anti-digoxigenin Ab (Roche) and colored with NBT and 5-bromo-4-chloro-3-indolyl-phosphate. The slides were counterstained with nuclear fast red and photographed by a pathologist not knowing the experimental groups.

Data are expressed as mean ± SD from at least three experiments and analyzed by one-way ANOVA followed by a Student-Neuman-Keuls test with significance defined as p < 0.05.

To determine whether an acute inflammatory challenge would induce PTX3 expression from lung epithelial cells, adult rats were challenged with LPS (5 mg/kg). LPS challenge induced a significant drop of blood pressure within 1 h. Following resuscitation, the blood pressure returned to the baseline but showed a time-dependent decrease afterward (Fig. 1,A). LPS challenge also induced a significant decrease in PaO2 (Fig. 1,B) and a profound increase of lung elastance (Fig. 1,C), both in a time-dependent fashion. Histological study showed mild hemorrhage and infiltration of inflammatory cells in alveoli, signs of early pathological changes of acute lung injury (Fig. 1 D; H&E) in LPS-challenged animals.

FIGURE 1.

LPS challenge induces PTX3 expression in rat lung epithelial cells in vivo. Adult rats were injected with LPS (5 mg/kg) i.v., followed by resuscitation and life-supporting ventilation. A, Mean arterial blood pressure. B, Artery blood PO2. C, Change of lung elastance. D, Representative slides from LPS-treated or sham-operated animals for lung pathology (H&E), PTX3 immunohistochemistry (IHC), and in situ hybridization (ISH). In the H&E staining panel, closed arrows and open arrowheads indicate the mild hemorrhage and infiltrated cells, respectively. The PTX3 IHC-positive staining (pink color) was seen mainly along the epithelial layer of alveolar walls. The PTX3 ISH-positive staining (dark blue) was similar to that of IHC (closed arrows: possible type II epithelial cells; open arrowheads: alveolar macrophages).

FIGURE 1.

LPS challenge induces PTX3 expression in rat lung epithelial cells in vivo. Adult rats were injected with LPS (5 mg/kg) i.v., followed by resuscitation and life-supporting ventilation. A, Mean arterial blood pressure. B, Artery blood PO2. C, Change of lung elastance. D, Representative slides from LPS-treated or sham-operated animals for lung pathology (H&E), PTX3 immunohistochemistry (IHC), and in situ hybridization (ISH). In the H&E staining panel, closed arrows and open arrowheads indicate the mild hemorrhage and infiltrated cells, respectively. The PTX3 IHC-positive staining (pink color) was seen mainly along the epithelial layer of alveolar walls. The PTX3 ISH-positive staining (dark blue) was similar to that of IHC (closed arrows: possible type II epithelial cells; open arrowheads: alveolar macrophages).

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The mRNA and protein levels of PTX3, TNF-α, and IL-1β were significantly increased in the LPS-treated lung tissues as determined by real-time RT-PCR or ELISA (data not shown). Using immunohistochemistry, we found a marked increase of PTX3 protein expression in the lungs from LPS-challenged animals, in comparison with that from sham controls. The PTX3-positive staining (pink color) was found mainly in the epithelial layer of the alveolar wall (Fig. 1,D; PTX3 IHC). The PTX3 gene expression in the lung tissues was determined by in situ hybridization. Similarly, the majority of positively stained cells (dark blue) were found along the alveolar wall with cuboidal shaped cells located at the corner of alveolar units, which may represent type II pneumocytes. A few cells inside the alveolar spaces were also positively stained that may be alveolar macrophages (Fig. 1 D; PTX ISH). Slides stained with sense probe had no staining (data not shown). These results demonstrated that PTX3 expression can be rapidly increased in lung epithelial cells in vivo in response to a proinflammatory stimulus.

In a recent microarray study, we found that TNF-α, but not LPS, induced PTX3 gene expression in human alveolar epithelial A549 cells (7). The opposite results from in vivo and in vitro studies promoted us to further examine the effects of these proinflammatory stimuli on PTX3 production from alveolar epithelial cells. We also extended our observation to airway epithelial cells. Confluent A549 and BEAS-2B cells were treated with TNF-α (20 ng/ml) for 4 h, and PTX3 gene expression was determined by quantitative real-time PCR. TNF-α significantly increased PTX3 gene expression in both cell types (Fig. 2,A). To determine whether the up-regulated PTX3 gene expression leads to an increase in PTX3 protein production, we measured PTX3 released into the culture medium. In addition to A549 and BEAS-2B cell lines, we also challenged primarily cultured human alveolar type II cells and small airway epithelial cells. TNF-α significantly increased PTX3 release from all of these cells (Fig. 2 B).

FIGURE 2.

TNF-α induces PTX3 gene expression and protein production in human lung epithelial cells. A, A549 and BEAS-2B cells were treated with either TNF-α (20 ng/ml) or LPS (10 μg/ml) for 4 h. PTX3 gene expression was detected by real-time PCR and expressed as a ratio against a housekeeping gene, HMBS. Data plotted are the mean ± SD from three independent experiments. B, A549 cell, primary human alveolar type II cells (ATII), BEAS-2B cells, and primary human small airway epithelial cells (SAEC) were treated as described above. C, A549, U937, and HMEC-1 cells were challenged with TNF-α and LPS as described above. PTX3 released into the culture medium was determined with ELISA and expressed as mean ± SD from three to four independent experiments (∗, p < 0.05; and ∗∗, p < 0.01 vs control). D, The responses of A549, U937, and HMEC-1 cells to TNF-α and LPS are expressed as fold of change compared with their controls.

FIGURE 2.

TNF-α induces PTX3 gene expression and protein production in human lung epithelial cells. A, A549 and BEAS-2B cells were treated with either TNF-α (20 ng/ml) or LPS (10 μg/ml) for 4 h. PTX3 gene expression was detected by real-time PCR and expressed as a ratio against a housekeeping gene, HMBS. Data plotted are the mean ± SD from three independent experiments. B, A549 cell, primary human alveolar type II cells (ATII), BEAS-2B cells, and primary human small airway epithelial cells (SAEC) were treated as described above. C, A549, U937, and HMEC-1 cells were challenged with TNF-α and LPS as described above. PTX3 released into the culture medium was determined with ELISA and expressed as mean ± SD from three to four independent experiments (∗, p < 0.05; and ∗∗, p < 0.01 vs control). D, The responses of A549, U937, and HMEC-1 cells to TNF-α and LPS are expressed as fold of change compared with their controls.

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We also challenged the cells with LPS for 4 h, but did not find an effect of LPS on either PTX3 gene expression (Fig. 2,A) or protein production (Fig. 2 B), even though we increased the dose of LPS from 1 μg/ml, the dose used in our previous study (7), to 10 μg/ml. We also repeated some of these experiments with 10% human serum to provide autologous soluble CD14 and soluble MD2, or in serum-free condition. Results were similar to that when cells were cultured with FBS (data not shown). We challenged A549 cells with IL-1β (4 ng/ml; 4 h). Similar to TNF-α treatment, IL-1β induced a significant increase of PTX3 gene and protein expression (data not shown). These results suggest that LPS-induced PTX3 in vivo is an indirect effect. In most of the subsequent studies, we used A549 cells to further elucidate the regulatory mechanisms of TNF-α-induced PTX3 expression in human lung epithelial cells.

We further compared PTX3 production in A549 cells with that in human U937 monocytes and HMEC-1 endothelial cells, in response to either TNF-α or LPS. Under the same stimulatory conditions, U937 cells responded to LPS more effectively than to that of TNF-α, whereas HMEC-1 cells responded to both TNF-α and LPS challenges similarly (Fig. 2,C). Although higher levels of PTX3 protein were observed in U937 and HMEC-1 cells (Fig. 2,C), the fold of increase of PTX3 was the highest in A549 cells in response to TNF-α stimulation than that in U937 and HMEC-1 cells (Fig. 2 D). Considering epithelial cells as one of the major cell types in the lung, PTX3 produced from this source may play an important role in inflammatory process.

We further studied the time and dose effects of TNF-α on PTX3 expression. An increase in PTX3 gene expression was found with TNF-α (20 ng/ml) stimulation, with the peak response at 4 h, which was reduced after 8 h (Fig. 3,A). We then stimulated cells with different concentrations of TNF-α for 4 h, and the peak of PTX3 gene expression was observed at 10 ng/ml TNF-α (Fig. 3,B). TNF-α-induced PTX3 protein release appears to be accumulated within the first 24 h treatment, and then remained at the peak level for at least another 12 h (Fig. 3,C). During a 24-h incubation period, 10–20 ng/ml TNF-α induced the maximum release of PTX3 (Fig. 3 D).

FIGURE 3.

Dose- and time-dependent regulation of PTX3 by TNF-α. A549 cells were treated with TNF-α (20 ng/ml) for various periods (A and C) or treated with different concentrations of TNF-α for 4 h (B) or 24 h (D). PTX3 gene expression was measured with real-time PCR (A and B), and PTX3 release was determined with ELISA (C and D), respectively. Data from three experiments are shown as mean ± SD.

FIGURE 3.

Dose- and time-dependent regulation of PTX3 by TNF-α. A549 cells were treated with TNF-α (20 ng/ml) for various periods (A and C) or treated with different concentrations of TNF-α for 4 h (B) or 24 h (D). PTX3 gene expression was measured with real-time PCR (A and B), and PTX3 release was determined with ELISA (C and D), respectively. Data from three experiments are shown as mean ± SD.

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To determine the transcriptional and/or translational regulation of PTX3 induced by TNF-α, cells were pretreated with either actinomycin D to block transcription, or with cycloheximide to inhibit the de novo protein synthesis. In the presence of actinomycin D (5 μg/ml), both the gene expression (Fig. 4,A) and protein release (B) of PTX3 induced by TNF-α were completely abolished. When cells were treated with cycloheximide (10 μg/ml), TNF-α-induced PTX3 protein release was completely blocked (Fig. 4 B), whereas the TNF-α-induced increase in PTX3 mRNA was not affected (A). Actinomycin D or cycloheximide treatment alone had no effect on basal levels of PTX3 mRNA and protein (data not shown). No cytotoxic effect was noted on cell viability at the concentrations used in the present study, as determined by XTT assay (data not shown). These results reveal de novo production of PTX3 in human lung epithelial cells induced by TNF-α.

FIGURE 4.

TNF-α-induced de novo expression of PTX3. A549 cells were pretreated with either actinomycin D (Act D; 5 μg/ml) or cycloheximide (CHX, 10 μg/ml) for 30 min, and then treated with TNF-α (20 ng/ml) for 4 h. PTX3 gene expression (A) and protein production (B) were determined as described above, and the mean ± SD from three experiments were plotted (∗, p < 0.05; ∗∗, p < 0.01 vs control).

FIGURE 4.

TNF-α-induced de novo expression of PTX3. A549 cells were pretreated with either actinomycin D (Act D; 5 μg/ml) or cycloheximide (CHX, 10 μg/ml) for 30 min, and then treated with TNF-α (20 ng/ml) for 4 h. PTX3 gene expression (A) and protein production (B) were determined as described above, and the mean ± SD from three experiments were plotted (∗, p < 0.05; ∗∗, p < 0.01 vs control).

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Multiple transcription factor binding elements (including AP-1, NF-κB, and NF-IL-6) have been identified in the promoter region of PTX3 gene, and NF-κB activation is crucial for TNF-α-induced PTX3 expression in endothelial cells and fibroblasts (27, 28). The activation of NF-κB pathway leads to the degradation of an inhibitor of NF-κB complex, IκBα; NF-κB subunits are freed from the complex in cytosol and move into the nucleus. We examined the translocation of NF-κB p65 subunit with immunofluorescent staining and the integrity of IκBα with Western blotting in the cells. TNF-α (20 ng/ml) induced a rapid NF-κB activation demonstrated by a rapid nuclear translocation of p65 and reduction of IκBα within 10 min (Fig. 5,A). However, preincubating cells with specific NF-κB inhibitors, either PDTC (10 μM) or CAPE (25 μM), had no significant effect on TNF-α-induced PTX3 gene expression (Fig. 5,B) and protein production (Fig. 5,C), even though PDTC effectively blocked TNF-α-induced IκBα degradation (Fig. 5 D). These experiments suggest that, although NF-κB was activated by TNF-α, it may not be responsible for TNF-α-induced PTX3 expression in human lung epithelial cells.

FIGURE 5.

TNF-α-induced NF-κB activation is not involved in regulated PTX3 expression. A, A549 cells were treated with TNF-α (20 ng/ml) for different times, and then were subjected to immunofluorescent staining to locate NF-κB p65, or subjected to immunoblotting to determine IκBα integrity. TNF-α induced p65 translocation from cytoplasm into the nucleus, and degradation of IκBα. Pretreatment of A549 cells with PDTC (10 μM) or CAPE (25 μM) did not significantly block PTX3 gene expression (B) and protein release (C) induced by TNF-α (20 ng/ml; 4 h). Data shown are the mean ± SD from three independent experiments. D, Representative blots show that PDTC blocked IκBα degradation induced by TNF-α (20 ng/ml; 10 min). GAPDH blot was used for loading control (∗, p < 0.05 vs all other groups).

FIGURE 5.

TNF-α-induced NF-κB activation is not involved in regulated PTX3 expression. A, A549 cells were treated with TNF-α (20 ng/ml) for different times, and then were subjected to immunofluorescent staining to locate NF-κB p65, or subjected to immunoblotting to determine IκBα integrity. TNF-α induced p65 translocation from cytoplasm into the nucleus, and degradation of IκBα. Pretreatment of A549 cells with PDTC (10 μM) or CAPE (25 μM) did not significantly block PTX3 gene expression (B) and protein release (C) induced by TNF-α (20 ng/ml; 4 h). Data shown are the mean ± SD from three independent experiments. D, Representative blots show that PDTC blocked IκBα degradation induced by TNF-α (20 ng/ml; 10 min). GAPDH blot was used for loading control (∗, p < 0.05 vs all other groups).

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One of the major downstream pathways for TNF-α-induced cell activation is MAPKs, which play an important role in cells for inflammatory response. TNF-α treatment rapidly increased phosphorylation of ERKs within 10 min, which reached the maximum at 30 min (Fig. 6,A). Increased phosphorylation of p38 MAPK was observed as early as 1 min after TNF-α treatment (Fig. 6,A). TNF-α-induced phosphorylation of ERKs and p38 MAPK was completely blocked by preincubating cells with PD98059 (10 μM) or SB203580 (10 μM), specific inhibitors for ERKs or p38 MAPK pathway, respectively (Fig. 6,D). However, neither PTX3 gene expression (Fig. 6,B) nor its protein production (Fig. 6 C) induced by TNF-α was significantly blocked by these inhibitors.

FIGURE 6.

Blocking ERKs and p38 MAPK did not affect TNF-α-induced PTX3 expression. A, TNF-α (20 ng/ml) induced phosphorylation of ERKs and p38 MAPK, with GAPDH as loading control, determined by immunoblotting. Incubation of A549 cells with 10 μM PD98059 (PD), an MEK-1 inhibitor, or SB203580 (SB), a p38 MAPK inhibitor 30 min before TNF-α challenge (20 ng/ml; 4 h) did not significantly block PTX3 gene expression (B) and protein production (C). The mean ± SD from three independent experiments are shown. D, TNF-α-induced increase in ERKs and p38 phosphorylation at 10 min was blocked by pretreatment of cells with PD98059 or SB203580, respectively (∗, p < 0.05 vs all other groups).

FIGURE 6.

Blocking ERKs and p38 MAPK did not affect TNF-α-induced PTX3 expression. A, TNF-α (20 ng/ml) induced phosphorylation of ERKs and p38 MAPK, with GAPDH as loading control, determined by immunoblotting. Incubation of A549 cells with 10 μM PD98059 (PD), an MEK-1 inhibitor, or SB203580 (SB), a p38 MAPK inhibitor 30 min before TNF-α challenge (20 ng/ml; 4 h) did not significantly block PTX3 gene expression (B) and protein production (C). The mean ± SD from three independent experiments are shown. D, TNF-α-induced increase in ERKs and p38 phosphorylation at 10 min was blocked by pretreatment of cells with PD98059 or SB203580, respectively (∗, p < 0.05 vs all other groups).

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Another member of MAPKs is JNK, one of the major downstream pathways for a variety of extracellular stresses. It has been shown that TNF-α induced JNK activation in human endothelial cells (29). In human lung epithelial cells, TNF-α treatment also increased JNK phosphorylation within 10 min (Fig. 7,A). Inhibition of JNK activation by preincubation of cells with SP600125 (25 μM), a specific JNK inhibitor (Fig. 7,D), completely abolished TNF-α-induced PTX3 gene expression (Fig. 7,B) and protein production (Fig. 7,C). JNKs consist of several isoforms, JNK1, JNK2, and JNK3, which may function differently. To further identify which isoform is necessary for TNF-α-induced PTX3 production, we incubated cells with siRNA specifically designed for JNK1 or JNK2 for 48 h. Each siRNA selectively reduced the targeted JNK isoform (Fig. 7,E). No cross-effect was observed between siRNAs against JNK1 and that against JNK2, suggesting that the siRNAs we developed are specific (Fig. 7,E). PTX3 released from the cells transfected with siRNA against either JNK1 or JNK2 was partially but significantly reduced, in comparison with that from the cells without transfection, or transfected with nonspecific control RNA (Fig. 7 F). These results strongly suggest that JNK is the major pathway for TNF-α-induced expression of PTX3 in human lung epithelial cells, and activations of both JNK1 and JNK2 are involved in mediating PTX3 production.

FIGURE 7.

TNF-α-induced PTX3 expression is mediated through JNK pathway. A, TNF-α (20 ng/ml) increased JNK phosphorylation, with GAPDH as loading control. JNK-specific inhibitor SP600125 (25 μM for 30 min) completely blocked TNF-α-induced PTX3 gene expression (B) and protein production (C). Data are the mean ± SD from three independent experiments (∗∗, p < 0.01 vs control). D, Pretreatment of cells with SP600125 blocked TNF-α-induced JNK phosphorylation at 10 min. E, Specific knockdown of JNK1 and JNK2 at protein levels by siRNA (25 nM). F, siRNA specific for either JNK1 or JNK2 reduced TNF-α-induced PTX3 protein release. Data plotted are the mean ± SD from three independent experiments (#, p < 0.05 vs all other groups; ∗∗, p < 0.01 vs cells treated with TNF-α alone or cells treated with TNF-α plus nonspecific RNA (NS)).

FIGURE 7.

TNF-α-induced PTX3 expression is mediated through JNK pathway. A, TNF-α (20 ng/ml) increased JNK phosphorylation, with GAPDH as loading control. JNK-specific inhibitor SP600125 (25 μM for 30 min) completely blocked TNF-α-induced PTX3 gene expression (B) and protein production (C). Data are the mean ± SD from three independent experiments (∗∗, p < 0.01 vs control). D, Pretreatment of cells with SP600125 blocked TNF-α-induced JNK phosphorylation at 10 min. E, Specific knockdown of JNK1 and JNK2 at protein levels by siRNA (25 nM). F, siRNA specific for either JNK1 or JNK2 reduced TNF-α-induced PTX3 protein release. Data plotted are the mean ± SD from three independent experiments (#, p < 0.05 vs all other groups; ∗∗, p < 0.01 vs cells treated with TNF-α alone or cells treated with TNF-α plus nonspecific RNA (NS)).

Close modal

The importance of PTX3 in the innate immune response as a soluble pattern recognition receptor and in the acute inflammatory responses as a potential mediator has drawn increasing attention. Although intensive studies have been done with transgenic animals (14, 18, 19), little is known about its regulatory mechanisms in the lung. Our results demonstrated that PTX3 can be induced in and released from lung epithelial cells by proinflammatory cytokines directly and by LPS indirectly. LPS-induced PTX3 production may participate in inflammatory regulation and contribute to lung injury by affecting tissue factor production (20, 30), complement activation (31), and clearance of apoptotic cells (21). Increased PTX3 has been reported in serum collected from critically ill patients (15). The early increase of PTX3 expression in lung epithelial cells could be one of the important sources for this molecule, which should be further studied as a potential biomarker for acute lung injury and multiorgan failure.

In comparison with classical pentaxins, one of the important features of PTX3 is that it is produced by macrophages, monocytes, and a variety of tissue cells; thus it may be important for the local inflammation. It has been shown that PTX3 can be induced in human fibroblasts (10) and endothelial cells (9, 32, 33) by TNF-α, IL-1β, or LPS (34). LPS also induced PTX3 in mouse peritoneal macrophages, but TNF-α or IL-1β failed to do so (35). The conidia of Aspergillus fumigatus induced PTX3 in human or mouse mononuclear phagocytes and dendritic cells, and to a lesser extent in endothelial cells, and had almost no effect on epithelial cells, fibroblasts, and lymphocytes (14). In our recent microarray study, PTX3 gene was found to be induced by TNF-α in alveolar epithelial A549 cells (7). In another microarray study with human bronchial epithelial BEAS-2B cells, PTX3 was listed among many other genes induced after Bordetella pertussis challenge (36). In the present study, we have found that human endothelial cells responded to both TNF-α and LPS. In contrast, in U937 cells, TNF-α induced less PTX3 than LPS did, which is opposite to what was observed in A549 cells. Therefore, the regulated expression of PTX3 in each cell type depends on the specific stimulus. Despite the lower basal level in A549 cells, PTX3 was more dramatically increased upon TNF-α stimulation in comparison with that in monocytes and endothelial cells. PTX3 production was induced in primary human type II pneumocytes and distal airway epithelial cells in response to TNF-α stimulation. IL-1β also induced PTX3 in A549 cells. Therefore, epithelial cells could be one of the major cell types producing PTX3 in the lung during acute inflammation, which could be an important host response to infections and inflammatory stimuli.

PTX3 production in lung epithelial cells is regulated at both transcriptional and posttranscriptional levels. TNF-α-induced PTX3 gene expression and protein production were completely abolished by pretreatment with actinomycin D, a potent transcription inhibitor. The presence of cycloheximide, an inhibitor for protein synthesis, abolished TNF-α-induced PTX3 release, but not the mRNA elevation. This is similar to the observation in human fibroblasts (10) but in contrast to the effect of cycloheximide in reducing LPS-induced PTX3 mRNA in macrophages (35). Therefore, the requirement of de novo protein synthesis for PTX3 gene expression also depends on the specific inflammatory stimulus and cell type.

It has been reported that the NF-κB is necessary for TNF-α- or IL-1β-induced PTX3 responses in human fibroblasts (28). NF-κB is also involved in LPS-induced PTX3 in macrophages (35). In the present study, NF-κB pathway was activated in lung epithelial cells; however, blocking NF-κB with its inhibitors did not abolish TNF-α-induced PTX3 expression (Fig. 5). The IFN-γ-mediated inhibitory effect on PTX3 expression induced by TNF-α or LPS in mouse macrophages was also NF-κB independent (35). Because multiple potential enhancer binding elements, such as NF-IL-6, AP-1, and Sp1, have been noted in PTX3 promoter (27, 28), the regulation of PTX3 gene expression through the binding elements, other than NF-κB site, should be further investigated.

In the present study, we noted that all three MAPK pathways were activated by TNF-α in lung epithelial cells. Pretreatment with specific inhibitors demonstrated that TNF-α-induced PTX3 expression was mainly through the JNK pathway, but not p42/44 ERKs and p38 MAP kinase. JNK has been shown to regulate expression of gene products involved in stress-related events (37). Using siRNA specifically against JNK1 or JNK2, we demonstrated that reducing the protein levels for each of them partially but significantly decreased TNF-α-induced PTX3 production. This NF-κB-independent and JNK-dependent regulation of PTX3 may be useful for selective regulation of PTX3 for therapeutic interventions.

PTX3 gene expression was induced by LPS in murine fibroblasts and peritoneal macrophages (35). We have observed no induction of PTX3 expression in both airway and alveolar epithelial cells in response to LPS challenge in vitro. It is known that A549 cells are hyporesponsive to LPS challenge (7). However, LPS did induce TNF-α (38) and MIP-2 (39) in primary cultured rat pneumocytes. Primary cultured human pneumocytes express TLR4 and responded to LPS stimulation to produce IL-8 (40). BEAS-2B cells also responded to LPS stimulation and produced GM-CSF and IL-8 (41). Therefore, the lack of PTX3 production suggests that LPS is not a direct inducer of this protein in human lung epithelial cells.

PTX3 gene expression was induced by LPS in mice in a variety of organs (42). In the present study, LPS also induced PTX3 gene expression in rat lungs. We further demonstrated that the expression of PTX3 gene and protein is along the epithelial layer of alveolar wall. We have found rapid increase of TNF-α and IL-1β gene and protein expression in the lung tissue after LPS challenge (data not shown). It is possible that these cytokines are intermediate mediators for LPS-induced PTX3 expression in lung epithelial cells in vivo. Indeed, we have previously reported that TNF-α is involved in LPS-induced MIP-2 production from rat lung pneumocytes (39).

In summary, lung epithelial cells could be an important source for PTX3 production. Pathogen products, such as LPS, may induce PTX3 expression through proinflammatory cytokines as mediators. The importance of PTX3 produced from lung epithelial cells in host defense and acute lung injury merits further investigation. Compared with known NF-κB-dependent PTX3 expression from other cell types, the JNK-dependent PTX3 expression in human alveolar epithelial cells may indicate the complex regulation of this molecule from different cell types by different stimuli.

We gratefully acknowledge Dr. Giuseppe Peri (Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy) for suggestions on PTX3 ELISA, and Dr. Jing Xu (University Health Network, Toronto, Ontario, Canada) for performing in situ hybridization.

The authors have no financial conflict of interest.

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.

2

Abbreviations used in this paper: PTX3, pentraxin 3; PDTC, pyrolidine dithiocarbamate; CAPE, caffeic acid phenylethyl ester; siRNA, small interfering RNA; HMEC, human microvascular endothelial cell.

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