Leukotriene B4 mediates histamine induction of NF-kappa B and IL-8 in human bronchial epithelial cells

Yosuke Aoki, Daoming Qiu, Guo Hua Zhao, and Peter N. Kao

Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, California 94305-5236

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In 16HBE transformed human bronchial epithelial cells, histamine stimulated interleukin (IL)-8 mRNA and protein secretion, and this histamine stimulation was inhibited by the H1-receptor antagonist diphenhydramine (DPH), by the inhibitor of 5-lipoxygenase-activating protein (FLAP) MK-886, by the 5-lipoxygenase inhibitor Zileuton, and by dexamethasone. Histamine stimulated bronchial epithelial cell production of leukotriene B4 (LTB4), and this production was inhibited by FLAP inhibitors MK-886 and L-655,238 and Zileuton. Histamine stimulated IL-8 luciferase reporter gene activity that was inhibited with DPH, dexamethasone, MK-886 and L-655,238, and Zileuton. The inhibition of IL-8 transcription and protein secretion by FLAP inhibitors and Zileuton was reversed with exogenous LTB4. There was increased IL-8 nuclear factor-kappa B (NF-kappa B) DNA-binding activity after histamine stimulation, and this was inhibited by DPH and MK-886. Cytoplasmic phospholipase A2 mRNA levels were also potently induced by histamine. Thus histamine stimulation of bronchial epithelial cells involves binding at H1 receptors, production of LTB4, activation of NF-kappa B and increased expression of IL-8.

inflammation; cytokine; transcription; Zileuton; phospholipase A2; nuclear factor-kappa B; interleukin-8

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

BRONCHIAL EPITHELIAL CELLS are capable of expressing a multitude of signaling cytokines (reviewed in Refs. 27 and 29), including interleukin (IL)-8, IL-6, tumor necrosis factor-alpha (TNF-alpha ), IL-1beta , and hematopoietic growth factors granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, colony-stimulating factor-1, IL-2 (1), and stem cell factor (33). These factors contribute to the development of a local immune response in the microenvironment of the airways, appropriate for host defense against foreign pathogens. However, excessive cytokine expression by bronchial epithelial cells likely contributes to inappropriate proliferation and activation of immune effector cells (8) and the cellular airway inflammation, which is a cardinal feature of asthma (27, 29).

IL-8 is a potent cytokine promoting the chemoattraction, proliferation, and activation of neutrophils (16, 21). IL-8 is expressed by a variety of cell types, including macrophages, neutrophils, lymphocytes, and epithelial and endothelial cells. The regulation of IL-8 expression involves transcriptional and posttranscriptional mechanisms and differs significantly between cell types (21). Epithelial and endothelial cells are capable of prompt and substantial expression of IL-8, and this induction is primarily at the level of transcription (21, 23). The critical transcription factor controlling the expression of IL-8 in epithelial and endothelial cells has been shown to be nuclear factor (NF)-kappa B (22). IL-8 expression in lung epithelial cells has been shown to be triggered by histamine (5) and also by the inflammatory cytokines TNF-alpha and IL-1beta (25, 31).

Histamine, released from mast cells in the lung, can exert proinflammatory and bronchoconstrictive effects and contribute to airway inflammation (4).

Leukotriene B4 (LTB4) has been shown to be produced by resting bronchial epithelial cells, and production of LTB4 is increased by inflammatory stimuli (14, 15, 24). Bronchial epithelial LTB4 acts as a powerful chemoattractant for leukocytes through mechanisms that involve upregulation of cell surface integrins (reviewed in Ref. 11). Neutrophils and macrophages, recruited to and activated at sites of inflammation, also produce LTB4. LTB4 synthesis involves lipid peroxidation by 5-lipoxygenase, and this process can generate reactive oxygen intermediates (20). The transcription factor NF-kappa B, implicated in the regulation of numerous inflammatory cytokines (3), may be activated by reactive oxygen intermediates (28). Brach et al. (6) showed that LTB4 caused transcriptional activation of IL-6 in monocytes, acting through transcription factors NF-IL-6 and NF-kappa B (6). Los et al. (20) analyzed the role of arachidonic acid metabolites involved in T cell activation through the CD28 receptor, and they identified an important role for 5-lipoxygenase, LTB4, and reactive oxygen intermediates in the enhancement of NF-kappa B DNA-binding activity and the transcriptional activation of IL-2.

Here, we have characterized an inflammatory signaling pathway in human bronchial epithelial cells that involves histamine acting upon H1 receptors, synthesis of LTB4, induction of transcription factor IL-8 NF-kappa B, and enhancement of IL-8 transcription and IL-8 protein secretion.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell culture. An SV40 large T antigen-transformed human airway epithelial cell line, 16HBE14o- (16HBE), that retains differentiated morphology and function of normal human airway epithelia (7) was from the laboratory of D. Gruenert (University of California, San Francisco, CA). The transformed cells were cultured in Eagle's minimum essential medium (BioWhittaker, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (BioWhittaker).

Cell stimulation and drug treatments. Monolayer epithelial cells grown to 80% confluency were stimulated for indicated times in culture medium containing various combinations of stimulants, including histamine (Sigma, St. Louis, MO), recombinant human TNF-alpha (Biosource, Camarillo, CA), recombinant human IL-1beta (PharMingen, San Diego, CA), and LTB4 (Calbiochem). Diphenhydramine (DPH) and cimetidine (Cim; Sigma) were used to examine histamine H1- and H2-receptor subtypes in bronchial epithelial cells. The following drug modulators of histamine-stimulated IL-8 gene expression were investigated: dexamethasone (Dex; Sigma), the 5-lipoxygenase-activating protein (FLAP) inhibitors (10) MK-886 (Calbiochem) and L-655,238 (Calbiochem), the 5-lipoxygenase inhibitor Zileuton (gift of Abbott, Abbott Park, IL; see Ref. 13), 3-isobutyl-1-methylxanthine (IBMX; Calbiochem), and cyclosporin A (Sandoz Research Institute, East Hanover, NJ).

Quantification of IL-8 protein and LTB4. IL-8 protein concentrations in culture supernatants of transformed human bronchial epithelial cells were measured using a human IL-8 sandwich enzyme-linked immunosorbent assay (ELISA) kit (Immunotech, Westbrook, ME). LTB4 concentrations in 16HBE culture supernatants were measured using a competitive human LTB4 enzyme immunoassay system (Amersham, Arlington Heights, IL). The assays were done following the instructions of the manufacturers and were measured in triplicate.

RNA isolation and analysis by reverse transcription-polymerase chain reaction. After stimulation, adherent epithelial cells were harvested by trypsinization, and total RNA was extracted using guanidinium isothiocyanate (RNAzol; Tel-Test, Friendswood, TX). Two micrograms of total RNA were used for reverse transcription (RT)-polymerase chain reaction (PCR) as described (1). Oligonucleotide primers for human IL-8, beta -actin, and cytosolic (c) phospholipase A2 (PLA2) were as follows: IL-8 sense, 5'-ATGACTTCCAAGCTGGCCGTGGCT-3'; IL-8 antisense, 5'-TCTCAGCCCTCTTCAAAAACTTCTC-3'; beta -actin sense, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; beta -actin antisense, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'; cPLA2 sense, 5'-CTCACACCACAGAAAGTTAAAAGAT-3'; cPLA2 antisense, 5'-AAATAAGTCGGGAGCCATAAA-3'. These primer pairs generate a 289-bp PCR product for IL-8, a 636-bp product for beta -actin, and a 306-bp product for cPLA2. The PCR was performed in a volume of 50 µl for 24-34 cycles using a GeneAmp PCR System 2400 (Perkin-Elmer, Foster City, CA) under the following conditions: denaturation at 94°C for 40 s, annealing at 60°C for 40 s, and extension at 72°C for 40 s. For PCR amplification of cPLA2, an annealing temperature of 56°C was used. The PCR products were analyzed in 1.0% agarose gels (GIBCO) and visualized by ethidium bromide staining on an ultraviolet transilluminator. Also, the PCR products on the gels were scanned using a densitometer (Electronic Dual Light Transilluminator; Alpha Innotech, San Leandro, CA) with which IL-8 and cPLA2 signals were normalized relative to the corresponding signals of beta -actin. For every RT-PCR experiment, multiple experiments were performed to identify the optimum number of cycles of amplification to avoid saturation of the PCR products.

Analysis of transcriptional activation of IL-8 gene expression. To analyze transcriptional regulation of IL-8 in airway epithelial cells, reporter gene assays were carried out using 16HBE cells stably transfected with a luciferase reporter plasmid in which luciferase expression is driven by the human IL-8 promoter. Briefly, the full human IL-8 promoter (-1481 to +44 relative to transcription start site of IL-8; see Ref. 23) was generated by PCR using a pair of oligonucleotide primers (sense, 5'-ATGT<UNL>CTCGAG</UNL>AATTCGTACCCAGGCATTATTTTATC-3'; antisense, 5'-TTGTCCTAG<UNL>AAGCTT</UNL>GTGTGCTCTGCTGTC-3'; the underlined sequences represent Xho I and Hind III restriction sites) and using as a template for amplification 1 µg of human genomic DNA, which had been extracted from white blood cells from a normal individual (Qiagen Genomic DNA extraction kit). The PCR product was 1.5 kb and showed the correct predicted sizes of DNA fragments upon restriction digest analyses with Xba I and Sca I. This PCR-amplified IL-8 promoter was then subcloned into the Xho I-Hind III site of the luciferase expression vector pCLN15Delta CX, generating a plasmid, pIL-8 luciferase, that consists of the human IL-8 enhancer sequence (-1481 to +44 relative to transcription start site of IL-8) linked to firefly luciferase cDNA and also containing a neomycin-resistant gene expressed under control of the constitutively active SV40 enhancer (1). The 16HBE cells grown to 50% confluency (2 × 105 cells) were then treated with 3 µg of pIL-8 luciferase linearized by Nde I digestion and 20 µl of lipofectamine (GIBCO) in a 1-ml reaction mixture. Selection pressure with G418 (0.3-0.8 mg/ml) was applied from 2 to 6 wk, and the surviving cells were tested for luciferase activity as described (1); cells were stimulated with 20 ng/ml phorbol 12-myristate 13-acetate for 6 h, and then the 16HBE cell population that showed the highest luciferase activity was established as a stably luciferase-transfected cell line, 16HBE/IL-8 luciferase, and subsequently used for studies of IL-8 transcriptional regulation in human airway epithelium.

Nuclear extract preparation and electrophoretic mobility shift assays. The DNA-binding activities of nuclear transcription factors were assayed by electromobility shift assays (EMSA). After 2 h of exposure to stimulants, nuclear proteins were extracted from a 100-mm plate of 16HBE cells (~3-4 × 106 cells). The cells were trypsinized and pelleted in a 1.5-ml microcentrifuge tube at 4,000 rpm for 5 min and then rinsed one time with phosphate-buffered saline. The nuclear extracts were prepared on ice and with ice-cold reagents at 4°C. Briefly, the cell pellet was resuspended with 300 µl of 10 mM HEPES, pH 7.6, 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, pH 8.0, 1 mM dithiothreitol (DTT), 0.2% (vol/vol) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 10 mM sodium fluoride, incubated for 20 min, and then centrifuged at 4,000 rpm for 5 min. The resultant nuclei were treated with 25 mM HEPES, pH 7.6, 50 mM KCl, 0.1 mM EDTA, and 10% glycerol (buffer C) supplemented with 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 10 mM sodium fluoride. A 1:9 volume of 3 M (NH4)2SO4 was added to the nuclear homogenates, incubated for 30 min, and then ultracentrifuged in a TLA-100.3 fixed-angle rotor (Beckman, Palo Alto, CA) at 75,000 rpm for 15 min at 4°C to pellet chromatin. The resultant supernatant containing extracted nuclear proteins was incubated with 1 vol of 3 M (NH4)2SO4 for 15 min and then the nuclear proteins were precipitated by centrifugation at 50,000 rpm for 10 min. The pellet of precipitated nuclear proteins was carefully resuspended with 100 µl of buffer C containing supplements and then dialyzed for 2 h against 100 ml of buffer C containing no supplements. The nuclear extract thus prepared typically contained 1-2 µg protein/ml based on Bradford dye-binding assay (Bio-Rad Laboratories, Hercules, CA). Nuclear proteins (5-10 µg) were incubated for 30 min at 25°C in 20 µl of binding buffer (25 mM HEPES, pH 7.6, 0.1 mM EDTA, 10% glycerol, 50 mM KCl, and 0.5 mM DTT) containing 1 µg of poly(dI-dC) and 2.5 pg of 32P-labeled oligonucleotide probe (~1 × 105 counts/min) for the NF-kappa B element (-84 to -68; 5'-agctTCGT<UNL>GGAATTTCC</UNL>TCTG-3') from the 5'-flanking region of the human IL-8 gene (23) and the NF-kappa B sequence in mouse immunoglobulin kappa -light chain enhancer (Ig-kappa B) element (5'-agctAAAGA<UNL>GGGACTTTCC</UNL>-3') from the 5'-flanking region of the mouse Ig-kappa light chain gene (3). Lowercase letters represent nucleotides added for radiolabeling purposes. These oligonucleotide probes and their complementary strands were designed to have 5'-agct overhangs, which, after annealing, were filled in using [alpha -32P]dCTP (Amersham, Arlington Heights, IL) and nonradioactive dATP, dTTP, and/or dGTP using Klenow DNA polymerase (Boehringer Mannheim, Indianapolis IN). In the antibody inhibition experiments, monoclonal antibody against p65 (Boehringer Mannheim) was incubated with nuclear extracts for 30 min at room temperature before the addition of radiolabeled probes. Protein-DNA complexes were resolved from free probe on 4% nondenaturing polyacrylamide gels in 0.5× Tris-borate-EDTA (pH 8.3) and visualized by fluorography of the dried gels.

Data and statistical analysis. Significance of the differences between the experimental conditions was determined by paired two-sample Student's t-test (Microsoft Excel). No statistical adjustments were made for multiple comparisons. Data shown are means ± SD.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Histamine induction and drug sensitivity of IL-8 mRNA and protein expression in 16HBE cells. 16HBE cells are SV40-immortalized tracheobronchial epithelial cells (7). The induction of IL-8 mRNA in 16HBE cells was analyzed by RT-PCR (Fig. 1A). For every RT-PCR experiment we performed, we optimized the number of cycles of PCR amplification to avoid saturation of the PCR products generated (Fig. 1B). The RT-PCR analysis of IL-8 mRNA induction shown in Fig. 1A was performed at 28 cycles of PCR amplification, which is within the linear range of PCR quantitation, as shown in Fig. 1B. In resting 16HBE cells, there was constitutive IL-8 mRNA detectable. Histamine induced a significant increase in IL-8 mRNA; for comparison, the stronger induction with the potent proinflammatory cytokines TNF-alpha and IL-1beta is also shown. Histamine induction of IL-8 mRNA was inhibited by the H1-receptor antagonist DPH but not by the H2-receptor antagonist Cim. Additionally, the FLAP inhibitor MK-886 (10) inhibited histamine induction of IL-8 mRNA. IBMX, a phosphodiesterase inhibitor, showed no inhibition, and cyclosporin A showed partial inhibition of histamine-induced IL-8 mRNA. The most potent inhibitor of IL-8 mRNA induction was Dex.


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Fig. 1.   Histamine induction and drug sensitivity of interleukin (IL)-8 mRNA and protein expression in 16HBE cells. 16HBE cells were nonstimulated (NS) or were stimulated with tumor necrosis factor-alpha (TNF-alpha ; 20 ng/ml), IL-1beta (100 U/ml), histamine (His; 2 mM), histamine + MK-886 (10 µM), histamine + diphenhydramine (DPH; 1 µM), histamine + cimetidine (Cim; 1 µM), histamine + 3-isobutyl-1-methylxanthine (IBMX; 10 mM), histamine + dexamethasone (Dex; 100 µM), and histamine + cyclosporin A (CsA; 100 ng/ml). A: IL-8 mRNA analyzed by RT-PCR (6-h stimulation, 28 cycles amplification). IL-8 and beta -actin mRNA were amplified using RT-PCR, and the ratio of IL-8 to beta -actin PCR products was determined from 3 independent experiments. In each experiment, the IL-8 PCR product generated in the NS condition is designated at 1.0; thus there is no SD for this condition. B: relationship of histamine-induced IL-8 RT-PCR product density to number of cycles of PCR amplification. Data shown are averages of 2 independent experiments. C: secreted IL-8 protein measured by ELISA (16-h stimulation). Data are derived from 3 independent experiments. Induction of IL-8 protein by histamine alone and its inhibition by DPH and Dex are statistically significant (** P < 0.01); inhibition of histamine induction by MK-886 is statistically significant (* P < 0.05). M, marker, 100-bp ladder.

When assayed by ELISA, histamine produced a significant increase in secreted IL-8 protein compared with nonstimulated bronchial epithelial cells (P < 0.01; Fig. 1C). There was also statistically significant inhibition of histamine-induced IL-8 secretion by DPH and Dex (P < 0.01) and by MK-886 (P < 0.05).

Transcriptional activation and drug sensitivity of the IL-8 gene in 16HBE cells. A stably transfected bronchial epithelial cell line was generated (16HBE/IL-8 luciferase) in which the IL-8 enhancer and promoter drive the expression of firefly luciferase gene. This transgenic bronchial epithelial cell line was used to compare the inflammatory stimulants histamine, TNF-alpha , and IL-1beta and to evaluate the effects of immunomodulating drugs on IL-8 transcription (Fig. 2A). The results generated with the stably transfected 16HBE/IL-8 luciferase bronchial epithelial cell line were replicated in transient transfections. Histamine triggered a significant increase in IL-8 luciferase activity; for comparison, the more potent stimulation by IL-1beta and TNF-alpha is shown (Fig. 2A). Histamine caused a dose-dependent increase in bronchial epithelial cell IL-8 luciferase expression (Fig. 2B), and the induction at 2 mM histamine was statistically significant (P < 0.05). Histamine at doses up to 10 mM did not induce any measurable adverse effects on the 16HBE/IL-8 luciferase cells; there were no significant changes in cell viability measured by trypan blue exclusion or in protein concentrations of whole cell extracts (data not shown). The histamine H1-receptor antagonist DPH (1 µM) caused a 30% inhibition in IL-8 luciferase activity, whereas the H2-receptor-antagonist Cim showed no inhibition at any concentration. At increasing doses of DPH, the inhibitory effect on histamine signaling was lost (Fig. 2C), suggesting that DPH might be acting as a partial agonist. These results imply that histamine activation of IL-8 transcription in bronchial epithelial cells involves the H1-receptor subtype.


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Fig. 2.   Transcriptional activation and drug sensitivity of IL-8 reporter gene in 16HBE cells. Transgenic cells, 16HBE/IL-8 luciferase, were stimulated for 6 h with the reagents shown. Whole cell extracts were assayed for luciferase activity in triplicate. In A-C, data are means of 3 independent experiments. A: relative IL-8 reporter gene activation by stimulants and effects of drugs; * P < 0.05. B: dose-dependent transcriptional activation by histamine. C: inhibition of histamine-stimulated IL-8 transcription by DPH but not by Cim. Cells were pretreated for 1 h with the indicated doses of DPH or Cim and then stimulated for 6 h with 2 mM histamine.

LTB4 production by histamine-stimulated 16HBE cells was inhibited by FLAP inhibitors and Zileuton. Nonstimulated 16HBE cells expressed no LTB4 into the culture supernatant (Fig. 3). Stimulation with histamine caused generation of detectable LTB4 in the culture supernatants (24 pg/ml; Fig. 3), and there was partial inhibition of LTB4 secretion with the FLAP inhibitors MK-886 and L-655,238 and potent inhibition with the 5-lipoxygenase inhibitor Zileuton.


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Fig. 3.   Secretion of leukotriene B4 (LTB4) by 16HBE cells in response to histamine and effects of 5-lipoxygenase-activating protein (FLAP) and 5-lipoxygenase inhibitors. 16HBE cells were either nonstimulated (NS) or were stimulated for 16 h in the presence of 2 mM histamine, histamine + MK-886 (1 µM), histamine + L-655,238 (1 µM), or histamine + Zileuton (1 µM), and 200 ml of the culture supernatant were tested for LTB4 using an ELISA assay.

LTB4 stimulated IL-8 transcriptional activation and protein secretion. Exogenous LTB4 measured in 16HBE/IL-8 luciferase cells was discovered to be a potent stimulator of IL-8 transcriptional activation (Fig. 4A). There was significant induction with 10 nM LTB4, and the activation induced with 100 nM LTB4 was greater than that with 2 mM histamine (compare Figs. 4A and 2B). In addition, stimulation for 6 h with exogenous LTB4 caused a dose-dependent increase in secreted IL-8 protein (Fig. 4B).


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Fig. 4.   LTB4 induction of IL-8 transcriptional activation and protein secretion and LTB4 rescue of histamine signaling inhibited by FLAP inhibitors and Zileuton. 16HBE/IL-8 luciferase cells were stimulated for 6 h, and luciferase activity and secreted IL-8 protein were measured. A: dose-dependent transcriptional activation with LTB4. B: dose-dependent increase in secreted IL-8 protein with LTB4. C: 16HBE/IL-8 luciferase cells were stimulated with 2 mM histamine for 6 h in the presence of increasing doses of the FLAP inhibitor MK-886, the FLAP inhibitor L-655,238, or the 5-lipoxygenase inhibitor Zileuton without (filled symbols) or with (open symbols) addition of exogenous LTB4 (10 nM, added 1 h earlier). D: inhibition of histamine-stimulated IL-8 protein secretion by Zileuton (10 mM) is reversed by exogenous LTB4 (10 nM). Data are means of 3 independent experiments.

Inhibition of histamine-stimulated IL-8 transcription and protein secretion by FLAP inhibitors and Zileuton was rescued with exogenous LTB4. The FLAP inhibitors MK-886 and L-655,238 (10) and the 5-lipoxygenase inhibitor Zileuton (13) each showed a dose-dependent inhibition of histamine-stimulated IL-8 luciferase activity (Fig. 4C). These results implicate FLAP and 5-lipoxygenase in histamine signaling, leading to IL-8 transcriptional activation. For each drug inhibitor, the ability of exogenous LTB4 (10 nM) to rescue IL-8 transcriptional activation was tested. It is evident that exogenous LTB4 was able to rescue the inhibition of histamine-stimulated IL-8 transcriptional activation caused by MK-886, by L-655,238, and also by Zileuton (Fig. 4C). In addition, in Fig. 4D we show that that Zileuton completely inhibited the effects of histamine stimulation on IL-8 protein secretion, and this Zileuton inhibition was reversed with exogenous LTB4. These results implicate LTB4 as an intracellular messenger molecule that acts downstream in the signaling pathway from the histamine H1 receptor, from FLAP, and from 5-lipoxygenase.

Induction and drug sensitivity of transcription factor IL-8 NF-kappa B in 16HBE cells. The critical regulators of IL-8 transcriptional activation in a glioblastoma cell line, T98G, and in Jurkat T cells have been identified as IL-8 NF-kappa B, NF-IL-6, and activator protein-1 (AP-1; see Ref. 22). Nuclear extracts were prepared from 16HBE cells stimulated with histamine, and the inductions of the IL-8 transcriptional regulators were investigated.

Stimulation of 16HBE cells with histamine caused induction of IL-8 NF-kappa B DNA-binding activity (Fig. 5A, compare nonstimulated vs. histamine). The histamine-induced NF-kappa B complex was diminished when 16HBE cells were stimulated in the presence of the DPH and the FLAP inhibitor MK-886. To characterize more precisely the IL-8 NF-kappa B DNA-binding activity induced by histamine, oligonucleotide cross-competition experiments were performed (Fig. 5B); there was complete inhibition of the specific IL-8 NF-kappa B EMSA complex with the self IL-8 NF-kappa B oligonucleotide, partial inhibition with the IL-2 NF-kappa B oligonucleotide, and no significant inhibition with the unrelated Sp-1 oligonucleotide. The effects of specific antisera to the NF-kappa B p65 subunit on the IL-8 NF-kappa B DNA-binding complex induced by histamine are shown in Fig. 5C. There was nearly complete obliteration of the IL-8 NF-kappa B complex with 200 ng of p65 antisera, and this effect was specific, as 200 ng of control mouse IgG had much less effect on the complex (Fig. 5C, lane 5 vs. lane 4). Under the conditions employed, the specific IgG effects were manifested as inhibition of the IL-8 NF-kappa B DNA-binding complex and not as a supershifted complex. These antibody results imply that the histamine-induced IL-8 NF-kappa B complex contains p65.


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Fig. 5.   Induction and drug sensitivity of IL-8 NF-kappa B DNA binding. 16HBE cells were stimulated for 2 h with reagents described in Fig. 2. Nuclear proteins were extracted and incubated with 32P-labeled oligonucleotide probes corresponding to the IL-8 NF-kappa B, and the Ig NF-kappa B site in the presence of poly(dI-dC). Protein-DNA complexes were resolved using nondenaturing acrylamide gel electrophoresis followed by fluorography. A: induction of IL-8 NF-kappa B by His and modulation by drugs; B: oligonucleotide cross-competitions demonstrating specificity of binding of IL-8 NF-kappa B complex; C: specific inhibition of IL-8 NF-kappa B with 20 and 200 ng of mouse monoclonal antibody (IgG) to NF-kappa B p65 subunit. Negative control incubation utilized 200 ng of nonspecific mouse IgG (mIgG). NE, nuclear extract; SP-1, specific promoter transcription factor-1.

Histamine stimulation also caused an induction of IL-8 AP-1 DNA-binding activity, and this induction was inhibited by DPH but not by MK-886 (data not shown).

Histamine potently induced cPLA2 mRNA in 16HBE cells. cPLA2 can be induced by inflammatory stimuli (reviewed in Ref. 34). Figure 6 showns the induction of cPLA2 mRNA after stimulation with TNF-alpha , IL-1beta , or histamine. It is apparent that histamine was the most potent stimulant for the induction of cPLA2 mRNA, and this induction was inhibited by DPH, MK-886, and Dex.


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Fig. 6.   Histamine induction of cytoplasmic phospholipase A2 (cPLA2) mRNA. 16HBE cells were stimulated as described in Fig. 1. cPLA2 mRNA was analyzed by RT-PCR, and normalization was performed by comparison with beta -actin as in Fig. 1A. * P < 0.05 and ** P < 0.01. Data are means ± SD from 3 independent experiments.

Taken together, these results demonstrate that histamine, acting at bronchial epithelial H1 receptors, activates intracellular signaling pathways involving FLAP and 5-lipoxygenase, production of LTB4, and induction of IL-8 NF-kappa B DNA-binding and IL-8 transcriptional activation.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have shown that histamine activates bronchial epithelial cell signaling pathways that involve synthesis of LTB4 and transcriptional activation of NF-kappa B, leading to secretion of IL-8. Histamine induction of IL-8 expression could be inhibited with an H1-receptor antagonist and with inhibitors of 5-lipoxygenase synthetic function. The 16HBE14o- cell line that we studied is a good model of the native epithelium, and its growth properties allowed us to characterize signaling pathways and transcriptional regulation that would not have been feasible using primary cultures of bronchial epithelium.

Our in vitro studies of the ability of histamine to induce IL-8 are relevant to the pathogenesis of airway inflammation in asthma. Bellini et al. (5) demonstrated that bronchial epithelial cells recovered from asthmatic but not from normal volunteers, when stimulated with 1 µM histamine, expressed lymphocyte chemoattractant activity that was partially due to IL-8. In airway inflammation, mast cells degranulate and release histamine in close proximity to bronchial epithelial cells. In immediate hypersensitivity reactions, the tissue concentration of histamine has been estimated to reach 1 mM (30). Thus the concentrations of histamine that we required to activate IL-8 expression in 16HBE cells in vitro may be achieved in vivo under conditions of airway inflammation characteristic of asthma.

Histamine was shown to trigger the expression of inflammatory cytokines IL-6 and fibronectin from BEAS-2B bronchial epithelial cells (26). These proinflammatory effects of histamine were mediated through H1 receptors and were associated with transient increases in intracellular calcium. In guinea pig tracheal epithelial cells and in BEAS-2B cells, histamine was shown to activate H1 receptors that coupled to pertussis toxin-insensitive G proteins, leading to accumulation of inositol phosphates (19). Leurs et al. (17) showed in Chinese hamster ovary cells that transfected H1 receptor coupled to four distinct intracellular signaling pathways: 1) elevation of intracellular calcium, 2) generation of inositol phosphates, 3) potentiation of cAMP accumulation, and 4) massive release of arachidonic acid. The coupling between the H1 receptor and each intracellular signaling pathway was dependent on several different G proteins. The massive release of arachidonic acid was likely due to stimulation of PLA2 (17).

Our experiments showed that the H1-receptorantagonist DPH could inhibit histamine-stimulated signaling leading to bronchial epithelial IL-8 expression. Increasing doses of DPH above 1 µM showed less suppression of histamine activation, consistent with DPH acting as a partial agonist at the H1 receptor (2, 12).

We observed a good correlation between the inhibitory effects of DPH, MK-886, and Zileuton upon histamine-stimulated IL-8 transcriptional activation, IL-8 mRNA levels, and IL-8 protein secretion. From this correlation, we conclude that the major level of regulation of IL-8 expression in bronchial epithelial cells occurs at the level of transcription, as has been described previously (25). It also justifies our use of the IL-8 luciferase reporter gene assay as a biologically relevant screening assay for evaluating immunomodulators of bronchial epithelial cell IL-8 expression.

The transcriptional induction of IL-8 in diverse cell types depends on activation of NF-kappa B, and IL-8 NF-kappa B activation can be augmented by activation of NF-IL-6 and AP-1 (22). Our experiments revealed that histamine could induce IL-8 NF-kappa B and AP-1 DNA-binding activities in 16HBE cells. The histamine-induced IL-8 NF-kappa B DNA-binding complex in bronchial epithelial cells contained immunoreactive p65, in agreement with the immunochemical identification of p65 in IL-8 NF-kappa B in glioblastoma and gastric carcinoma cell lines (22). Although DPH inhibited histamine induction of both IL-8 NF-kappa B and AP-1 DNA-binding activities, MK-886 inhibited only NF-kappa B binding.

In our studies, MK-886 and Zileuton inhibited histamine-stimulated IL-8 transcription and IL-8 protein secretion. MK-886 inhibition of IL-8 NF-kappa B and not of AP-1 DNA-binding activity correlated with the inhibition of IL-8 transcription and IL-8 expression, underscoring the essential requirement for activation of NF-kappa B for bronchial epithelial cell IL-8 expression. The inhibitory effects of MK-886 and Zileuton implicate 5-lipoxygenase signaling and lipid signaling intermediates such as LTB4 in histamine induction of IL-8 transcription. We demonstrated that 16HBE cells produced LTB4 after stimulation with histamine. Further support that 5-lipoxygenase signaling is involved in the histamine-induced activation of IL-8 NF-kappa B was provided with the alternative FLAP inhibitor L-655,238, which also caused inhibition of IL-8 transcriptional activation. Compellingly, the inhibition of histamine signaling with FLAP inhibitors and Zileuton could be rescued with exogenous LTB4. Our conclusion is that histamine stimulates 16HBE cell production of endogenous LTB4, which acts downstream to activate NF-kappa B and IL-8 transcription; FLAP inhibitors and Zileuton diminish the production of endogenous LTB4, decreasing IL-8 transcription, and exogenous LTB4 can restore downstream activation of NF-kappa B and IL-8.

Activation of 5-lipoxygenase can generate reactive oxygen intermediates capable of activating NF-kappa B (3, 6, 20). Los et al. (20) analyzed T lymphocyte activation triggered through the CD28 receptor and showed a role for 5-lipoxygenase and LTB4 in activation of NF-kappa B DNA-binding activity and expression of IL-2. The FLAP inhibitor MK-886 potently inhibited NF-kappa B DNA-binding activity and IL-2 secretion triggered by anti-CD28 and anti-CD3 activation (20). MK-886 showed no inhibitory effects at the level of induced DNA binding of the transcription factors NF of activated T cells or AP-1 (20).

Recently, LTB4 has been identified as an activating ligand of the ryanodine-receptor intracellular calcium- release channel (32). Striggow and Ehrlich (32) showed that 100 nM LTB4 was sufficient for full activation of the ryanodine receptor purified from canine cerebellar microsomes and reconstituted into planar lipid bilayers. Interestingly, arachidonic acid, a precursor for synthesis of LTB4, was found to be an inhibitor of the inositol 1,4,5-trisphosphate (IP3)-gated intracellular calcium channel. The authors concluded that the differential effects of arachidonic acid and LTB4 on the IP3 receptor and ryanodine receptor would provide an additional level of complexity in calcium signaling in cells that contained both forms of intracellular calcium channels (32). In neutrophils loaded with the fluorescent calcium indicator quin 2, LTB4 caused a rapid rise in intracellular calcium due both to influx from the extracellular medium and to efflux from intracellular stores (18).

A cell-surface membrane receptor for LTB4 has recently been cloned from retinoic acid-differentiated HL-60 cells (35). The cDNA predicts a seven-span receptor with limited sequence similarities to the human IL-8 receptor and formyl-peptide receptor (35). Expression of the LTB4-receptor cDNA in heterologous cells showed that 100 nM LTB4 induced a rapid increase in intracellular calcium and IP3 accumulation (35). These increases were partially inhibited by pertussis toxin, implying that the LTB4 receptor couples to intracellular signaling pathways through pertussis-sensitive and -insensitive G proteins (35). Northern analysis of LTB4-receptor expression showed expression in leukocytes, spleen, and thymus, with no expression detected in lung tissues.

LTB4 has also recently been shown to be a direct intracellular activating ligand for the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR; see Ref. 9). LTB4 was shown to specifically activate PPARalpha -driven transcription and also to bind directly to a fusion protein consisting of bacterial glutatathione S-transferase fused to the ligand-binding domain of PPARalpha .

The potent induction of cPLA2 mRNA identifies cPLA2 as an additional gene target of histamine induction. The induction of cPLA2 mRNA is likely followed by translation into protein. However, we believe that increases in cPLA2 protein will occur too late to contribute significantly to histamine-stimulated production of LTB4 and NF-kappa B and IL-8 transcriptional activation at 6 h. Nevertheless, upregulation of cPLA2 by histamine likely increases the later generation of arachidonic acid substrates for 5-lipoxygenase, as described by Leurs et al. (17) in H1 receptor-transfected Chinese hamster ovary cells. In this manner, histamine stimulation could upregulate its own signaling pathway in a positive feedback loop. This might be the basis for the increased sensitivity to histamine (1 µM) described in epithelial cells derived from airways of asthmatics but not from normal volunteers (5).

We propose that LTB4, produced endogenously by bronchial epithelial cell 5-lipoxygenase or exogenously from activated macrophages or neutrophils, activates bronchial cell transcription factor NF-kappa B and inflammatory cytokine gene expression. This activation might involve transient increases in calcium mediated through intracellular ryanodine receptors (32) or possibly through plasma membrane, G protein-coupled LTB4 receptors (35). Alternatively, LTB4 might activate NF-kappa B through mechanisms related to the direct intranuclear activation of the PPARalpha transcription factor (9).

Our findings that histamine acts through H1 receptors to stimulate bronchial epithelial cell synthesis of LTB4, which induces IL-8 expression, suggest that therapeutic strategies to decrease airway inflammation should include efforts directed at histamine-triggered and leukotriene-mediated intracellular inflammatory signaling pathways.

    ACKNOWLEDGEMENTS

We thank Glenn D. Rosen for helpful discussions and review of the manuscript.

    FOOTNOTES

This work was supported by grants from the California Affiliate of the American Lung Association, the Donald E. and Delia B. Baxter Foundation, and National Institute of Allergy and Infectious Diseases Grants K04-AI-01147 and R01-AI-39624 to P. N. Kao. Y. Aoki received salary support from Saga Medical School, Saga, Japan.

Address for reprint requests: P. N. Kao, Pulmonary and Critical Care Medicine, Stanford Univ. Medical Center, Stanford, CA 94305-5236.

Received 10 October 1997; accepted in final form 6 March 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 274(6):L1030-L1039
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