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- and 
-defensin on cytokine production by cultured human bronchial epithelial cells
1Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan; 2Second Department of Internal Medicine, Oita University Faculty of Medicine, Oita, Japan; and 3Third Department of Internal Medicine, Miyazaki Medical College, Miyazaki, Japan
Submitted 5 March 2004 ; accepted in final form 15 November 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-defensins, mainly present in neutrophils in the lung, have a cytotoxic effect and induce IL-8 production from airway epithelial cells. Although 
-defensins are expressed in epithelial cells in various tissues, including lung, there are no reports of their effects on cytokine synthesis in airway epithelial cells. The aim of the present study was to determine the effects of both - and -defensins on the cytokine production, transcription factor binding activity, and cytotoxicity in primary cultured human bronchial epithelial cells (HBECs). We used human neutrophil peptide-1 (HNP-1; -defensin) and human -defensin-2 (HBD-2) to stimulate HBECs. The results showed that treatment of HBECs with HNP-1, but not HBD-2, increased IL-8 and IL-1 mRNA expression in a dose-dependent manner and also enhanced IL-8 protein secretion and NF-
B DNA binding activity. The 24-h treatments with >20 µg/ml of HNP-1 or >50 µg/ml of HBD-2 were cytotoxic to HBECs. These results suggest that - and -defensins have different effects on cytokine synthesis by airway epithelial cells, and we speculate that they play different roles in inflammatory lung diseases.
human neutrophil peptide-1; human -defensin-2; interleukin-8; nuclear factor-B; cytotoxicity
- and -defensins. Six -defensin peptides have been identified. Human neutrophil peptide (HNP)-1 to -4 are mainly present in neutrophils and in certain lymphocyte subsets. Human defensin (HD)-5 and HD-6 are primarily expressed in intestinal Paneth cells, female reproductive tract, and respiratory tract (2). The -defensin subfamily consists of four members, called human -defensin (HBD)-1 to -4 (2). HBD-1 is constitutively expressed in epithelial cells of the urinary and respiratory tracts (34). HBD-2 is found in epithelia of the inner and outer surfaces of the human body, such as skin and the respiratory and gastrointestinal tracts (2). HBD-3 is expressed in the skin, tongue, and respiratory tract (14). HBD-4 is expressed in the testis, gastric antrum, uterus, neutrophils, thyroid gland, lung, and kidney (13). It is known that the activities of -defensins strongly depend on their concentrations. The microbicidal concentration of -defensin ranges between 1 and 100 µg/ml and that of -defensins ranges between 0.1 and 50 µg/ml (2, 43).
Although most studies on defensins have focused on their direct antimicrobial activity, recent studies (32, 41, 44) have suggested that defensins also contribute to the regulation of host adaptive immunity (e.g., chemoattractant for dendritic cells and T cells). At concentrations exceeding 50 µg/ml, -defensins induce cytotoxic effects and cytokine production in epithelial cells (1, 41).
In a series of studies, we reported that -defensins HNP-1, -2, and -3, and -defensins, especially HBD-2, were increased in plasma or bronchoalveolar lavage fluid (BALF) of patients with various inflammatory lung diseases (4–6, 16, 17, 19, 27). In patients with diffuse panbronchiolitis (DPB), a disease that affects the respiratory bronchioles and causes severe obstructive ventilatory impairment, we showed that the levels of several cytokines such as IL-1, TNF-, IL-8, and regulated on activation, normal T cell expressed, and secreted (RANTES) were increased in BALF (20, 28). In addition, the levels of IL-8 correlated significantly with those of -defensins in BALF of these patients (6). In cystic fibrosis, in which elevated levels of -defensins in sputum have also been identified (39), high levels of TNF-, IL-1, IL-8, and transforming growth factor (TGF)-1 were found in BALF or sputum (8, 37). In vitro studies have also demonstrated that -defensins induced the release of IL-8 from airway epithelial cells (40, 42). These findings suggest that defensins may be associated with the release of certain inflammatory cytokines from airway epithelial cells. However, there have only been a few reports so far about the effects of -defensins (40, 42) and no report about the effects of -defensins on the synthesis of inflammatory cytokines in airway epithelial cells.
The aim of the present study was to determine the effects of - and -defensin on the inflammatory cytokine synthesis and their cytotoxic effects in airway epithelial cells.
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MATERIALS AND METHODS |
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Cell culture. Primary cultured human bronchial epithelial cells (HBECs) obtained from three different donors were purchased from Clonetics and grown to monolayers in tissue culture flasks at 37°C in a 5% CO2-humidified atmosphere. HBECs were maintained in BEGM. Hydrocortisone was removed from this medium before treatment with defensins and during the time of the study. All experiments were performed at either the third or the fourth passage.
Stimulation of cells with defensins.
For the analysis of cytokine mRNA expression, HBECs grown to confluence (
1.0 x 106/dish) in 60-mm cell culture dishes were incubated for 6 h with culture medium alone (control) or 12.5–50 µg/ml of each defensin. On the basis of the results of cytokine mRNA expression, we selected the concentrations of each defensin for other experiments: 50 µg/ml of HNP-1 and 12.5 µg/ml of HBD-2. To assess the cytokine production at protein level, experiments were performed in 24-well plates. When HBECs were 90–100% confluent (6.0 x 104/well), cells were incubated for 24 h with medium alone or HNP-1 or HBD-2 solution. To assess the intracellular production of IL-1 by the immunocytochemical method, HBECs grown to near confluence on cell culture slides were incubated for 24 h with medium alone or HNP-1 or HBD-2 solution. For EMSA, HBECs grown to confluence in 60-mm dishes were incubated for 1 or 2 h with medium alone or HNP-1 or HBD-2 solution. To assess the cytotoxicity of the defensins, HBECs grown to confluence in 96-well plates were separately incubated for 24 h with 0–50 µg/ml of HNP-1 or HBD-2.
RNase protection assay.
Total RNA was isolated from HBECs after treatment using a single-step phenol/chloroform extraction procedure (Isogen; Nippongene, Toyama, Japan). Cytokine mRNA levels were determined with the RPA RiboQuont multiple system (Pharmingen, San Diego, CA) based on the instructions provided by the supplier. The customized template sets for human RANTES, TNF-, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-1, macrophage chemoattractant protein-1 (MCP)-1, IL-8, TGF-1, and VEGF were used. Internal controls included ribosomal protein L32 and GAPDH. In brief, 10 µg of total cellular RNA was hybridized overnight to the [-32P]UTP-labeled riboprobes that had been synthesized from the supplied template sets. Single-stranded RNA and free probes remaining after hybridization were digested by RNase A and T1 mixture. The protected RNA was then phenolized, precipitated, and analyzed on a 5% denaturing polyacrylamide gel. After electrophoresis, the gel was dried and subjected to autoradiography. The quantity of protected labeled RNA was determined using densitometry. Results were normalized to the expression of the internal control, GAPDH.
ELISA measurements.
GM-CSF, IL-1, IL-8, and TGF-1 levels of supernatants from HBECs were measured by immunoenzymometric assay using EASIA kit (BioSource Europe, Nivelles, Belgium). Lower limits of detections were 50 pg/ml for GM-CSF, 33 pg/ml for IL-1, 7 pg/ml for IL-8, and 8 pg/ml for TGF-1. All measurements were performed in triplicate, and values reported are the means of five measurements.
Immunocytochemistry.
Cells were fixed with acetone for 10 min, and immunocytochemistry was performed by the alkaline phosphatase anti-alkaline phosphatase method using rabbit anti-human IL-1 polyclonal antibody (AbCam Limited, Cambridge, UK). This antibody recognizes not only mature 17-kDa IL-1 but also nondenaturated (native) 31-kDa IL-1 precursor. Negative control studies were performed by using irrelevant rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
EMSA.
The nuclear proteins were extracted using the CelLytic NuCLEAR Extraction kit (Sigma-Aldrich, St. Louis, MO). EMSA was performed using Gel Shift Assay Systems (Promega, Madison, WI) and [
-32P]ATP-labeled oligonucleotides containing the NF-B consensus binding sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3'). Nuclear extracts (10 µg) were incubated in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris·HCl, and 50 µg/ml poly(dI-dC)] with 1.75 pmol of either specific or nonspecific competitor oligonucleotides for 10 min before addition of labeled probe. After incubation at room temperature for 20 min, samples were separated on a 5.5% polyacrylamide and 5% glycerol gel in Tris-glycine buffer. Gels were dried under vacuum and autoradiographed. HeLa nuclear extract (Promega) was used as positive control.
AlamarBlue reduction assay. After the 24-h incubation with HNP-1 or HBD-2, cells were treated for 3 h with 10-fold diluted AlamarBlue (Serotec, Oxford, UK) with hydrocortisone-free BEGM. Cell viability was measured at 540 nm excitation and 620 nm emission. The results were compared with the nonexposed control cells.
Statistical analysis. Data are expressed as means ± SE. The minimum number of replicates for all measurements was at least three. For RNase protection assay and AlamarBlue reduction assay, differences among various groups were compared by one-way ANOVA. The post hoc test for a multiple comparison was Fisher's protected least significant differences test. For ELISA, differences between control and defensin-treated samples were compared by matched pair t-test. Significance was assumed at P < 0.05.
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RESULTS |
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and IL-8 mRNA expression in HBECs treated with HNP-1 was increased in a dose-dependent manner as shown in the densitometric analysis of these bands (Fig. 2). Figure 2 also shows that these two mRNA levels when treated with 50 µg/ml of HNP-1 were significantly higher than those of untreated HBECs (P < 0.05, respectively). TNF-, GM-CSF, MCP-1, TGF-1, and VEGF mRNA expression was not affected by the HNP-1 treatment. Some of the candidate mRNAs were constitutively expressed in the HBD-2 group, but these levels were not different from those in untreated cells (Figs. 1 and 2). RANTES mRNA was not detected in either group.
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, IL-8, and TGF-1 in supernatants of HBECs incubated for 24 h with medium alone, 50 µg/ml of HNP-1, or 12.5 µg/ml of HBD-2. IL-8 production of HNP-1-treated HBECs was significantly higher than in untreated control cells (P < 0.05). The levels of other cytokines were not different from the controls.
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production.
Immunocytochemical studies using anti-IL-1 antibody showed weak immunoreactive IL-1 expression on HBECs after a 24-h incubation with medium alone (Fig. 4B) or 12.5 µg/ml of HBD-2 (Fig. 4D). In contrast, markedly increased expression was observed when incubated with 50 µg/ml of HNP-1 (Fig. 4B).
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B binding activity.
Representative autoradiographs of EMSA to detect NF-B binding activity in nuclear extracts of HBECs after a 1-h incubation with medium alone (control), 50 µg/ml of HNP-1, or 12.5 µg/ml of HBD-2 are shown in Fig. 5. NF-B binding activity in HNP-1-treated HBECs was higher than in control but not detectable with HBD-2. Similar results were obtained when these experiments were repeated three times after either a 1- or 2-h incubation (data not shown). Specific binding was abolished by competition with excess unlabeled oligonucleotide specific for NF-B, whereas the nonspecific competitor was ineffective.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-defensin) induced IL-8 and IL-1 mRNA expression, IL-8 protein production, and NF-B binding activity on HBECs, whereas HBD-2 (-defensin) did not induce these responses. It has been reported that IL-8 is a potent neutrophil attractant that can induce the release of -defensins from neutrophils (6). We have reported that the BALF levels of -defensins in patients with DPB were highly elevated compared with healthy subjects and correlated with the BALF levels of IL-8 (26). Also, van Wetering et al. (41) demonstrated that -defensin induced IL-8 synthesis in airway epithelial cells, which was confirmed in the present study. Considered together, these findings suggest that -defensins may contribute to lung neutrophilic inflammation through IL-8 synthesis in airway epithelial cells.
We have previously shown that the BALF levels of IL-1, TNF-, and RANTES as well as IL-8 were increased in patients with DPB (20, 28). Therefore, we examined the expression of these cytokines as the major candidates released by the defensin-treated HBECs. IL-1 is produced by various cell types, including epithelial cells (7), and plays an important role in mediating the local and systemic immunoresponses (22). In the present study, although HNP-1 induced mRNA expression and intracellular expression of IL-1, it did not induce IL-1 protein secretion in the cell supernatants. Several cell types have the capacity to produce the IL-1 precursor; however, its release is predominantly limited to monocytes and macrophages (10). IL-1 is translated from the mRNA into the immature form, pro-IL-1 (3), and then IL-1-converting enzyme (ICE) cleaves the pro-IL-1 to release the mature form into the extracellular environment (38). In the present study, we observed prominent expression of IL-1 in the cells treated with HNP-1 using anti-IL-1 antibody that could recognize the pro-IL-1. This indicates that IL-1 was actually translated from the mRNA into the immature IL-1 in the HNP-1-treated HBECs. Proteolytic enzymes have also been shown to cleave pro-IL-1 into a form similar in size and specific activity to the ICE-processed IL-1 (11, 33). These proteases are released at inflammatory and tissue damage sites by a variety of immune cells (15, 25). The discrepancy between IL-1 mRNA expression and its protein secretion noted in the present study might be explained by the lack of these proteases in the in vitro environment. We reported the presence of high levels of BALF IL-1 in patients with DPB and that treatment with macrolides reduced these IL-1 levels in correlation with a reduction in BALF neutrophils (28). Therefore, we speculate that HNP-1 could be associated with IL-1 synthesis and mediate the inflammation as well as IL-8 in the lung.
We also examined the mRNA expression or protein secretion of other inflammatory cytokines such as TNF-, MCP-1, and GM-CSF, but our results showed no significant response of these cytokines in HBECs. Van Wetering et al. (43) showed that -defensins increased the levels of epithelial cell-derived neutrophil activator-78, MCP-1, and GM-CSF proteins released from type II pneumocyte-like A549 cells but not from HBECs. In addition, IL-8 mRNA expression in A549 cells was increased after a 6-h incubation with -defensin. In our current study, although we chose the similar time course for HBECs in accordance with their results, mRNA expression of TNF-, TGF-1, and VEGF as well as MCP-1 and GM-CSF and production of TGF-1 and GM-CSF were not induced by -defensin. This might be one reason why these transient signals were not induced in our study, suggesting that the synthesis of these cytokines, when treated with -defensins, depends on the cell types of lung epithelium. In the present study, NF-B DNA binding activity was enhanced by HNP-1. NF-B is a pleiotropic transcription factor expressed in various cell types after stimulation and/or cell activation by a wide variety of stimuli. It is also known to play an important role in IL-8 (32) and IL-1 synthesis (18). Although our results did not show a direct relationship between NF-B and IL-8 or IL-1, our results suggest that NF-B could participate in increasing IL-8 and IL-1 expression in lung epithelial cells in response to -defensin. On the other hand, we did not find any HBD-2 (-defensin)-induced cytokine synthesis and NF-B binding activity in HBECs. These findings suggest that - and -defensin have different effects on transcriptional regulation of inflammatory cytokine genes in airway epithelial cells. Defensins are known to bind to the lipid bilayer where they form voltage-dependent, ion-permeable membrane channels (24), but there are no reports about defensin-related receptors on airway epithelial cells. Further studies are needed to clarify the intracellular mechanism mediating defensin-induced cytokine response.
Each HNP-1 and HBD-2 at a concentration of 50 µg/ml contained trace amounts of endotoxin in this study. We have reported that a much larger dose of LPS (1 µg/ml) had a much smaller effect on HBECs (12), suggesting that the endotoxin contamination of the defensin was not responsible for the cytokine mRNA induction. Therefore, we conclude that endotoxin was not a critical factor in HBEC stimulation and speculate that the endotoxin component of the defensins cannot explain the cytokine production by HBECs exposed to defensins.
We demonstrated that the 24-h treatment with >20 µg/ml of HNP-1 and >50 µg/ml of HBD-2 was cytotoxic to HBECs. Several reports demonstrated that -defensins had a cytotoxic effect on airway epithelial cells due to cytolysis (1, 31, 40). In the present study, we found that -defensin, in addition to -defensin, also had cytotoxic effects on airway epithelial cells. Aarbiou et al. (1) also reported that HNP-1 to HNP-3 increased cell proliferation at concentrations of 4–10 µg/ml and that HNP-1 increased cell proliferation at a concentration of 2 µg/ml for A549 cells. They showed that HNPs mediated cell proliferation via the mitogen-activated protein kinase signaling pathway. Murphy et al. (29) also reported that HNP-1 to HNP-3 increased proliferation of murine fibroblasts and retinal epithelial cells. However, our results showed that both HNP-1 and HBD-2 at concentrations ranging from 0 to 50 µg/ml did not induce HBEC proliferation. These differences may in part be explained by the differences in cell types used, HBECs and A549 cells (smaller airway epithelium).
In this study, although the synthetic HBD-2 we used did not significantly affect cytokine expression in HBECs, we have established in the previous study (17) that HBD-2 has high antimicrobial activity against Escherichia coli, suggesting that this synthetic product has bioactivity.
In conclusion, we have demonstrated in the present study that only -defensin stimulates the expression of inflammatory cytokines, particularly IL-8 and IL-, by HBECs. Our results further suggest that -defensin, but not -defensin, could play differential roles in this cytokine synthesis that contributes to inflammatory lung diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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