help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Arima, M.
Right arrow Articles by Schleimer, R. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Arima, M.
Right arrow Articles by Schleimer, R. P.
Am. J. Respir. Cell Mol. Biol., Volume 21, Number 6, December 1999 684-692

Expression of Interleukin-16 by Human Epithelial Cells
Inhibition by Dexamethasone

Masafumi Arima, Jim Plitt, Cristiana Stellato, Carol Bickel, Shinji Motojima, Sohei Makino, Takeshi Fukuda, and Robert P. Schleimer

Johns Hopkins Asthma and Allergy Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; and Dokkyo University School of Medicine, Mibu, Tochigi, Japan


    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Production of chemoattractants by bronchial epithelial cells may contribute to the local accumulation of inflammatory cells in patients with bronchial asthma and other pulmonary diseases. Recently, interleukin (IL)-16 (lymphocyte chemoattractant factor) was reported to be a potent chemotactic stimulus for CD4+ T lymphocytes and eosinophils, the types of leukocyte found in the proximity of bronchial epithelium in asthmatic individuals. To test the possibility that bronchial epithelial cells produce IL-16, we analyzed RNA and culture supernatants from the human bronchial epithelial cell line BEAS-2B, using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. BEAS-2B constitutively expressed IL-16 messenger RNA (mRNA) and protein; IL-16 expression was significantly upregulated in a concentration-dependent manner within 24 h by stimulation with histamine, IL-1beta , or tumor necrosis factor (TNF)-alpha whereas interferon-gamma did not significantly increase IL-16. Findings in BEAS-2B cells were confirmed in primary bronchial epithelial cells. Using TA cloning, IL-16 was cloned from BEAS-2B airway epithelial cells. Sequence analysis confirmed its near identity with lymphocyte-derived IL-16. The combination of IL-1beta and TNF-alpha had an additive effect on IL-16 expression. This combination of cytokines also had a priming effect on histamine-induced IL-16 mRNA expression, which was observed within 24 h and which increased to at least 48 h after stimulation. The IL-16 expression induced by histamine and combined cytokines was significantly inhibited by pretreatment with the protein synthesis inhibitor cycloheximide (10 µg/ml). Pretreatment with dexamethasone also significantly suppressed the expression of IL-16, in a concentration-dependent manner. Sputum samples from asthmatic subjects were found to have higher levels of IL-16 than were samples from subjects with other pulmonary inflammatory diseases. These findings suggest that bronchial epithelial cells have the capacity to produce IL-16 after stimulation with histamine, IL-1beta , and TNF-alpha , and raise the possibility that epithelium-derived IL-16 may play a role in recruitment of eosinophils and CD4+ T lymphocytes in the airways. Downregulation of IL-16 expression by dexamethasone suggests that glucocorticoids may inhibit airway inflammation partly by suppressing the synthesis of inflammatory cytokines including IL-16.


    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Inflammatory changes in airway mucosa have been implicated in the pathogenesis of bronchial asthma. Mucosal invasion by activated eosinophils has been particularly recognized, and eosinophil products are suspected of playing an important role in the pathogenesis of asthma and other inflammatory diseases of the airways (1). CD4+ T cells have also been shown to be recruited to airway mucosa and bronchoalveolar lavage fluid (BALF) in asthmatic individuals (2). The importance of CD4+ T cells is indicated by in vivo studies in animal models of asthma in which airway hypersensitivity and eosinophilia were lacking in CD4+ T-cell-deficient mice after antigen challenge (3, 4). Conversely, normal rats receiving transfers of sensitized CD4+ T cells developed into late-phase responders, with infiltration of eosinophils following antigen challenge (5). It has been suggested that the role of CD4+ T cells in asthma may be mediated by release of cytokines including interleukin (IL)-3, IL-4, and IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are involved in the proliferation, prolongation of survival, activation, and migration of eosinophils (2, 6). Thus, the recruitment of eosinophils and CD4+ T cells into the airway may be a crucial occurrence in asthmatic individuals. Recent studies have suggested that airway inflammation may be perpetuated by bronchial epithelial cells themselves. Epithelial cells have been shown to produce numerous inflammatory mediators, such as platelet activating factor (12) and prostaglandins (13). Bronchial epithelial cells also produce a wide variety of proinflammatory cytokines, such as IL-1, IL-6, IL-8, GM-CSF, tumor necrosis factor (TNF), macrophage chemotactic protein (MCP)-1, and regulated on activation, normal T cell expressed and secreted (RANTES) (12). Production of cytokines and chemoattractants by bronchial epithelial cells may contribute to the local accumulation of inflammatory cells in patients with bronchial asthma and other airway inflammatory diseases.

Recently, IL-16 has been reported to be a potent chemotactic stimulus for CD4+ T lymphocytes (17) and eosinophils (18). IL-16 is a 14-kD protein that is released within 2-4 h from eosinophils (19) and CD8+ T cells stimulated with histamine or serotonin (20, 21). Cruikshank, and coworkers recently identified IL-16 and macrophage inflammatory protein (MIP)-1alpha in BALF at 6 h after antigen challenge of asthmatic subjects, and showed that the great majority of the lymphocyte chemoattractant activity in the BALF was attributable to IL-16 and MIP-1alpha (22). Bellini and associates have reported observing chemotactic activity for CD4+ T cells in the supernatants of primary bronchial epithelial cell cultures isolated from patients with bronchial asthma following in vitro stimulation with histamine (10-6 M) for 48 h (23), and speculated that IL-16 might have been responsible for this activity. To test the possibility that bronchial epithelial cells produce IL-16, we analyzed messenger RNA (mRNA) and supernatants from human bronchial epithelial cells, using reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay (ELISA). We found in the study reported here that epithelial cells express IL-16 mRNA and protein, and that it is increased in a concentration-dependent manner 24 h after stimulation with histamine, IL-1beta , and TNF-alpha , but not interferon (IFN)-gamma . The combination of IL-1beta and TNF-alpha showed an additive effect, and expression of IL-16 mRNA was significantly suppressed by the glucocorticoid dexamethasone.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Reagents

The following reagents were purchased from the indicated sources: Hanks' F12 medium (Biowhitaker, Walkersville, MD), Dulbecco's modified Eagle's medium (DMEM), Ca2+- and Mg2+-free Hanks' balanced salt solution (HBSS), fetal calf serum (FCS), penicillin/streptomycin solution, agarose, trypsin/ethylenediaminetetraacetic acid (EDTA) solution, reverse transcription reagents kit (Moloney murine leukemia virus [MMLV] reverse transcriptase, 5× buffer, dithiothreitol [DTT]), ribonuclease (RNase) inhibitor, PCR reagents kit (10× buffer, Taq polymerase, MgCl) (Gibco BRL, Gaithersburg, MD), RNAzol B (Tel-Test Inc., Friendswood, TX), oligo-deoxythymidine (oligo-dT) (Boehringer-Mannheim, Indianapolis, IN), chloroform, isopropanol (Fisher Scientific, Fernwood, NJ), pGEM-T vector system (Promega Corp., Madison, WI), and plasmid Midi Kit and QIAquick Gel Extraction Kit (Qiagen Inc., Chatsworth, CA). Primers for IL-16 and beta -actin were generated by Bioserve Biotechnologies, Inc. (Laurel, MD). Dexamethasone and beta -estradiol were purchased from Sigma Chemical Co. (St. Louis, MO) and stored as 0.1 M stock solutions in dimethylsulfoxide (DMSO) at -20°C. Human recombinant TNF-alpha , IL-1beta , and IFN-gamma were purchased from R&D Systems (Minneapolis, MN) and were stored as 1 µg/ml stock solutions in phosphate-buffered saline supplemented with 0.1% bovine serum albumin (BSA) at -20°C.

Cell Culture

The study utilized the BEAS-2B cell line (24), which was derived from human bronchial epithelium transformed by an adenovirus 12-SV40 hybrid virus. This cell line was generously supplied by Dr. Curtis Harris of the National Institutes of Health, Bethesda, MD. BEAS-2B cells were cultured in F12/DMEM supplemented with 5% FCS, penicillin (100 U/ml), and streptomycin (100 mg/ml) in 25-cm2 tissue culture flasks at 37°C in 5% CO2. Only cells that had reached 80-90% confluence were used for experiments. Viability of cells, assessed by staining with erythrosin B, was always more than 98% of cells harvested, and viability was more than 80% for cells treated with cycloheximide. Additional studies utilized NHBE 4683 normal human bronchial epithelial cells (Lot No. 17714; Clonetics Corp.), which were obtained from a 60-yr-old female donor. NHBE 4683 cells were cultured in a complete medium (bronchial/tracheal epithelial cell growth medium) that is a modified light harvesting complex-9 formulation, and which was supplemented with bovine pituitary extract (7.5 mg/ml), hydrocortisone (0.5 µg/ml), human epithelial growth factor (0.5 µg/ml), epinephrine (0.5 mg/ml), transferrin (10 mg/ml), insulin (0.5 mg/ml), retinoic acid (0.1 µg/ml), triiodothyronine (6.5 µg/ml), gentamycin (50 mg/ ml), and bovine serum albumin, fatty acid free (50 mg/ml).

Preparation of mRNA and RT-PCR Analysis

A total of 5-8 × 106 cells/flask were detached by incubation in 0.05% trypsin and 0.53 mM EDTA-4Na in HBSS. Total mRNA was then extracted from the cells with the RNAzol B extraction technique (25), and was stored as a 1 µg/µl stock solution in diethylpyrocarbonate-treated water at -80°C. An RT-PCR procedure was performed to determine relative quantities of mRNA for IL-16, using an adaptation of methods described elsewhere (26, 27). Briefly, the first-strand complementary DNA (cDNA) was synthesized from mRNA, using oligo-dT primer. A 20-µl reaction volume mix, containing 1 µg mRNA, 2.5 pmol oligo-dT primer, 4 µl of 5× synthesis buffer (250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, pH 8.3), 10 mM DTT, 2 mM deoxynucleotide triphosphate (dNTP) mixture, 20 U RNase inhibitor, and 100 U MMLV reverse transcriptase, was incubated at 37°C for 30 min in an Omnigene thermocycler (Hybaid, Holbrook, NY). At the end of this incubation, the reaction was stopped by heating at 99°C for 5 min, and the reaction mixuture was then cooled to room temperature (RT). Five microliters of cDNA from the previous reaction were amplified in a 50-µl PCR reaction volume containing 5 µl of 10× PCR buffer (200 mM Tris-HCl, 500 mM KCl, 2 mM MgCl2, pH 8.4), 200 µM dNTP mixture, 20 pmol of each primer for IL-16, 1 U Taq DNA polymerase, and 30 µl H2O. Each cycle consisted of a 1 min denaturation at 94°C, 1 min annealing at 60°C, and 2 min extension at 72°C in the thermocycler.

To verify that equal amounts of RNA were added in each PCR within an experiment, primers for the housekeeping gene beta -actin were used in each experiment. For IL-16 and beta -actin gene products, the optimum number of cycles was determined experimentally, and was defined as the number of cycles that would produce a detectable concentration that was well below saturating conditions. The IL-16 PCR primers consisted of base pairs 1,504-1,893 of the coding region of the IL-16 gene, and had the sequences 5'-ATGCCCGACCTCAACTCC-3' and 5'-CTAGGAGTCTCCAGCAGC-3'; the expected PCR product size is 389 bp. The primers for beta -actin were 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3' and 5'-CTAGAAGCATTGCGGTGGACGATGGAGGG-3'; the expected PCR product size is 600 bp. The PCR product was subjected to electrophoresis at 100 V on a 1.5% agarose gel in TAE buffer. PCR products were detected by ethidium bromide staining. PCR products in electrophoretic analyses were quantified by video densitometry, using a gel documentation system configured by UVP (San Gabriel) interfaced with a Macintosh Performa computer (Apple, Inc., Cupertino, CA) containing Image 1.53 software (NIH Public Software, National Institutes of Health, Bethesda, MD). The level of IL-16 expression was quantified by calculating the ratio of densitometric readings of the bands for IL-16 and beta -actin from the same cDNA. beta -Actin densitometric values did not usually differ by more than 1.5-fold. Changes in IL-16 expression were expressed as a percent of the level in unstimulated control cells.

Assessment of the Expression of IL-16 mRNA in Epithelial Cells

To assess the effect of stimuli on IL-16 gene expression, we treated epithelial cell monolayers with control medium, histamine (10-8, 10-7, 10-6, 10-5 M), TNF-alpha (1, 10, 100 ng/ml), IL-1beta (1, 10, 100 ng/ml), or IFN-gamma (1, 10, 100 ng/ml), or with combinations of these stimuli. For time- course experiments, cultures were harvested after stimulation with either medium alone or histamine (10-6 M), IL-1beta (10 ng/ml), and TNF-alpha (100 ng/ml) for 3, 6, 12, 24, or 48 h. To assess the protein synthesis-dependence of cytokine-induced IL-16 expression, we extracted total mRNA from cells pretreated for 2 h with either medium alone or with the protein synthesis inhibitor cycloheximide (10 µg/ml) before stimulating the cells with the combination of histamine (10-6 M), IL-1beta (10 ng/ml), and TNF-alpha (100 ng/ml). To assess the effect of glucocorticoids on IL-16 gene expression, cells were pretreated for 24 h with dexamethasone (10-11, 10-9, 10-7 M) or an equivalent amount of DMSO diluent before stimulation. beta -Estradiol (10-7 M) was used as a control steroid. Specific IL-16 and beta -actin mRNAs were visualized as PCR products in electrophoresis gels appropiate for the expected molecular sizes of these mRNAs. When IL-16 or beta -actin cDNA was amplified through 28 PCR cycles, a linear correlation was observed between the quantity of input RNA and the optical density (OD) of the PCR product (data not shown).

ELISA Analysis of IL-16 in Epithelial Cell Supernatants

BEAS-2B and NHBE 4683 cells were grown to near confluence (n = 4) and stimulated with histamine (10-6 M), IL-1beta (100 ng/ml), and TNF-alpha (100 ng/ml). IL-16 in the culture supernatant was measured at various time points up to 48 h after stimulation, as well as in supernatant from unstimulated cells, by using a solid-phase sandwich ELISA (BioSource International, Inc.). The sensitivity of this assay was approximately 5 pg/ml.

Analysis of IL-16 mRNA Stability

To investigate the stabilizing effect of histamine, IL-1, or TNF-alpha on IL-16 mRNA, we incubated near-confluent cultured BEAS-2B cells with histamine (10-7 M) and IL-1beta (10 ng/ml) or TNF-alpha (100 ng/ml) for 48 h. Immediately after the incubation, cells were incubated with actinomycin D (5 µg/ml). The level of expression of IL-16 mRNA was investigated immediately and after 3, 10, and 20 h.

Sequence Analysis of PCR Products

The TA cloning method was utilized to clone PCR products from the BEAS-2B cell line with the pGEM-T vector system (28, 29). The purified clone products from BEAS-2B cells were sequenced by using SP6 and T7 primers with the fluorescent dideoxy method on an automated DNA sequencer (Model 373a; Perkin Elmer Corp., Foster City, CA). All consensus sequences were generated with Sequencer Software (Gene Codes Corp., Ann Arbor, MI). The consensus sequence of four of six clones derived from the RT- PCR products of RNA from BEAS-2B cells was identical with that of the published sequence of the coding region of the lymphocyte IL-16 gene (17). However, in two separately cloned cDNA products we observed a missing codon (-CAG-), as also described elsewhere (30).

Analysis of IL-16 Proteins in Sputum

A group of 23 patients with chronic symptoms of bronchial asthma (14 men and nine women) were examined (Table 1). All patients were treated at the Department of Medicine and Clinical Immunology of Dokkyo University School of Medicine, and were enrolled after informed consent was obtained. The patients' mean age was 36 yr; the patients ranged in age from 25 to 69 yr. Asthma was defined according to the international consensus report on the diagnosis and management of asthma in patients who had had episodic attacks of dyspnea with wheezing and a fluctuation in FEV1 or in peak expiratory flow of more than 20%. The patients were classified according to severity of asthma: eight cases were classified as mild asthma, defined as dyspnea attacks less often than three times a week, and 15 cases were classified as moderate asthma, defined as dyspnea attacks more often than three times a week. No cases were classified as severe asthma, defined as daily dyspnea attacks that required administration of corticosteroids. All of the patients were taking bronchodilators only. Sputum was collected during symptom-free states after inhalation of 10% saline, using a DeVilbiss 646 nebulizer for 3-5 min. As a disease control, sputum samples were collected from 10 patients with diffuse panbronchiolitis and seven patients with bronchiectasis. They were 10 men and 7 women ranging in age from 19 to 62 yr (Table 1).

                              
View this table:
[in this window]
[in a new window]
 

TABLE 1
Patient characteristics

Preparation of Sputum

Sputum samples were treated as described previously (31). Briefly, sputum was expectorated into a Petri dish and transferred to a tube after the watery portion was removed. The sputum was mixed with a vortex mixer for 1 min, after a 3-fold volume of physiologic saline was added. Samples were then centrifuged at 4°C for 30 min at 40,000 × g. The supernatant was collected through a 4-µm filter (Biofield, Tokyo, Japan) and is referred to as the sputum extract. The sputum extracts were stored at -20°C until assay for IL-16.

Statistical Analysis

Data are expressed as mean ± SD. Statistical analysis of between-group comparisons was done through analysis of variance, with post hoc analysis through Fisher's protected least-squares difference test). A value of P < 0.05 was considered significant.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Densitometric analysis of RT-PCR products showed that BEAS-2B cells exhibited basal expression of IL-16 mRNA (Figure 1). Activation of BEAS-2B cells with TNF-alpha or IL-1beta for 24 h increased IL-16 mRNA expression in a concentration-dependent manner. The maximum IL-16 induction was observed at a concentration of 100 ng/ml of TNF-alpha and 10 ng/ml of IL-1beta . Expression of IL-16 mRNA did not significantly change after 24 h of incubation of BEAS-2B cells with IFN-gamma (Figure 1C). The combination of IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml) caused an additive upregulation of IL-16 mRNA at 24 h (Figure 2). When combined with the other cytokines, IFN-gamma failed to show a significant additive or synergistic effect (not shown). Histamine-induced expression of IL-16 mRNA was magnified by combination of histamine with IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml); in this case, even the lowest concentration of histamine tested (10-7 M) induced significant expression of IL-16 mRNA (Figure 3). These results indicate that signaling mechanisms for IL-1beta , TNF-alpha , and histamine may be distinct. Time-course experiments showed that expression of IL-16 mRNA was increased by 24 h and continued to rise at 48 h after stimulation with the combination of histamine (10-6 M), IL-1beta (10 ng/ml), and TNF-alpha (100 ng/ml) (Figure 4).


View larger version (25K):
[in this window]
[in a new window]
  		Figure 1.   (A) Upregulation of IL-16 mRNA expression by TNF-alpha in BEAS-2B cells. Top: Densitometric analysis (mean ± SD, n = 6) of RT-PCR products shows that TNF-alpha increased IL-16 expression in a concentration-dependent manner after 24 h of incubation. Bottom: Representative electrophoresis showing induction of expression of IL-16 mRNA by TNF-alpha . (B) Upregulation of IL-16 mRNA expression by IL-1beta in BEAS-2B cells. Top: Densitometric analysis (mean ± SD, n = 6) of RT-PCR products shows that IL-1beta increased IL-16 expression in a concentration-dependent manner after 24 h of incubation. Bottom: Representative electrophoresis showing expression of IL-16 induced by IL-1beta . (C) Lack of upregulation of IL-16 mRNA expression by IFN-gamma in BEAS-2B cells. Top: Densitometric analysis (mean ± SD, n = 6) of RT-PCR products. Bottom: Representative electrophoresis. * P < 0.05; **P < 0.01 compared with control.


View larger version (24K):
[in this window]
[in a new window]
  		Figure 2.   Additive effect of IL-1beta (10 ng/ml) and TNF-alpha (100 ng/ml) on upregulation of IL-16 mRNA expression in BEAS-2B cells. Top: Densitometric analysis (mean ± SD, n = 5) of RT- PCR products shows that the combination of IL-1beta and TNF-alpha increased IL-16 expression at 24 h. Bottom: Representative electrophoresis showing expression of IL-16 by stimulation with indicated combinations of IL-1beta and TNF-alpha . * P < 0.05; ** P < 0.01 compared with control.


View larger version (36K):
[in this window]
[in a new window]
  		Figure 3.   Additive effect of IL-1beta and TNF-alpha with histamine on upregulation of IL-16 mRNA. Top: Densitometric analysis (mean ± SD, n = 6) of RT-PCR products shows additivity of IL-1beta and TNF-alpha with histamine-induced expression of IL-16 mRNA after 24 h of incubation. Bottom: Representative electrophoresis. * P < 0.05; ** P < 0.01 compared with control.


View larger version (33K):
[in this window]
[in a new window]
  		Figure 4.   Kinetics of induction of IL-16 mRNA in unstimulated cells (open circles) and cells stimulated with histamine (10-6 M) and IL-1beta (10 ng/ml) (closed circles). Top: Densitometric analysis (mean ± SD, n = 3) of RT-PCR products. *P < 0.05 compared with control. Bottom: Representative electrophoresis.

Expression of IL-16 mRNA at 48 h after stimulation was significantly inhibited by pretreatment with the protein synthesis inhibitor cycloheximide (10 µg/ml), indicating that protein synthesis may be required for transcriptional induction of IL-16 mRNA (Figure 5A). To determine whether activating stimuli increased IL-16 mRNA indirectly via stabilization, we stimulated BEAS-2B cells with histamine, IL-1beta , and TNF-alpha for 48 h and then treated them with actinomycin D to block further transcription. The level of mRNA was assayed immediatedly and then periodically over a 20-h period. Figure 5B shows that the decay of mRNA was similar in unstimulated and stimulated cells. Half-lives of IL-16 mRNA in unstimulated and histamine-stimulated cells were 8.3 and 15.7 h, respectively. The half-life after stimulation with TNF-alpha was 6.3 h, which was not influenced by histamine treatment. The half-life of IL-1beta -induced IL-16 mRNA was 3.2 h, which was prolonged to 8.2 h by histamine treatment (Figure 5B). The expression of IL-16 mRNA was significantly suppressed by dexamethasone in a concentration-dependent manner after a 48 h incubation (Figure 6). The maximum effect was observed at a dexamethasone concentration of 10-7 M, whereas treatment with beta -estradiol or DMSO alone had no effect on mRNA expression (Figure 6).


View larger version (29K):
[in this window]
[in a new window]
 

View larger version (23K):
[in this window]
[in a new window]
  		Figure 5.   (A) Effect of a 2-h preincubation with cycloheximide (10 µg/ml) on IL-16 mRNA levels 48 h after stimulation with the combination of histamine (10-6 M ), IL-1beta (10 ng/ml), and TNF-alpha (100 ng/ml). Top: Densitometric analysis (mean ± SD, n = 3) of RT-PCR products shows that cycloheximide pretreatment inhibited histamine- and cytokine-induced IL-16 mRNA expression. * P < 0.05 compared with control. Bottom: Representative electrophoresis. (B) Influence of histamine on decay kinetics of IL-16 mRNA in BEAS-2B cells. Cells were stimulated for 48 h as indicated, treated with actinomycin D (5 µg/ml), and harvested for mRNA analysis at the indicated times. ** P < 0.01 versus stimulated cells in the absence of histamine.


View larger version (38K):
[in this window]
[in a new window]
  		Figure 6.   Effect of a 24-h preincubation with steroids on IL-16 mRNA expression induced by histamine (10-6 M), IL-1beta (10 ng/ ml), and TNF-alpha (100 ng/ml). Top: Densitometric analysis (mean ± SD, n = 3) of RT-PCR products shows that dexamethasone inhibited IL-16 mRNA expression in a concentration-dependent manner. * P < 0.05 compared with control. Bottom: Representative electrophoresis.

To confirm the mRNA results and extend the findings to primary bronchial epithelial cells, we used an ELISA to measure IL-16 protein in supernatants of both BEAS-2B and primary bronchial epithelial cells. IL-16 levels increased in a time-dependent manner for up to 48 h in supernatants from stimulated cells. Unstimulated cells produced low levels of IL-16 throughout the experiment (Figure 7A). Dexamethasone reduced levels of IL-16 protein in supernatants of both BEAS-2B and NHBE 4683 cells that had been stimulated, but did not influence baseline levels of IL-16 protein (Figure 7B).


View larger version (18K):
[in this window]
[in a new window]
 

View larger version (18K):
[in this window]
[in a new window]
  		Figure 7.   (A) Levels of IL-16 protein in supernatants from cultured epithelial cells after stimulation with histamine (10-6 M), IL-1beta (100 ng/ml), TNF-alpha (100 ng/ml), or medium. Data for both BEAS-2B (stimulated, open circles; nonstimulated, closed squares) and NHBE 4683 (stimulated, closed circles; nonstimulated, open squares) cells are shown as mean ± SEM (n = 4). ** P =< 0.01; * P =< 0.05 compared with control cultures. (B) Effect of dexamethasone on production of IL-16 by epithelial cells (open bars: BEAS-2B cells, closed bars: NHBE 4683 cells). The data are given as mean ± SEM (n = 3). ** P =< 0.01, * P =< 0.05 compared with control cultures.

To determine whether IL-16 is increased in asthma, we collected sputum from 23 subjects with bronchial asthma and 17 controls with other airway diseases including panbronchiolitis and bronchiectasis. IL-16 levels in sputum extracts of asthmatic subjects were 639 ± 89 pg/ml, which was significantly higher (P < 0.05) than levels in sputum extracts from control-disease subjects, which were 189 ± 90 pg/ml. There were modestly higher levels in sputum from pateints with moderate asthma (683 ± 55 pg/ml) than in those with mild asthma (558 ± 86 pg/ml) (P = 0.003).

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In the present study, human bronchial epithelial cells were found through a PCR-based assay and a specific ELISA to constitutively express IL-16 mRNA and protein. Sequence analysis of six independently isolated clones of IL-16 RNA from epithelial cells revealed identity of four of the clones to the reported sequence of lymphocyte-derived IL-16 mRNA (17). Two clones were missing one codon previously described as being deleted in some human IL-16 alleles by Baier and colleagues (30). IL-16 mRNA expression was significantly increased by stimulation with histamine, IL-1beta , or TNF-alpha in a concentration-dependent manner, whereas IFN-gamma did not significantly increase IL-16 mRNA. The combination of IL-1beta , histamine, and TNF-alpha caused a significant increase in IL-16 mRNA expression. Recently, Laberge and coworkers reported the expression of IL-16 mRNA in human bronchial epithelial cells in vivo as detected with immunohistochemistry and in situ hybridization (32, 33). Two other reported sources of IL-16 are CD8+ T cells (20, 21) and eosinophils (19) isolated from normal subjects. CD8+ T cells release IL-16 within 4 h after stimulation with histamine at high concentrations (10-4-10-2 M ) (21). Eosinophils release IL-16 by 24 h after culture with GM-CSF (19). In the same series of studies, it was reported that CD8+ T cells and freshly isolated eosinophils constitutively express IL-16 mRNA. Because histamine did not upregulate IL-16 mRNA expression in CD8+ T cells during a 4 h period in these studies, it was concluded that preformed IL-16 was secreted independently of transcriptional upregulation. We observed both constitutive and inducible expression of IL-16 mRNA and IL-16 protein in BEAS-2B and primary bronchial epithelial cells.

Pleiotropic proinflammatory cytokines such as IL-1beta , TNF-alpha , and IFN-gamma have been observed in airway mucosa or BALF during symptomatic asthma or after antigen challenge in asthmatic patients (2, 34). These cytokines have thus been suggested to potentially play an important role in bronchial inflammation. TNF-alpha and/or IL-1beta induce airway epithelial cells to express and/or release IL-6, GM-CSF, and the chemokines IL-8, MCP-1, granulocyte chemoattractant (GRO), RANTES, and MCP-4 (16, 38- 40). Interestingly, IL-1beta and TNF-alpha are themselves produced in small amounts by bronchial epithelial cells (41- 43). The present study demonstrated that TNF-alpha and IL-1beta also induce IL-16 in epithelial cells. Studies with specific blockers suggest a role for IL-1 and TNF in airway hyperreactivity in guinea pigs (44) and mice (45). Although IFN-gamma may also be involved in airway inflammation, we found no significant effect of IFN-gamma on the expression of IL-16 mRNA. We detected IL-16 in the sputum of subjects with asthma as well as subjects with other airway inflammatory diseases. Whether the source of this IL-16 is epithelial cells has not been established. CD4+ T cells are also involved in other airways diseases, including pulmonary sarcoidosis, hypersensitivity pneumonitis, allergic bronchopulmonary aspergillosis, eosinophilic pneumonias, and others.

Dexamethasone profoundly inhibited the expression of IL-16 induced by the combination of histamine, IL-1beta , and TNF-alpha . An important molecular mechanism of steroid action in inhibiting cytokine production is now considered to be direct interaction between the activated glucocorticoid receptor and transcription factors such as NF-kappa B or activator protein-1 (AP-1), which are heterodimeric transcription factors derived from the c-jun and fos oncogene subfamilies (46). These factors are proteins that bind to regulatory sequences, usually in the 5' upstream promoter region of target genes, to regulate gene transcription. Expression of the c-fos protooncogene was observed in bronchial epithelium of asthmatic but not normal subjects (49), and NF-kappa B activation has been demonstrated in human lung and in airway epithelial cells (50). Activation of AP-1 was observed in human lung after stimulation with TNF-alpha and IL-1beta (51). It is thus conceivable that upregulation of IL-16 expression results from activation of these transcription factors by IL-1beta and/or TNF-alpha . Recently, Laberge and associates showed that treatment with steroid in vivo reduced IL-16 production in human subjects (33).

Takizawa and colleagues have reported that histamine (10-6-10-3 M) induces the release of IL-6, IL-8, and GM-CSF in epithelial cells at 6 h after stimulation, without an increase in steady-state levels of IL-6 mRNA, suggesting that some epithelial cytokines may be present intracellularly and may be secreted after stimulation with histamine (55). However, histamine is also known to regulate the expression of some cytokine genes. Histamine was found to enhance IL-1alpha -induced expression of IL-1beta and IL-6 mRNA in mononuclear cells (56, 57). Histamine also promoted expression of endothelin in epithelial cells (58), and has been known to suppress lipopolysaccharide-induced TNF-alpha expression (59). Our results suggest that histamine modulates the expression of IL-16 at the transcriptional level, directly or indirectly, in bronchial epithelial cells. Histamine was found by others to increase the expression of steady-state c-fos mRNA in airway smooth-muscle cells (60) and in bovine chromaffin cells (61). Induction of c-fos and activation of AP-1 by histamine may thus be involved in the transcription of cytokine genes, including that for IL-16. Suppression of IL-16 gene upregulation by the protein synthesis inhibitor cycloheximide indicates that de novo protein synthesis may be involved in the activation of IL-16 gene transcription, supporting a model in which a secondary protein, like c-fos, is involved in the histamine effect. Similarly, the suppressive effect of dexamethasone on IL-16 mRNA expression could result from blockage of AP-1 and other transcription factors.

In conclusion, the present study showed that bronchial epithelial cells have the capacity to express IL-16 after stimulation with histamine, IL-1beta , and TNF-alpha , and that glucocorticoids inhibit its expression. This finding, and the observation that IL-16 is present in the sputum of asthmatic subjects, suggests that epithelial-derived IL-16 protein may be involved in airway mucosal inflammation, and especially in the accumulation of eosinophils and CD4+ T cells that is observed in patients with bronchial asthma, and could be a target of the antiinflammatory effects of glucocorticoids.

    Footnotes

Address correspondence to: Robert P. Schleimer, Ph.D., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. 

(Received in original form January 27, 1999 and in revised form June 2, 1999).

Abbreviations: complementary DNA, cDNA; granulocyte-macrophage colony-stimulating factor, GM-CSF; interleukin-16, IL-16; messenger RNA, mRNA; regulated on activation, normal T cell expressed and secreted, RANTES; tumor necrosis factor-alpha , TNF-alpha .

Acknowledgments: The authors thank Ms. Bonnie Hebden for assistance in the preparation of the manuscript.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

1. Sur, S., C. R. Adolphson, and G. J. Gleich. 1993. In Allergy Principles and Practice, 4th ed. E. Middleton, C. E. Reed, E. F. Ellis, J. N. F. Adkinson, J. W. Yunginger, and W. Busse, editors. Mosby, St. Louis. 169-200.

2. Corrigan, C. J., and A. B. Kay. 1990. CD4 T-lymphocyte activation in acute severe asthma: relationship to disease severity and atopic status. Am. Rev. Respir. Dis. 141: 970-977 [Medline].

3. Gavett, S. H., X. Chen, F. Flinkelman, and M. Wills-Karp. 1994. Depletion of murine CD4+ T lymphocytes prevents antigen-induced airway hyperreactivity and pulmonary eosinophilia. Am. J. Respir. Cell Mol. Biol. 10: 587-593 [Abstract].

4. Nakajima, H., I. Iwamoto, S. Tomoe, R. Matsumura, H. Tomioka, K. Takatsu, and S. Yoshido. 1992. CD4+ T-lymphocytes and interleukin-5 mediate antigen-induced eosinophil infiltration into the mouse trachea. Am. Rev. Respir. Dis. 146: 374-377 [Medline].

5. Watanabe, A., H. Mishima, P. M. Renzi, L. Xu, Q. Hamid, and J. Martin. 1995. Transfer of allergic airway responses with antigen-primed CD4+ but not CD8+ T cells in brown norway rats. J. Clin. Invest. 96: 1303-1310 .

6. Hamid, Q., M. Azzawi, S. Ying, R. Moqbel, A. J. Wardlaw, C. J. Corrigan, B. Bradley, S. R. Durham, J. V. Collins, P. K. Jeffery, D. J. Quint, and A. B. Kay. 1991. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J. Clin. Invest. 87: 1541-1546 .

7. Broide, D. H., and G. S. Firestein. 1991. Endobronchial allergen challenge in asthma. Demonstration of cellular source of granulocyte macrophage colony-stimulating factor by in situ hybridization. J. Clin. Invest. 88: 1048-1053 .

8. Kato, M., M. C. Liu, B. A. Stealey, B. Friedman, L. M. Lichtenstein, S. Permutt, and R. P. Schleimer. 1992. Production of granulocyte/macrophage colony-stimulating factor in human airways during allergen-induced late-phase reactions in atopic subjects. Lymph. Cytokine Res. 11: 287-292 .

9. Krishnaswamy, G., M. C. Liu, S. N. Su, M. Kumai, H. Q. Xiao, D. G. Marsh, and S. K. Huang. 1993. Analysis of cytokine transcripts in the bronchoalveolar lavage cells of patients with asthma. Am. J. Respir. Cell Mol. Biol. 9: 279-286 .

10. Ebisawa, M., M. C. Liu, T. Yamada, M. Kato, L. M. Lichtenstein, B. S. Bochner, and R. P. Schleimer. 1994. Eosinophil transendothelial migration induced by cytokines: II. The potentiation of eosinophil transendothelial migration by eosinophil-active cytokines. J. Immunol. 152: 4590-4597 [Abstract].

11. Ebisawa, M., T. Yamada, C. Bickel, D. Klunk, and R. P. Schleimer. 1994. Eosinophil transendothelial migration induced by cytokines: III. Effect of the chemokine RANTES. J. Immunol. 153: 2153-2160 [Abstract].

12. Holtzman, M. J., B. Ferdman, A. Bohrer, and J. Turk. 1991. Synthesis of the 1-0-Hexadecyl molecular species of platelet-activating factor by airway epithelial and vascular endothelial cells. Biochem. Biophys. Res. Commun. 177: 357-364 [Medline].

13. Churchill, L., F. H. Chilton, J. H. Resau, R. Bascom, W. C. Hubbard, and D. Proud. 1989. Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells. Am. Rev. Respir. Dis. 140: 449-459 [Medline].

14. Marini, M., E. Vittori, J. Hollemborg, and S. Mattoli. 1992. Expression of the potent inflammatory cytokines, granulocyte-macrophage-colony-stimulating factor and interleukin-6 and interleukin-8, in bronchial epithelial cells of patients with asthma. J. Allergy Clin. Immunol. 82: 1001-1009 .

15. Churchill, L., B. Friedman, R. P. Schleimer, and D. Proud. 1992. Production of granulocyte-macrophage colony-stimulating factor by cultured human tracheal epithelial cells. Immunology 75: 189-195 [Medline].

16. Stellato, C., L. A. Beck, G. A. Gorgone, D. Proud, T. J. Schall, S. J. Ono, L. M. Lichtenstein, and R. P. Schleimer. 1995. Expression of the chemokine RANTES by a human bronchial epithelial cell line: modulation by cytokines and glucocorticoids. J. Immunol. 155: 410-418 [Abstract].

17. Cruikshank, W. W., D. M. Center, N. Nisar, B. Natke, A. C. Theodore, and H. Kornfeld. 1994. Molecular and functional analysis of a lymphocyte chemoattractant factor: association of biologic function with CD4 expression. Proc. Natl. Acad. Sci. USA 91: 5109-5113 [Abstract/Free Full Text].

18. Rand, T. H., W. W. Cruikshank, D. M. Center, and P. F. Weller. 1991. CD4-mediated stimulation of human eosinophils - lymphocyte chemoattractant factor and other CD4-binding ligands elicit eosinophil migration. J. Exp. Med. 173: 1521-1528 [Abstract/Free Full Text].

19. Lim, K. G., H.-C. Wan, P. T. Bozza, M. B. Resnick, D. T. W. Wong, W. W. Cruikshank, H. Kornfeld, D. M. Center, and P. F. Weller. 1996. Human eosinophils elaborate lymphocyte chemoattractants. J. Immunol. 156: 2566-2570 [Abstract].

20. Laberge, S., W. W. Cruikshank, H. Kornfeld, and D. M. Center. 1995. Histamine-induced secretion of lymphocyte chemoattractant factor from CD8+ T cell is independent of transcription and translation: evidence of constitutive protein synthesis and strage. J. Immunol. 155: 2902-2910 [Abstract].

21. Laberge, S., W. W. Cruikshank, D. J. Beer, and D. M. Center. 1996. Secretion of IL-16 (lymphocyte chemoattractant factor) from serotonin-stimulated CD8+ T cells in vitro. J. Immunol. 155: 310-315 .

22. Cruikshank, W. W., A. Long, R. E. Tarpy, H. Kornfeld, M. P. Carroll, L. Teran, S. T. Holgate, and D. M. Center. 1995. Early identification of interleukin-16 (lymphocyte chemoattractant factor) and macrophage inflammatory protein-1alpha (MIP1alpha ) in bronchoalveolar lavage fluid of antigen-challenged asthmatics. Am. J. Respir. Cell Mol. Biol. 13: 738-747 [Abstract].

23. Bellini, A., H. Yoshimura, E. Vittori, M. Marini, and S. Mattoli. 1993. Bronchial epithelial cells of patients with asthma release chemoattractant factors for T-lymphocytes. Am. J. Respir. Cell Mol. Biol. 13: 738-747 .

24. Roddel, R. R., Y. Ke, B. I. Gerwin, M. G. McMenamin, J. F. Lechner, R. T. Su, D. E. Brash, O. B. Park, J. S. Rhim, and C. C. Harris. 1988. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV4-hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res. 48: 1904-1909 [Abstract/Free Full Text].

25. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159 [Medline].

26. Gendelman, H. E., R. M. Friedman, S. Jope, L. M. Baca, J. A. Turpin, G. Dveksler, M. S. Metzer, and C. Dieffenbach. 1990. A selective defect of interferon-alpha production in human immunodeficiency virus-infected monocytes. J. Exp. Med. 172: 1433-1442 [Abstract/Free Full Text].

27. Wynn, T. A., I. Eltoum, A. W. Cheever, F. A. Lewis, W. C. Gause, and A. Sher. 1993. Analysis of cytokine mRNA expression during primary granuloma formation induced by eggs of Schistosoma mansoni. J. Immunol. 151: 1430-1440 [Abstract].

28. Marchuk, D., M. Drumm, A. Saulino, and F. Collins. 1991. Construction of T-vectors, a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res. 19: 1156-1160 [Free Full Text].

29. van Willingen, G., J. Donath, E. G. Lapetina, and J. W. Akkerman. 1995. Identification of alpha-subunits of trimeric GTP-binding proteins in human platelets by PCR. Biochem. Biophys. Res. Commun. 214: 254-262 [Medline].

30. Baier, M., A. Werner, N. Bannert, K. Metzner, and R. Kurth. 1995. HIV suppression by interleukin-16. Nature 378: 563-567 [Medline].

31. Adachi, T., S. Motojima, A. Hirata, T. Fukuda, and S. Makino. 1995. Eosinophil viability-enhancing activity in sputum from patients with bronchial asthma: contributions of interleukin-5 and granulocyte/macrophage colony stimulating factor. Am. J. Respir. Crit. Care Med. 151: 618-623 [Abstract].

32. Laberge, S., P. Ernst, O. Chaffar, W.W. Cruikshank, H. Kornfield, D. M. Center, and Q. Hamid. 1997. Increased expression of interleukin-16 in the bronchial mucosa of subjects with atopic asthma. Am. J. Respir. Cell Mol. Biol. 17: 193-202 [Abstract/Free Full Text].

33. Laberge, S., S. R. Durham, O. Ghaffar, S. Rak, D. M. Center, M. Jacobson, and Q. Hamid. 1997. Expression of IL-16 in allergen-induced late-phase nasal responses and relation to topical glucocorticoid treatment. J. Allergy Clin. Immunol. 100: 569-574 [Medline].

34. Marini, M., E. Avoni, J. Hollemborg, and S. Mattoli. 1992. Cytokine messenger RNA profile and cell activation in bronchoalveolar lavage fluid from nonatopic patients with symptomatic asthma. Chest 102: 661-669 [Abstract/Free Full Text].

35. Ying, S., D. S. Robinson, V. Varney, Q. Meng, A. Tsicopoulos, R. Moqbel, S. R. Durham, A. B. Kay, and Q. Hamid. 1991. TNFalpha mRNA expression in allergic inflammation. Clin. Exp. Allergy 21: 745-750 [Medline].

36. Gosset, P., A. Tsicopoulos, B. Wallaert, C. Vannimenus, M. Joseph, A.-B. Tonnel, and A. Capron. 1991. Increased secretion of tumor necrosis factor-alpha and interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction. J. Allergy Clin. Immunol. 88: 561-571 [Medline].

37. Calhoun, W. J., K. Murphy, C. A. Stevens, N. N. Jarjour, and W. W. Busse. 1992. Increased interferon-gamma and tumor necrosis factor-alpha in bronchoalveolar lavage (BAL) fluid after antigen challenge in allergic subjects. Am. Rev. Respir. Dis. 145: A638 .

38. Cromwell, O., Q. Hamid, C. J. Corrigan, J. Barkans, Q. Meng, P. D. Collins, and A. B. Kay. 1992. Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement of IL-1beta and tumour necrosis factor-alpha . Immunology 77: 330-337 [Medline].

39. Becker, S., J. Quay, H. S. Koren, and J. S. Haskill. 1994. Constitutive and stimulated MCP-1, GRO alpha, beta, and gamma expression in human airway epithelium and bronchoalveolar macrophages. Am. J. Physiol. 266: L278-L286 [Abstract/Free Full Text].

40. Stellato, C., P. Collins, H. Li, J. White, P. D. Ponath, W. Newman, D. Soler, C. Bickel, M. Liu, B. Bochner, T. Williams, and R. Schleimer. 1997. Production of the novel C-C chemokine MCP-4 by airway cells and comparison of its biological activity to other C-C chemokines. J. Clin. Invest. 99: 926-936 [Medline].

41. Mattoli, S., S. Miante, F. Calabro, M. Mezzetti, A. Fasoli, and L. Allegra. 1990. Bronchial epithelial cells exposed to isocyanates potentiate activation and proliferation of T cells. Am. J. Physiol. (Lung Cell Mol. Physiol.) 259: L320-L327 [Abstract/Free Full Text].

42. Devalia, J. L., A. M. Campbell, R. J. Sapsford, C. Ruszak, D. Quint, P. Godard, J. Bousquet, and R. J. Davies. 1993. Effect of nitrogen dioxide on synthesis of inflammatory cytokines by human bronchial epithelial cells in vitro. Am. J. Respir. Cell Mol. Biol. 9: 271-279 .

43. Ohkawara, Y., K. Yamauchi, Y. Tanno, G. Tamura, H. Ohtani, H. Nagura, K. Ohkuda, and T. Takashima. 1992. Human lung mast cells and pulmonary macrophages produce tumor necrosis factor-alpha in sensitized lung tissue after IgE receptor triggering. Am. J. Respir. Cell Mol. Biol. 7: 385-392 .

44. Okada, S., H. Inoie, K. Yamauchi, H. Iijima, Y. Ohkawara, T. Takishima, and K. Shirato. 1995. Potential role of interleukin-1 in allergen-induced late asthmatic reactions in guinea pigs: suppressive effect of interleukin-1 receptor antagonist on late asthmatic reaction. J. Allergy Clin. Immunol. 95: 1236-1245 [Medline].

45. Lukacs, N. W., R. M. Strieter, S. W. Chensue, M. Widmer, and S. L. Kunkel. 1995. TNF-alpha mediates recruitment of neutrophils and eosinophils during airway inflammation. J. Immunol. 154: 5411-5417 [Abstract].

46. Yang-Yen, H.-F., J.-C. Chambard, Y.-L. Sun, T. Smeal, T. J. Schmidt, J. Drouin, and M. Karin. 1990. Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62: 1205-1215 [Medline].

47. Schule, R., P. Rangarajan, S. Kliewer, L. J. Ransone, J. Bolado, N. Yang, I. M. Verma, and R. M. Evans. 1990. Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell 62: 1217 [Medline].

48. Ray, A., and K. E. Prefontaine. 1994. Physical association and functional antagonism between the p65 subunit of transaction factor NF-kappa B and the glucocorticoid receptor. Proc. Natl. Acad. Sci. USA 91: 752-756 [Abstract/Free Full Text].

49. Demoly, P., N. Basset-Segin, O. Chanez, A. M. Campbell, C. Gauthier-Rouviere, P. Godard, F. B. Michel, and J. Bousquet. 1992. c-Fos proto- oncogene expression in bronchial biopsies of asthmatics. Am. J. Respir. Cell Mol. Biol. 7: 128-133 .

50. Jany, B., R. Betz, and R. Schreck. 1995. Activation of the transcription factor NF-kappa B in human tracheobronchial epithelial cells by inflammatory stimuli. Eur. Respir. J. 8: 387-391 [Abstract].

51. Adcock, L. M., H. Shirasaki, C. M. Gelder, M. J. Peters, C. R. Brown, and P. J. Barnes. 1994. The effects of glucocorticoids on phorbol ester and cytokine stimulated transcription factor activation in human lung. Life Sci. 55: 1147-1153 [Medline].

52. Adcock, I. M., C. R. Brown, H. Chirasaki, and P. J. Barnes. 1994. Effects of dexamethasone on cytokine and phorbol ester stimulated c-fos and c-jun DNA binding and gene expression in human lung. Eur. Respir. J. 7: 2117-2123 [Abstract].

53. Siebenlist, U., G. Franzuso, and R. Brown. 1994. Structure, regulation and function of NF-kappa B. Annu. Rev. Cell Biol. 10: 405-455 .

54. Muegge, K., T. M. Williams, J. Kant, M. Karin, R. Chiu, A. Schmidt, U. Siebenlist, H. A. Young, and S. K. Durum. 1989. Interleukin-1 costimulatory activity on the interleukin-2 promoter via AP-1. Science 246: 245-251 .

55. Takizawa, H., T. Ohtoshi, T. Kikutani, H. Okazaki, N. Akiyama, M. Sato, S. Shoji, and K. Ito. 1995. Histamine activates bronchial epithelial cells to release inflammatory cytokines in vitro. Int. Arch. Allergy Immunol. 108: 260-267 [Medline].

56. Vannier, E., and C. A. Dinarello. 1993. Histamine enhances interleukin (IL)-1-induced IL-1 gene expression and protein synthesis via H2 receptors in peripheral blood mononuclear cells: Comparison with IL-1 receptor antagonist. J. Clin. Invest. 92: 281-289 .

57. Vannier, E., and C. A. Dinarello. 1994. Histamine enhances interleukin (IL)-1-induced IL-6 gene expression and protein synthesis via H2 receptors in peripheral blood mononuclear cells. J. Biol. Chem. 269: 9952-9956 [Abstract/Free Full Text].

58. Ackerman, V., S. Carpi, A. Bellini, G. Vassalli, M. Marini, and S. Mattoli. 1995. Constitutive expression of endothelin bronchial epithelial cells of patients with symptomatic and asymptomatic asthma and modulation by histamine and interleukin-1. J. Allergy Clin. Immunol. 96: 618-627 [Medline].

59. Vannier, E., L. C. Miller, and C. A. Dinarello. 1991. Histamine suppresses gene expression and synthesis of tumor necrosis factor-alpha via histamine H2 receptors. J. Exp. Med. 174: 281-284 [Abstract/Free Full Text].

60. Panettieri, R. A., P. A. Yadvish, A. M. Kelly, N. A. Rubinstein, and M. I. Kotlikoff. 1990. Histamine stimulates proliferation of airway smooth muscle and induces c fos expression. Am. J. Physiol. 259: L365-L371 [Abstract/Free Full Text].

61. Farin, C. J., and V. Holt. 1990. Mechanisms involved in the transcriptional activation of proenkephalin gene expression in bovine chromaffin cells. J. Biol. Chem. 265: 19116-19121 [Abstract/Free Full Text].




This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
T. Kikuchi, J. D. Shively, J. S. Foley, J. M. Drazen, and D. J. Tschumperlin
Differentiation-dependent responsiveness of bronchial epithelial cells to IL-4/13 stimulation
Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L119 - L126.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
K. Honda, M. Arima, G. Cheng, S. Taki, H. Hirata, F. Eda, F. Fukushima, B. Yamaguchi, M. Hatano, T. Tokuhisa, et al.
Prostaglandin D2 Reinforces Th2 Type Inflammatory Responses of Airways to Low-dose Antigen through Bronchial Expression of Macrophage-derived Chemokine
J. Exp. Med., August 18, 2003; 198(4): 533 - 543.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
F. F. Little, E. Lynch, G. Fine, D. M. Center, and W. W. Cruikshank
Tumor Necrosis Factor-{alpha}-Induced Synthesis of Interleukin-16 in Airway Epithelial Cells: Priming for Serotonin Stimulation
Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 354 - 362.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
F. Gantner, K. Sakai, M. W. Tusche, W. W. Cruikshank, D. M. Center, and K. B. Bacon
Histamine H4 and H2 Receptors Control Histamine-Induced Interleukin-16 Release from Human CD8+ T Cells
J. Pharmacol. Exp. Ther., October 1, 2002; 303(1): 300 - 307.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
G. Cheng, M. Arima, K. Honda, H. Hirata, F. Eda, N. Yoshida, F. Fukushima, Y. Ishii, and T. Fukuda
Anti-Interleukin-9 Antibody Treatment Inhibits Airway Inflammation and Hyperreactivity in Mouse Asthma Model
Am. J. Respir. Crit. Care Med., August 1, 2002; 166(3): 409 - 416.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
W. Y. Almawi and O. K. Melemedjian
Molecular mechanisms of glucocorticoid antiproliferative effects: antagonism of transcription factor activity by glucocorticoid receptor
J. Leukoc. Biol., January 1, 2002; 71(1): 9 - 15.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
G. Cheng, T. Ueda, F. Eda, M. Arima, N. Yoshida, and T. Fukuda
A549 Cells Can Express Interleukin-16 and Stimulate Eosinophil Chemotaxis
Am. J. Respir. Cell Mol. Biol., August 1, 2001; 25(2): 212 - 218.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed