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* Departments of Pulmonology and
Pathology, Leiden University Medical Center, The Netherlands; and
Department of Pathology, University Hospital Nijmegen, The Netherlands
Correspondence: Jamil Aarbiou, Dept. of Pulmonology, C3-P, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands. E-mail: j.aarbiou{at}lumc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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1-proteinase inhibitor, the defensin-induced cell proliferation was not affected. These data suggest that neutrophil defensins may possibly be involved in epithelial repair in the airways by inducing lung epithelial cell proliferation.
Key Words: antimicrobial peptides • wound repair • epidermal growth factors • 1-proteinase inhibitor
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Repair of injured epithelium is often accompanied by an influx of inflammatory cells such as neutrophils and macrophages. Neutrophils contain and produce several products, including elastase, cathepsin G, defensins, and reactive oxygen species, which are released upon stimulation. These products are not only involved in defense against a broad spectrum of microorganisms, but upon release may also cause epithelial cell injury [4 ]. Besides their injurious potential, neutrophil products may also be involved in the subsequent repair process [5 6 7 8 9 ]. In vivo studies in rats demonstrated that neutrophil depletion resulted in decreased epithelial proliferation and repair following ozone-induced epithelial injury [5 ]. Data from in vitro studies also support a role for neutrophils in epithelial repair, as the neutrophil products lactoferrin and H2O2 increase epithelial cell proliferation [6 , 7 ] and differentiation [8 ]. In addition, neutrophil defensins have been shown to increase proliferation of mouse retinal epithelial cells and fibroblasts in vitro [9 ]. This is of potential importance in view of the abundance of neutrophil defensins in the neutrophil [10 ] and their presence in airway secretions [11 ].
Human neutrophil defensins, also called human neutrophil peptides (HNP), are small (3.5–4 kDa), cationic polypeptides that belong to the -defensin subfamily. They are present in large amounts in azurophilic granules and are released upon neutrophil activation [10
]. Four homologous members of the neutrophil defensins (HNP1–4) have been identified so far. Neutrophil defensins were originally identified as broad-spectrum antimicrobial peptides. Subsequent studies have shown that neutrophil defensins also display cytotoxic activity to eukaryotic cells and a variety of other pro- and anti-inflammatory activities (reviewed in ref. [12
]). Defensins have now been shown to be active in regulating a variety of processes, including complement activation [13
], chemotaxis of human immature dendritic cells, CD4+/CD45RA+ and CD8+ human T cells [14
], and monocytes [15
]. In vitro studies also revealed marked effects of defensins on epithelial cells, as defensins induce cytotoxicity [16
, 17
], interleukin (IL)-8 [18
], and SLPI expression [19
] and increase adhesion of Haemophilus influenzae [20
]. Some of the proinflammatory activities of defensins appear to be inhibited by defensin-inhibitory components, such as the serpin serine proteinase inhibitors 1-proteinase inhibitor (1-PI) and 1-antichymotrypsin [18
, 20
, 21
].
Based on the possible involvement of neutrophils in epithelial repair processes and the observation that human neutrophil defensins increase proliferation of murine fibroblasts and retinal epithelial cells [9
], we hypothesized that neutrophil defensins increase proliferation of human lung epithelial cells. Therefore, the aim of the present study was to examine the effect of neutrophil defensins on proliferation of cultured epithelial cells, to explore the possible involvement of EGF receptor activation and the downstream mitogen-activated protein (MAP) kinase signaling pathway in this process, and to determine the effect of 1-PI on defensin-induced cell proliferation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Generation of anti-HNP mouse monoclonal antibodies (mAb)
Anti-HNP mAb were generated using conventional hybridoma technology. Briefly, female Balb/c mice were immunized subcutaneously with a mixture of native and glutaraldehyde-cross-linked HNP-1 in Freund’s complete adjuvant and boosted with HNP-1 in Freund’s incomplete adjuvant. Four days following an intrasplenal injection with HNP-1, splenocytes were harvested and fused with SP2/0 myeloma cells. Hybridomas producing antibodies specific for HNP1–3 were subcloned by limiting dilution, tested for positivity by enzyme-linked immunosorbent assay using purified HNP1–3 as antigen, and screened for specificity by Western blot analysis of neutrophil granule extracts and immunohistochemistry on formalin-fixed bronchial tissue (data not shown). Antibodies from clone HNP-E3 (immunoglobulin G class) were purified from culture supernatant by protein G affinity chromatography using the ÄKTAprime system (Amersham Pharmacia Biotech, Upsala, Sweden).
Cell culture
Cells from the A549 human lung carcinoma cell line (a cell line with type II alveolar epithelial cell characteristics) and the NCI-H292 human mucoepidermoid tumor cell line were obtained from the American Type Culture Collection (Manassas, VA). The cells were cultured in tissue culture flasks at 37°C in a 5% CO2-humidified atmosphere in RPMI 1640 (Gibco, Grand Island, NY), supplemented with 2 mM L-glutamine, 200 U/ml penicillin, 200 µg/ml streptomycin (all BioWhittaker, Walkersville, MD), and 10% heat-inactivated fetal calf serum (FCS; Gibco).
Cell proliferation
Cell proliferation was assessed using automated cell counting, 5-bromo-2-deoxyuridine (BrdU) incorporation or by measuring mitochondrial activity using (4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT).
Cell counting
A549 and NCI-H292 cell cultures were seeded at a concentration of 30,000 cells/cm2 and were cultured overnight in 12- or 24-well plates (Costar, Cambridge, MA) in complete RPMI medium. After washing with prewarmed phosphate-buffered saline (PBS), the cells were starved for growth factors by overnight incubation in serum-free medium followed by incubation for 24 or 48 h in the same medium alone (negative control) or supplemented with HNP1–3, HNP-1, reduced and alkylated HNP-1 (red-HNP-1), transforming growth factor (TGF-; Sigma Chemical Co., St. Louis, MO), or FCS. Cell counting was performed after harvesting the cells using a cell scraper (Greiner, Alphen a/d Rijn, The Netherlands), and cell numbers were determined using an automated cell counter (Casy-1, Schärve System, Germany). To study the role of EGF receptor activation and the downstream MAP kinase signaling pathway, cells were incubated with 10 µM of the EGF receptor tyrosine kinase inhibitor tyrphostin AG1478 (Calbiochem, La Jolla, CA) or 25 µM of the MAP kinase kinase (MEK) inhibitor U0126 (Promega, Madison, WI) for 1 h, followed by incubation with HNP1–3 or TGF- in the presence of AG1478 or U0126. Cytotoxic effects of AG1478 and U0126 were excluded by trypan blue exclusion assays. In other experiments, HNP1–3 was preincubated with 2 µg/ml polymixin-B (PB; Sigma Chemical Co.) or equimolar concentrations of 1-PI (Laboratoire Français de Fractionnement et des Biotechnologies, Lille, France) 1 h before the addition to cells. The effect of HNP-specific mAb on HNP1–3-induced cell proliferation was assessed by preincubation of HNP1–3 (58 µg/ml) with anti-HNP mAb (2.2 mg/ml) for 1 h at 37°C, followed by dilution to a final concentration of 8 µg/ml HNP1–3 in medium and removal of anti-HNP/HNP1–3 aggregates by centrifugation prior to addition to cells.
BrdU incorporation
For assessment of A549 cell proliferation by BrdU incorporation, A549 cells were seeded and starved for growth factors, as described above, on 12-well tissue-culture plates (Costar). Following incubation of the cells with various stimuli for the indicated time periods, 10 mM BrdU (Sigma Chemical Co.) was added, and cells were incubated for 1 h. Next, the cells were washed twice in PBS and fixed in 70% ethanol (v/v) for at least 1 h. BrdU incorporation was assessed using immunocytochemistry [24
]. Briefly, cells were permeabilized with 1 M hydrochloric acid followed by subsequent washes with 0.1 M sodiumtetraborate and PBS. BrdU incorporation was demonstrated by incubation with a mouse anti-BrdU mAb (kindly provided by Prof. Dr. F. C. S. Ramaekers, Dept. of Molecular Cell Biology, University of Maastricht, The Netherlands), followed by incubation with a peroxidase-labeled rabbit anti-mouse polyclonal antibody (Dako, Glostrup, Denmark). BrdU incorporation was visualized using Nova RED (Vector Laboratories, Burlingame, CA), and the percentage BrdU-positive nuclei was calculated.
MTT assay
A549 cells were incubated, as described above, in 96-well plates (Costar). After a 24-h incubation of A549 cells in 100 µl RPMI medium containing the stimulus, 50 µl of a 5 mg/ml MTT (Sigma Chemical Co.) solution in PBS was added, and cells were incubated at 37°C for 2 h [25
]. The cells were then lysed by addition of 100 µl per well extraction buffer [20% (w/v) SDS, 50% (v/v) N,N-dimethyl formamide, 2% (v/v) acetic acid, pH 4.7]. After overnight incubation with extraction buffer, the optical density (OD) at 562 nm was measured.
Statistical analysis
Results are expressed as mean ± SEM. Data obtained from three separate experiments were analyzed for statistical difference by the Student’s t-test for paired samples. For individual experiments performed in triplicate, the unpaired t-test was used. Differences were considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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; however, no differences were observed between the different time periods.
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1-PI on defensin-induced A549 cell proliferation1-PI has been shown to modulate selected defensin-induced activities. To assess the effect of 1-PI on defensin-induced cell proliferation and cytotoxicity, HNP1–3 was incubated at proliferation-inducing (8 µg/ml) and cytotoxic (50 µg/ml) concentrations with equimolar concentrations of 1-PI before addition to A549 cells (Fig. 6
). 1-PI alone at low concentrations (equimolar to 8 µg/ml HNP1–3) did not affect A549 cell proliferation; high concentrations (equimolar to 50 µg/ml HNP1–3) resulted in a small but significant increase in A549 cell proliferation. A549 cell proliferation induced by 8 µg/ml HNP1–3 was not significantly affected by preincubation with equimolar concentrations of 1-PI. Incubation with concentrations of 1-PI equimolar to 50 µg/ml HNP1–3 not only inhibited defensin-induced A549 cell cytotoxicity (ref. [21
] and this study; data not shown), but also resulted in a 2.9-fold increase of A549 cell numbers.
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as a control (Fig. 7A
). AG1478 completely blocked TGF--induced A549 cell proliferation, and defensin-induced A549 cell proliferation was not affected. To assess a possible involvement of the MAP kinase signaling pathway downstream of the EGF receptor in defensin-induced cell proliferation, A549 cells were preincubated with the MEK inhibitor U0126 for 1 h prior to addition of HNP1–3 or TGF- (Fig. 7B)
. Unlike AG1478, U0126 completely inhibited defensin-induced A549 cell proliferation. These findings indicate that although defensin-induced cell proliferation is not EGF receptor-dependent, this induction may be mediated via the downstream MAP kinase signaling pathway.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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1-PI on defensin-induced cell proliferation were assessed to investigate whether this specific property of defensins can be modulated. While defensin-induced cytotoxicity was inhibited by 1-PI, the defensin-induced cell proliferation was not.
Previous studies have shown that neutrophil defensins enhance proliferation of murine fibroblasts and retinal epithelial cells [9
]. The present study extends these findings by showing that neutrophil defensins also enhance proliferation of human lung epithelial cells. We observed that this effect is dependent on structural integrity of the defensins and is not affected by 1-PI. Various studies have demonstrated the involvement of the EGF receptor and the MAP kinase signaling pathway in (epithelial) cell proliferation [26
]. MEK-mediated activation of extracellular regulated kinase-1/2 was found to be especially important for epithelial cell proliferation. Our results show that defensin-induced epithelial cell proliferation is not dependent on EGF receptor activation but appears to be mediated via the MAP kinase signaling pathway downstream of the EGF receptor. Other studies have also provided evidence for a role of neutrophil products in epithelial cell proliferation. Hagiwara et al. [6
] have demonstrated that lactoferrin and lactoferrin-derived peptides show growth-promoting activities toward the rat intestinal epithelial cell line IEC-18. It has been suggested that another neutrophil-derived antimicrobial protein, the porcine cathelicidin PR-39, is involved in wound repair, as it induced mesenchymal cell expression of syndecans, which are heparan-sulfate proteoglycans that show growth-promoting activities [27
]. A third neutrophil product that has been implicated in cell proliferation is H2O2. In an in vitro study using a rabbit lens epithelial cell line, Ohguro et al. [7
] demonstrated that H2O2 induces cell proliferation. Similar to neutrophil defensins, the nature of the H2O2 effects on epithelial cells is concentration dependent. At high concentrations (1 mM), H2O2 is cytotoxic, whereas at low concentrations (1 nM–1 µM), it induces cell proliferation. Furthermore, Goldkorn et al. [28
] have shown that H2O2 activates the EGF receptor, suggesting a role for EGF receptor signaling in H2O2-induced cell proliferation. However, we found that the EGF receptor is not involved in neutrophil defensin-induced cell proliferation. Taken together, these data illustrate that stimulation of neutrophils may lead to the release of potentially cytotoxic mediators that at low concentrations, may contribute to restoration of epithelial integrity.
The present results were obtained using highly purified preparations of neutrophil defensins that were isolated from granules derived from peripheral blood human neutrophils. Therefore, it is unlikely that contaminants in the defensin preparations are responsible for the observed effects on cell proliferation. Contribution of LPS as a contaminating factor to defensin-induced cell proliferation was excluded by demonstrating that PB did not affect the defensin activity. Furthermore, we have demonstrated that LPS from Escherichia coli and Pseudomonas aeruginosa did not affect A549 cell proliferation, which makes a role of LPS in defensin-induced cell proliferation unlikely (data not shown). Effects of possible minor mitogenic contaminants could also be excluded, as the observed increase in cell numbers was fully blocked by HNP-specific mAb. The activity of defensins appeared to depend on an intact structure of defensins, as reduction and alkylation of defensins resulted in complete loss of the growth-promoting activity. Finally, defensin-induced cell proliferation is not likely to be a mere consequence of the cationic character of the peptide, as the cationic peptide protamine was devoid of growth-promoting activity. It is unknown whether neutrophil products that are released by stimulated neutrophils concomitant with defensins affect defensin-induced cell proliferation.
Based on structural differences, human defensins can be divided into two subfamilies: the - and ß-defensins [10
]. Neutrophil defensins belong to the subfamily of -defensins, whereas epithelial cells secrete ß-defensins. Understanding the mechanism that underlies neutrophil defensin-induced epithelial cell proliferation is hampered by the fact that no receptor has been identified for these defensins. ß-Defensins have been shown to use the chemokine receptor CCR6 to cause chemoattraction of CCR6-bearing memory T cells and immature dendritic cells [29
]. It has recently been shown that neutrophil defensin-induced chemotaxis of T and dendritic cells is pertussis toxin sensitive, suggesting the involvement of G-protein-mediated signaling [14
]. Therefore, it is possible that neutrophil defensins also use G-protein-coupled chemokine receptors to exert their various actions. The chemokine IL-8 has been implicated in the induction of cell proliferation and migration of epidermal and colon epithelial cells [30
31
32
], suggesting a role for chemokine receptors in epithelial cell proliferation. Further experiments are required to delineate the signal transduction pathways involved in defensin-induced epithelial cell proliferation.
What are the implications of the present findings? Neutrophilic inflammation is generally considered a beneficial response to potential, harmful intruders such as microorganisms. Neutrophils are involved in eradicating microorganisms, cause tissue injury, and may aid in restoration of epithelial integrity after injury. Neutrophilic inflammation may be harmful because of the tissue injury associated with sustained and extensive neutrophilic inflammation. Indeed, neutrophilic inflammation in the lung has been shown to be associated with chronic bronchitis [33
], chronic obstructive pulmonary disease [2
, 34
], cystic fibrosis [35
], and various other inflammatory lung diseases. We [17
] and others [16
] have previously shown that defensins at high concentrations cause epithelial injury and expression of proinflammatory genes in airway epithelial cells. In contrast, the present results indicate that at low concentrations, defensins may contribute to epithelial reconstitution by enhancing cell proliferation, considered as one of the crucial phases in the repair process. Therefore, the effect of neutrophil defensins is dependent on local defensin concentrations that may be determined by the extent of neutrophil inflammation, clearance mechanisms, and defensin-inhibitory substances. Whereas in healthy individuals neutrophil defensin concentrations in the epithelial lining fluid are low, these concentrations may markedly increase through neutrophil-dominated, inflammatory processes. Indeed, high (up to 1.6 mg/ml) concentrations of neutrophil defensins have been shown to be present in purulent secretions of patients with chronic bronchitis [21
] and cystic fibrosis [36
]. In addition, bronchoalveolar lavage fluid collected from patients with active pulmonary tuberculosis contains high levels of HNP1–3 (1.25±0.31 µg/ml) [37
], indicating that the concentration in the epithelial lining fluid is ten- to 100-fold higher. Defensin-binding substances that are present in the epithelial lining fluid may differentially affect the various activities of defensins, as shown in the present study by the ability of 1-PI to selectively block defensin-induced epithelial cell death, allowing high, potentially cytotoxic concentrations of defensins to induce proliferation. In line with this observation, Murphy et al. [9
] have demonstrated that serum albumin (bovine) is also capable of enabling cell proliferation by defensin concentrations that are otherwise cytotoxic. Therefore, the range of defensin concentrations that may induce cell proliferation in vivo may be higher than predicted based on studies using purified defensins alone.
One previous report, which has not yet been confirmed further, indicated that rabbit neutrophil defensins increase wound healing in the skin [38 ]. This observation is in line with our results and suggests that defensins also enhance airway epithelial wound repair. Although defensin-induced cell proliferation may be beneficial to the host defense, it is possible that when the local defensin concentration is inadequately controlled, excessive and prolonged stimulation of cell proliferation may also have detrimental effects. An association between neutrophil influx and epithelial cell proliferation in human lung disease is suggested by the observation that bronchial biopsies obtained from chronic bronchitis patients showed an increased proportion of proliferating epithelial cells as compared with normal individuals and asthmatic patients [39 ]. As other studies showed that airway neutrophilia is more prominent in patients with chronic bronchitis compared with patients with asthma or healthy controls [33 , 40 ], epithelial cell proliferation and neutrophil influx may be associated features in chronic bronchitis. It is tempting to speculate that defensins are involved in the occurrence of epithelial abnormalities in chronic bronchitis, such as squamous and mucus cell metaplasia, which are often associated with neutrophilic inflammation [2 ].
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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1-PI. The defensin-induced cell proliferation appears to be mediated via an EGF receptor-independent activation of the MAP kinase signaling pathway.
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ACKNOWLEDGEMENTS |
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Received January 9, 2001; accepted February 13, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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) or 48 h (•). For comparison, cells treated with medium containing 10% FCS showed a 3.2- and 4.0-fold increase in cell numbers at 24 and 48 h, respectively (data not shown). Data represent mean ± SEM of three separate experiments. *, P < 0.05 versus serum-free medium-treated cells.
