Nasal polyposis is defined as a chronic inflammatory disease of the upper airways, with the polyps originating from the paranasal sinus mucosa. The etiology and pathogenesis of nasal polyposis are still understood poorly. As airway epithelial cells are the first physical barrier against airborne particles containing bacteria or lipopolysaccharide (LPS) from the commensal flora in the nasopharynx, or from the environment, the continuous exposure to these agents may initiate chronic immune inflammatory responses. It was reported recently that airway epithelial cells could produce several chemokines, including IL-8, RANTES, and eotaxin, which contribute directly or indirectly to the local accumulation of inflammatory cells (Momoi et al.,1999; Bals and Hiemstra,2004). Nasal mucosal epithelia are also part of the upper airway epithelial wall, which should share the same immunopathogenesis mechanism as other airway epithelial cells. However, the function of nasal mucosal epithelia in the formation of nasal polyps remains unclear.
IL-18, originally identified as IFN-γ-inducing factor, is first produced as a 24-kDa inactive precursor (pro-IL-18) that can be cleaved by Caspase-1 to generate an 18-kDa, biologically active protein (Gu et al.,1997). It is now well established that IL-18 plays predominant roles in the regulation of immune responses in vivo (Hoshino et al.,2001). These include participating in the regulation of innate and acquired immunity (Tsutsui et al.,2004; Li et al.,2009), promoting both T helper cell Type 1 and Type 2 (Th1, Th2) responses (Pollock et al.,2003), regulating the secretion of various cytokines, such as IFN-γ, IL-4, and GM-CSF, and enhancing Fas-ligand (FasL) expression (Stober et al.,2001; Matsui et al.,2008). IL-18 is also capable of enhancing airway remodeling, increasing serum IgE levels, and promoting allergen-induced eosinophil influx into the airways in mice asthma models (Wild et al.,2000). IL-18 is expressed by a variety of immune and nonimmune cells; immature (i.e., pro-IL-18) and mature forms of IL-18 are also detected in patients with Crohn's disease and lupus nephritis (Kanai et al.,2001; Liang et al.,2006). However, to our knowledge, the expression and distribution of IL-18 in normal nasal mucosa and nasal polyps has not yet been investigated. Moreover, little is known about the expression and regulation of IL-18 within airway epithelial cells.
Thus, in this study, we examined the expression of IL-18 in nasal mucosa and nasal polyps. In addition, as a model to investigate the regulation of Th1 and Th2 cytokines, and IL-18 secretion, we evaluated the capacity of A549, which is often used as the representative of airway epithelial cell line, to release IL-18, IFN-γ, and IL-4 in response to LPS and IL-4. These factors, to some extent, mimic infection and inflammation. Thus, evaluating their expression will provide new insights into the pathophysiological roles of IL-18 in the pathogenesis of nasal polyps formation and in the potential role of nasal epithelial cells in this process.
MATERIALS AND METHODS
Cell Culture and Harvest
A549, the representative of human airway epithelial cell line, was purchased from the Cell Resource Center, IBMS, CAMS/PUMC and cultured in DMEM supplemented with 10% fetal bovine serum. Cells were plated in six-well plates at a density of 3 × 106 mL−1 and cultured for 48 hr. After overnight starvation, the cells were treated with LPS (1 μg/mL) (Sigma) or IL-4 (50 ng/mL) (PeproTech), respectively, for 3, 6, 12, or 24 hr. The cells were then harvested, and the total cell lysate was collected with the lysis buffer (50 mM Tris hydrochloride, pH 8.0, 150 mM sodium chloride, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride).
Subjects and Samples Collection
Twenty adult patients with nasal polyps and eight normal controls were included in the study. The investigation was approved by the local ethics committee, and informed written consent was obtained from all the patients who entered the study. The diagnosis of nasal polyposis was established on medical history and clinical examinations, including nasal endoscopic examinations and computed tomographic scans of nasal fossa and paranasal sinuses. Nasal polyp samples were obtained from individuals who had been recommended for functional endoscopic sinus surgery. Nasal mucosa of control samples was obtained from the middle turbinate of patients with septal deviation or the middle turbinate bulb during septal plastic surgery. All patients were asked to stop general and/or local nasal treatment 1 month before surgery. Each sample was divided into two parts. One part was homogenized immediately in the lysis buffer (1 mL/100 mg wet weight of tissues) and quick frozen for Western blot analysis. The other was fixed in a 10% neutral formaldehyde solution, embedded in paraffin, and divided into 4-μm sections for hematoxylin–eosin (H&E) staining and immunohistochemistry.
The sample sections were processed routinely and stained with H&E. To determine the degree of eosinophil infiltration, the sections were examined with a light microscope (400× magnification), and the number of eosinophils and total inflammatory cells in the subepithelial connective tissue in five fields was counted independently by two individuals.
Formalin-fixed and paraffin-embedded segments were subjected to immunohistochemical analysis, as described previously (Liu et al.,2010). Briefly, tissue sections were deparaffinized, rehydrated, and treated with 3% hydrogen peroxide to quench endogenous peroxidase activity, and 10% normal goat serum to block nonspecific binding. Sections were then incubated overnight at 4°C with primary polyclonal antibodies [anti-IL-18 (1:300) from KPL, Japan; anti-IL-4 (1:2,000) from Abcam, UK; and anti-IFN-γ (1:1,000) from Abcam, UK] followed by incubation with appropriate horseradish peroxidase-labeled secondary antibodies (goat anti-mouse IgG or goat anti-rabbit IgG). After washing in a phosphate-buffered saline solution, sections were stained with 3,3′-diaminobenzidine. Negative controls were prepared using PBS in place of the primary antibodies.
Western Blot Analysis
Proteins from extracts of normal nasal mucosa and nasal polyps (30 μg) and A549 cells (40 μg) were denatured and separated on a 12% SDS-polyacrylamide gel electrophoresis gel and analyzed by Western blotting with β-actin (1:5,000; Sigma) and IL-18 (1:1,000; KPL) mouse polyclonal antibody or IL-4 (1:2,000; Abcam) and IFN-γ (1:100; Boster) rabbit polyclonal antibody. The results were visualized using a chemiluminescent substrate kit (SuperSignal West Pico Trial Kit; Pierce Biochemicals, Rockford, IL). The intensities of the detected bands were quantified using Totallab v2.01 software. Relative intensities of target protein signals were obtained by dividing the intensities of the target protein signals by those of the β-actin signals.
Data are presented as mean ± SD. Paired sets of Western blotting data were compared with the Kruskal–Wallis H and Mann–Whitey U-tests. Paired t-test was used in A549 cell culture data analysis. Differences were considered statistically significant at a P-value of <0.05.
Clinical and histological characteristics of the 20 patients are given in Table 1. Histological characteristics of H&E-stained sections were observed through the tissue densities of eosinophils and other infiltrating inflammatory cells. On the basis of the histopathologic findings, nasal polyp patients were divided into two groups as follows: the noneosinophilic nasal polyp (NEosNP) group (Fig. 1A), characterized by eosinophil infiltration constituting less than 10% of the total inflammatory cell infiltrate in the polyps, and the eosinophilic nasal polyp (EosNP) group (Fig. 1B), in which eosinophils constituted more than 20% of the total inflammatory cells. The eosinophil tissue density was increased significantly in the eosinophilic group (563 ± 272 mm−2) when compared with the noneosinophilic group (47 ± 35 mm−2) (P < 0.001). Nominal numbers of inflammatory cells were observed in the normal sinus mucosa.
Table 1. Clinical and histological characteristics of patients
Male, N (%)
Age, median (IQ range)
No. with AR, N (%)
Eos infiltration, % median (IQ range)
EosNP, eosinophilic nasal polyps; NEosNP, noneosinophilic nasal polyps; AR, allergic rhinitis; N, normal nasal mucosa.
IL-18, IFN-γ, and IL-4 Were Upregulated in the Nasal Mucous of Nasal Polyps
To elucidate if IL-18, IFN-γ, or IL-4 are involved in the formation of nasal polyps, immunohistochemical staining analysis was performed to observe the distribution of IL-18, IFN-γ, and IL-4 in normal nasal mucosa and nasal polyps. As shown in Fig. 2, in both the normal sinus mucous and nasal polyps, IL-18, IFN-γ, and IL-4 immunoreactivity was observed in the epithelium, goblet cells, ciliated cells, submucosal glandular cells, and the inflammatory cells (Fig. 2A–I). Western blot analysis was carried out to further explore the expression of IL-18, IFN-γ, and IL-4 in normal nasal mucosa compared with nasal polyps. As shown in Fig. 3A, the nasal polyp tissue expressed much more IL-18, IFN-γ, and IL-4 than the normal samples. Moreover, the intensity of the IL-18, IFN-γ, and IL-4 bands in the EosNP was higher than that in NEosNP. In addition, the 18-kDa mature form of IL-18 was only detected in nasal polyps but was absent in normal nasal mucous. For optical density values obtained by densitometric analysis, the densities of IL-18, IFN-γ, and IL-4 in the nasal polyps were higher than in their normal counterparts and were higher in the eosinophilic group than in the noneosinophilic group (Fig. 3B–D). There was a statistically significant difference between the groups (P < 0.01). These data demonstrate that the expression of IL-18, IFN-γ, and IL-4 is increased in the nasal polyps, especially in the EosNP.
The Expression of IL-18, IFN-γ, and IL-4 in A549 Cells Treated With LPS
As airway epithelia are the first physical barrier against antigens, airway epithelial cells may play a central role in the immune inflammatory responses. We examined whether airway epithelial cells (A549) express IL-18, IFN-γ, and IL-4 in response to LPS. We found that the expression of IL-18, IFN-γ, and IL-4 increased after LPS (1 μg/mL) treatment (Fig. 4A). The 18-kDa mature form of IL-18 could only be detected after LPS treatment, but was absent in the untreated control cells. Densitometric analysis indicated that the density of IL-18 and IFN-γ increased significantly (mainly after 3 and 6 hr) after treatment with LPS for the indicated time (3, 6, 12, and 24 hr) (Fig. 4B–D). The density level of IL-18 and IFN-γ decreased after LPS treatment for 12 and 24 hr compared with that at 3 and 6 hr, but was still significantly increased compared with the control. The density of IL-4 gradually increased after treatment with LPS for the indicated time points (3, 6, and 12 hr). The expression level of IL-4 decreased after LPS treatment for 24 hr compared with 3, 6, and 12 hr, but was still significantly increased compared with the control.
The Expression of IL-18 and IFN-γ in A549 Cells Treated With IL-4
Our results indicated that IL-4 was upregulated both in NEosNP and EosNP patients. To investigate if IL-4 can regulate the secretion of cytokines from airway epithelial cells and thus promote the formation of nasal polyps, we examined the expression of IL-18 and IFN-γ in airway epithelial cells (A549) following treatment with IL-4. We found that IL-18 and IFN-γ expression was lower after IL-4 (50 ng/mL) treatment for 3 hr (Fig. 5A). The 18-kDa mature form of IL-18 could not be detected after IL-4 treatment. The optical density measurements obtained by densitometric analysis (Fig. 5B,C) indicated that the density of IL-18 increased after IL-4 treatment for 3 hr, but then decreased gradually with increasing treatment time, and became weaker than the control after 24 hr. The density of IFN-γ increased after treatment with IL-4 for 3 hr, but it was no significant difference compared with controls, and after 6 hr, it significantly decreased gradually.
Histological studies indicate that nasal polyps are composed of epithelial cells and edematous connective tissue, with many inflammatory cells that accumulate in the subepithelial layer, in which predominant eosinophilia is observed in more than 80% of all cases (Bachert et al.,2000). In the other 20% of patients with nasal polyps, however, few or even no eosinophils are observed. Furthermore, it is reported that the incidence of neutrophilic polyps in the Asian population is higher (40%), with a lower incidence of eosinophilia (41.7–65.2%) (Bunnag et al.,1984). This may be the result of different pathogenic mechanisms. In this study, our data are consistent with a previous report that 40% of patients lack eosinophils. Thus, we divided the nasal polyp patients into the noneosinophilic and eosinophilic groups, and detected the expression of IL-18 in normal nasal mucosa and in polyps from these two different groups, to investigate the potential mechanisms of IL-18 in the etiology and the formation of nasal polyps.
IL-18 is a multifunctional cytokine with structural similarities to the IL-1 cytokine family. Previous studies have demonstrated that IL-18 can function in regulating a variety of chemokines, including IFN-γ, GM-CSF, and IL-13, and enhances chronic airway inflammation and airway remodeling in the OVA-induced asthma mouse model (Yamagata et al.,2008). It has also been shown that LPS activates IL-1β-converting enzyme that might contribute to the secretion of IL-18 (Abu Elhija et al.,2008). Monteleone et al. (1999) and Pizarro et al. (1999) have shown that the 18-kDa mature form of IL-18 is abundant in intestinal mucosal biopsies from inflammatory bowel disease patients, but is absent in normal controls. Faust et al. (2002) also found that IL-18 was expressed in tubular epithelial cells, and the mature IL-18 was upregulated in parallel with the severity of nephritis. The expression of IL-18 in normal nasal mucosa and nasal polyps, however, has not been investigated before. In this study, we found that the expression of IL-18 was clearly enhanced in nasal polyps compared with normal controls. Furthermore, the 18-kDa mature form of IL-18 was only detected in nasal polyps, but not in normal nasal mucous. We also found that the 24-kDa inactive precursor was expressed at a higher level in EosNP than in NEosNP. This indicates that IL-18 plays an important role in the formation of nasal polyps.
The pathogenesis of nasal polyps, including how eosinophils and other inflammatory cells accumulate in the subepithelial layer, remains a puzzle. As the sinonasal tract is exposed constantly to microorganisms and airborne contaminants, and nasal epithelial cells are the first layer of cells that keep the growth of “normal flora” in check and clear microbes in the nasopharynx. Thus, we hypothesize that nasal epithelial cells can release different chemokines in response to different antigens and play a central role in the initiation of immune inflammatory responses. In this study, we found that IL-18 was expressed in A549 airway epithelial cells in response to stimulation by LPS and IL-4. More specifically, the 18-kDa mature form of IL-18 was detected in A549 cells after stimulation by LPS, but was absent in normal controls, whereas no mature form of IL-18 was found in A549 cells in response to IL-4. We have also proved that airway epithelial cells can release other cytokines such as IL-4, IFN-γ react to the presence of LPS and the intensity of IL-18, FN-γ decreased when A549 cells response to IL-4. Taken together, this indicates that IL-18, IL-4, and IFN-γ can be secreted by nasal epithelial cells and inflammatory cells after exposure to antigens; airway epithelial cells play an important role in the formation of nasal polyps and contribute to the infiltration of inflammatory cells to the subepithelial layer.
In this study, we also found that eosinophils from nasal polyps are capable of expressing both Th1- and Th2-type cytokines. This is based on the observation that IFN-γ and IL-4 are more intensely expressed in eosinophilic polyps compared with noneosinophilic polyps. Our results are consistent with a recent study by Nunes et al. (2009) showing higher IL-4 messenger RNA transcription in eosinophilic than in noneosinophilic groups. Other previous researches have also been reported that nasal polyps express a mixture of Th1 and Th2 cytokines (Bachert et al.,2002; Danielsen et al.,2006). Therefore, eosinophils are likely to contribute to tissue remodeling and progression of nasal polyps. And, this may also be the reason why a large number of EosNP patients do not have clinical allergy symptoms.
In this study, the expression of IL-18, IFN-γ, and IL-4 was clearly enhanced in nasal polyps compared with normal controls. This indicates that they constitute a complex cytokine network and play important roles in the formation of nasal polyps. To date, accumulating evidence suggests a positive relationship among IL-18, IL-4, and IFN-γ. For example, Th1 cells produce larger amounts of Th1 cytokines such as IFN-γ and TNF-α when stimulated additionally through their IL-18R by OVA plus IL-18 (Yoshimoto et al.,1998). A recent study on adaptive immunity unveiled that IL-18 has the potential to stimulate Th1 cells to produce Th2 cytokines, such as IL-3, IL-9, and IL-13 (Sugimoto et al.,2004). Furthermore, conventional Th cells, in collaboration with NK cells in vivo, have the capacity to release IL-4 in response to IL-18 plus IL-2 (Tsutsui et al.,2004). Taken together, these findings suggest that IL-18, IFN-γ, and IL-4 exert a combined effect on inflammatory reactions in the formation of nasal polyps.
In conclusion, this study demonstrates that nasal polyps exhibit high levels of IL-18, both in epithelial cells and inflammatory cells. In addition, the expression of IL-18 in A549 airway epithelial cells can be regulated by LPS and IL-4. All the data suggest that nasal epithelial cells may act as an initiator for immune inflammatory responses to secrete IL-18, and IL-18 is likely to play an essential role in the development of nasal polyps. However, further investigations on the mechanisms of the pathogenesis of nasal polyps are required.
The authors thank H. Zhang and Xiulan Zhao for their kind technical assistance.