Expression of lipocortins in human bronchial epithelial cells: effects of IL-1β , TNF-α, LPS and dexamethasone

In this study, we investigated the expression of lipocortin I and II (annexin I and I in the human bronchial epithelium, both in vivo and in vitro. A clear expression of lipocortin I and II protein was found in the epithelium in sections of bronchial tissue. In cultured human bronchial epithelial cells we demonstrated the expression of lipocortin I and II mRNA and protein using Northern blotting, FACScan analysis and ELISA. No induction of lipocortin I or II mRNA or protein was observed after incubation with dexamethasone. Stimulation of bronchial epithelial cells with IL-1β, TNF-α or LPS for 24 h did not affect the lipocortin I or II mRNA or protein expression, although PGE2 and 6-keto-PGF1α production was significantly increased. This IL-1β- and LPS-mediated increase in eicosanoids could be reduced by dexamethasone, but was not accompanied by an increase in lipocortin I or II expression. In human bronchial epithelial cells this particular glucocorticoid action is not mediated through lipocortin I or II induction.


Introduction
The bronchial epithelium is considered to play an important role in initiating and perpetuating inflammatory and immunological reactions by production of a variety of inflammatory mediators. Therefore, it is thought that human bronchial epithelial cells (HBEC) may play a role in inflammatory pulmonary diseases such as asthma. Upon in vitro stimulation with inflammatory agents such as interleukin-l[ (IL-113), tumour necrosis factor-a (TNF-a) and lipopolysaccharide (LPS), HBEC are able to produce several cytokines, such as IL-1, interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte-macrophage colonystimulating factor (GM-CSF) and monocyte chemoattractant factor-1 (MCP-1). -Furthermore, HBEC exposed to various stimuli in vitro release several arachidonic acid metabolites, including 15-hydroxyeicosatetraenoic acid , prostaglandin E2 (PGE2) and 6-keto-prostaglandin F= . 4 '5 In addition to these pro-inflammatory properties, HBEC could also be involved in anti-inflammatory reactions through the production of potential anti-inflammatory proteins, e.g. lipocortins.
Lipocortin I and II (annexin I and II) are members of the annexin family of Cai+-depen dent phospholipid-binding proteins. Biological evidence suggests that at least some members of this family are glucocorticoid inducible proteins with anti-inflammatory properties7 It has been proposed that lipocortins I and II mediate part of the immunosuppressive activity of glucocorticoids by inhibiting phospholipase A2 (PLA2) activity, hereby preventing eicosanoid production. 8 In some recent studies, however, the induction of lipocortin I and II by glucocorticoids was not obseeeed. [9][10][11] Other biological functions for lipocortins have also been reported, such as the regulation of cell differentiation and growth, and a role in the central nervous system and neuroendocrine system (reviewed in Reference 12).
Animal studies have suggested that the lung is a rich source of lipocortin I. [13][14][15][16] In the human lung, lipocortin synthesis has been described in blood leucocytes and alveolar macrophages. 7' In cultured human tracheal submucosal gland cells production of lipocortin-like proteins have been found. 19 However, to our knowledge, no data are available on the presence of lipocortins in HBEC.
The aim of this study was to investigate the expression of lipocortin I and II in HBEC and in the human bronchial epithelial cell line BEAS 2B, and to examine whether these potential antiinflammatory proteins could, be induced by glucocorticoids. Furthermore, we investigated the effect of inflammatory agents such as IL-I, TNFa and LPS on the lipocortin expression. Finally, we studied whether the effects of dexamethasone Bronchi were obtained from patients undergoing one passage in DMEM/F12 with supplements, as surgery for lung cancer. Only bronchial tissue described for HBEC cells. distant from the tumour and having a normal macroscopic appearance was used. Freshly Immunofluorescence and immunoperoxidase removed tissue samples were collected in cold stainings of cells and tissue: Immunostainings sterile HEPES buffered RPMI (GIBCO, Paisley, were performed with a rabbit polyclonal anti-UK), supplemented with penicillin G sodium body against lipocortin I, kindly provided by Dr (100 U/ml; Gist-Procades, Delft, The Nether-R.B. Pepinsky and a mouse monoclonal antilands) and streptomycin sulphate (0.1 mg/ml; body against lipocortin II (Oncogene Science Biochrom KG, Berlin, Germany) for transport to Inc., Manhasset, NY). All antibody reagents were the laboratory. Tissue samples were cut into diluted in PBS containing (w/v) 0.5% BSA and pieces, washed with cold phosphate buffered 0.1% sodium azide. Normal mouse serum, saline (PBS), and incubated either overnight at normal rat serum and two subclass specific anti-4C or l h at 37C in HEPES buffered RPMI bodies were used as negative controls. containing 0.1% protease XlV (Sigma, St Louis, MO), penicillin G sodium (100 U/ml) and strep-Cells. For fluorescence activated cell scan tomycin sulphate (0.1 mg/ml). Subsequently, (FACScan) experiments cells were detached with epithelial cells were gently scraped from the 0.02% EDTA, fixed for 40 min at room temperatissue samples, washed twice in culture medium ture (RT) with Permea-Fix Reagent (Ortho Diagand plated onto 35-mm dishes at a density of nostic Systems, Beerse, Belgium)and washed for 2.5 x 105 cells/dish. The Netherlands), penicillin G sodium and streptomycin sulphate (0.1 mg/ml). Cells were char-Tissue. Bronchi specimens for immunohistoacterized as epithelial cells by immuno-chemistry, from one female and two male nonfluorescence staining using a mouse monoclonal asthmatic patients, were quickly frozen in liquid antibody directed against a number of human nitrogen and stored at -80C. Six lam frozen cytokeratins (CK-1; DAKOpatts, Glostrup, bronchi sections were fixed in acetone for 1 min Denmark). At least 99% of the isolated cells at RT and incubated with anti-lipocortin I or II stained positive for cytokeratin (n 5).
antibodies for 60 min in a humidified chamber. The sections were then washed twice with PBS/ Human bronchial epithelial cell line: BEAS 2B is Tween (0.1%) and incubated for 60 min with a SV-40 transformed human bronchial epithelial horseradish peroxidase (HRP)-conjugated swine cell line, which was kindly provided by Dr J. anti-rabbit-IgG or HRP-conjugated rabbit anti-Lechner (Inhalation Toxicology Research Insti-mouse-IgG for detection of lipocortin I and II, tute, Albuquerque, NM). 2 Cells were maintained respectively (DAKOpatts). After three washing in a keratinocyte growth medium containing procedures with PBS/Tween, peroxidase activity bovine pituitary extract, EGF, penicillin G sodium was measured using diaminobenzidine tetraand streptomycin sulphate (KGM; GIBCO). 21  experiments were performed on days 7 through dehydrogenase (GAPDH) probe was a 0.7 kb 10 of primary culture of HBEC. Twenty-four h EcoRI-PstI fragment. 25 The IL-8 probe was a 1.3 before treatment with cytokines and/or dex-kb EcoRI fragment, kindly provided by Dr T. J. amethasone the medium was replaced by a basal Stoof (Department of Dermatology, VU Hospital, medium consisting of DMEM/F12 without hydro-Amsterdam, The Netherlands). cortisone or other supplements to prevent influ- The intensity of the lipocortin I, lipocortin II, ence of endogenous steroids. IL-I (20 ng/ml; IL-8 and GAPDH mRNA signals on the auto-UBI, Lake Placid, NY), TNF-a (20 ng/ml; UBI), radiograph were scanned with a handscanner LPS (10 or 100 btg/ml; Difco Laboratories, Detroit, (Colorscanner 2, 24 Highscreen, WCrselen, MI) and/or dexamethasone (10 -M; Duchefa bv., Germany) at a resolution of 100 dots per inch.
Haarlem, The Netherlands) were added to the Computer software described by Koning  Northern blotting and hybridization were performed as described previously. 24 The lipocortin Production of arachidonic acid metabolites.. The I and II probes were a 1.3 and a 0.9 kb EcoRI arachidonic acid metabolites PGE2 and 6-ketofragment, respectively, kindly provided by PGFla were measured by radioimmunoassay as Dr B. P. WaRner (Biogen Research Corp., Camdescribed previously, is Arachidonic acid metabobridge, MA). The glyceraldehyde-3-phosphatelite release was normalized to total RNA content. Statistical analysis.. The Mann-Whitney U test was used to assess significant differences in PGE2 and 6-keto-PGFla production, and in lipocortin I and II mRNA expression in cell cultures under different conditions of incubation. A p-value of less than 0.05 was considered significant.

Results
Lipocortin I and II expression in vivo: Immunoperoxidase staining of bronchi sections (n 3) with anti-lipocortin I and II antibodies showed a strong expression in the bronchial epithelium (Fig. 1). Similar staining patterns were observed for lipocortin I and II. Positive staining for lipocortin I and II was also found in the epithelial cells of the submucosal glands.
Lipocortin I and II expression in vitro: In cultured HBEC and in BEAS 2B cells lipocortin expression was studied using FACScan and Northern blot analysis. With FACScan analysis we found that more than 99% of BEAS 2B cells were positive for both intracellular lipocortin I and II (Fig. 2). Fifty and 80% of cultured HBEC (n 3) were positive for intracellular lipocortin I and II, respectively. A representative experiment is shown in Fig. 2  concentrations of IL-1], TNF-a, LPS and dexaand 6-keto-PGFl production was measured, methasone. Incubation for 24h with IL-1], and we studied whether the expected inhibition TNF-a, LPS or dexamethasone did not sig-of stimulated eicosanoid production by dexanificantl affect lipocortin I or II mRNA expres-methasone correlated with an induction of liposion in cultured HBEC or in BEAS 2B cells (Figs cortin I and II. Incubation of HBEC (n 5) 3A and 3B). IL-8 mRNA was used as a positive for 24h with either IL-I[, or TNF-a or LPS sigcontrol, as it has been shown previously to nificantly increased basal PGE2 and 6-ketoincrease upon stimulation with IL-1] or LPS, and PGFI production (p < 0.01) (Fig. 4). PGE2 to decrease upon incubation with dexa-and 6-keto-PGF production in one representamethasone. Figure 3B shows that IL-8 mRNA tive culture of HBEC is shown in Table 1  in the human bronchus are in agreement with those in the airway epithelium of the rat, studied HBEC were incubated for 24 h with IL-11 (20 ng/ml), TNF-(20 ng/ml), LPS (100 gg/ml) and dexamethasone (Dex)(10 -6 M).
by Fava et al. 29 They demonstrated the presence bpGE2 and 6-keto-PGFl= production are expressed as ng.ml-l.mg total of lipocortin I in the rat epithelium of nasal, NA. tracheal and bronchial airways, and in the ductal epithelium of various glands. Production of lipo-mRNA (Fig. 4) or lipocortin II mRNA (data not cortin-like proteins in cultured human tracheal shown), submucosal .land cells has been described by Jacquot eta/. 9 In the foetal and neonatal human Analysis of the inducibility of lipocortin I and II lung, lipocortin I immunostaining was found in mRNA by dexamethasone: To evaluate the effect the bronchiolar epithelium as early as 12 weeks, of dexamethasone on lipocortin I and II mRNA beginning with the largest airways, and by 24 expression more precisely we cultured HBEC weeks extending distally to the bronchioloalveoand BEAS 2B cells for up to 5 days without lar portals. In bovine bronchial epithelial cells a glucocorticoids. The cells were then incubated differential expression of lipocortin I and II was with different concentrations of dexamethasone found. 1 Lipocortin I was expressed in the cili-(10 -, 10-9, 10-8, 10-7, and 10 -M) and with ated cells, whereas lipocortin II was expressed in 10 5 M dexamethasone for various lengths of the basal cells. We did not find such a differential times (2, 4, 6, 10, 24 and 48h). Both the effects expression of lipocortin I and II in human of dexamethasone on cells cultured in a bronchi in our studies.

medium of DMEM/F12 without supplements and
The bronchial epithelium is considered to play on cells in a medium of DMEM/F12 supplean important role in inflammatory and immunomented with insulin, transferrin, EGF, NaiSeO, logical reactions by production of a variety of and charcoal-stripped FCS, but without hydroinflammatory mediators as can be obseeeed in cortisone were examined. Under none of these inflammatory pulmonary diseases such as asthma.
conditions dexamethasone affected the expres-In addition to this role, the bronchial epithelium sion of lipocortin I or II mRNA (data not could also be involved in anti-inflammatory reacshown), tions through the production of anti-inflammatory proteins, e.g. lipocortins. In this study we found a high expression of lipocortin I and II in Discussion the human bronchial epithelium. A physiological We demonstrated in this study that lipocortin role has been proposed for lipocortin I recently. and II are expressed in cultured HBEC, a Lipocortin I may function as a 'barrier' to inapbronchial epithelial cell line and in the epithepropriate inflammatory and autoimmune responlium in bronchi sections. Immunoperoxidase ses at specific sims around the body. 7 Following stainings of human bronchi sections with anti-an inflammatory stimulus, stress-induced stimulalipocortin I and II antibodies showed a clear tion of the hypothalamic-pituitary-adrenal axis expression in the bronchial epithelium and in (HPA) axis may result in the release of cortisol, the epithelial cells of the submucosal glands. In which in turn leads to the production of lipocultured HBEC and in the BEAS 2B cell line, we cortin I. The lipocortin-binding molecules on demonstrated the presence of lipocortin I and II monocytes and neutrophils arriving at tissue sims mRNA and protein by Northern blotting and are expected to become saturated, with resultant FACScan analysis, respectively. Lipocortin I and II moderation of migratory and/or pro-inflamma-mRNA and protein expression in the HBEC was tory activities. Others have speculated from not affected by incubation of the cells with IL-results in animal studies that epithelial cell 113, TNF-a, LPS or dexamethasone. The HBEC damage and loss, as seen in asthmatics, could were able to respond to these stimuli, as the decrease availability of these protective properties production of PGE2 and 6-keto-PGFl signifi-of lipocortin I. 1 Increased inflammation would cantly increased upon incubation with IL-113, thereby be predicted.

TNF-a or LPS. This increased production was
We did not observe an induction of lipocortin  32 For the induction experiments we used both HBEC, just a few days after isolation and after culturing for 1 or 2 weeks. Other studies point to the importance of culture conditions in studying synthesis of lipocortins by cultured cells. 3x In our experiments we have used charcoal-stripped FCS in the culture medium so that endogenous steroids, which might influence lipocortin synthesis, are removed. We have incubated epithelial cells for up to 5 days in steroid-free medium prior to the addition of dexamethasone, to ensure that the cells were in an unstimulated condition at the beginning of the experiment. As growth factors, such as EGF, are thought to induce lipocortin synthesis, we have also performed experiments, in which cells were cultured in a basal medium without growth factors. In all cases we found a clear mRNA and protein expression of lipocortin I and II in HBEC, but no induction of lipocortins by dexamethasone after incubation for various lengths of times with different concentrations. After culturing for 5 days in steroid free medium, lipocortin I mRNA was expressed constitutively at approximately 90% of the lipocortin I mRNA level in complete medium, containing hydrocortisone. Vishwanatha et al. found in bovine bronchial epithelial cells, that the constitutive expression of lipocortin I was 20% of the lipocortin I level in medium containing hydrocortisone after culturing for 5 days. x6 These studies indicate that there is a difference between species in the inducibility of lipocortins by glucocorticoids.
In summary, we found a high expression of lipocortin I and II in the bronchial epithelium compared to the underlying layers in human bronchi sections and demonstrated that glucocorticoids do not increase the expression of lipocortin I and II in HBEC. Our study indicates, that decrease in PGE2 and 6-keto-PGFl production in HBEC upon incubation with glucocorticoids is not mediated by increased expression of lipocortins.