 Stimulates Fractalkine Expression in Human Bronchial
Epithelial Cells and Regulates Mononuclear Cell Adherence
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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Bronchial epithelial cells may contribute to airway inflammation by releasing chemokines and expressing surface membrane molecules involved in the adhesion of leukocytes. We
found that interferon (IFN)-
stimulates expression of fractalkine, a potent chemoattractant for monocytes and T lymphocytes, in a time- and concentration-dependent manner by
normal human bronchial epithelial cells in culture. Enhanced
expression of fractalkine messenger RNA was confirmed by
both reverse transcription/polymerase chain reaction and
Northern blotting. IFN- also stimulated fractalkine protein
production and most of the protein was found in cell lysates.
The adherence of blood mononuclear cells to the monolayers
of bronchial epithelial cells stimulated with IFN- was partly
inhibited by an antifractalkine antibody. An antibody against
intercellular adhesion molecule-1 was similarly effective in inhibiting the adhesion. Fractalkine protein levels in bronchoalveolar lavage fluids from patients with inflammatory diseases
correlated positively with mononuclear cell counts in the fluids. The bronchial epithelium in a biopsy specimen of lung
cancer was stained positively by immunofluorescent staining
for fractalkine. We conclude that IFN- stimulates fractalkine expression by bronchial epithelial cells, which may play an important role in inflammatory responses by recruiting mononuclear leukocytes to the bronchial epithelium.
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Introduction |
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Materials and Methods
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On the basis of the number and arrangement of the conserved cysteine residues, chemokines have been subdivided
into two subfamilies, the CXC and CC chemokines, whereas
fractalkine (CX3C) and lymphotactin (C) are unique relatives (1). Fractalkine shares high homology with the CC family of chemokines, but has an insert of three amino acids between the two NH2-terminal cysteine residues, conferring a
CX3C structural motif (2). Fractalkine was first identified as a transmembrane molecule expressed on the surface of
endothelial cells stimulated with interleukin-1
or tumor
necrosis factor-
(2). Bacterial lipopolysaccharide was also
found to stimulate endothelial fractalkine production (3,
4). Expression of fractalkine is demonstrated in other
types of cells, such as neurons, astrocytes, dendritic cells,
and intestinal epithelial cells (5). The specific receptor
for fractalkine, CX3CR1, is expressed on monocytes, T lymphocytes, and natural killer cells (11), and this chemokine
induces adhesion and chemotaxis of these cells (2, 11).
Bronchial epithelial cells have the capacity to recruit and activate inflammatory cells by expressing cell-surface adhesion molecules and releasing chemokines (14). Expression of intercellular adhesion molecule (ICAM)-1, for instance, is considered to play a key role in airway inflammation and hyperresponsiveness (15), and enhanced production of inflammatory cytokines in bronchial epithelial cells is demonstrated in patients with asthma (16). We hypothesized that fractalkine may mediate a part of inflammatory responses by the control of leukocyte trafficking at the bronchial epithelium, and we addressed the possibility of fractalkine production in human bronchial epithelial cells and secretion of this chemokine into the bronchoalveolar trees in pathologic situations. The role of this chemokine in the adherence of blood mononuclear cells to cultures of bronchial epithelial cells was also studied.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Cell Culture
Normal human bronchial epithelial cells were obtained from Clonetics (San Diego, CA) and cultured in type I collagen-coated plates using small airway cell basal medium (SABM) supplemented with 30 µg/ml bovine pituitary extract, 0.5 µg/ml hydrocortisone, 0.5 µg/ml human recombinant epidermal growth factor, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5 µg/ml insulin, 0.1 µg/ml retinoic acid, 6.5 µg/ml triiodothyronine, 50 µg/ml gentamicin, 50 µg/ml amphotericin-B, and 500 µg/ml fatty acid-free bovine serum albumin (Clonetics). Cells were subcultured by incubating with Hanks' balanced salt solution (HBSS) containing 0.025% trypsin and 0.01% ethylenediaminetetraacetic acid at room temperature for 5 min, and the following experiments were performed using the cells of third to fifth passage.
When the cells reached about 80% confluence, the medium
was replaced with fresh SABM containing only 10% human serum and antibiotics, and after 24 h the cultures were stimulated
with 0.05 to 100 ng/ml interferon (IFN)- for up to 48 h.
RNA Extraction, Reverse Transcription/Polymerase Chain Reaction, and Northern Blot
Total RNA was isolated from bronchial epithelial cells using an RNeasy total RNA isolation kit (Qiagen, Hilden, Germany). Single-strand complementary DNA (cDNA) for a polymerase chain reaction (PCR) template was synthesized from 1 µg of total RNA using a primer oligo(dT)12-18 (GIBCO BRL, Rockville, MD) and M-Mulv reverse transcriptase (RT) (GIBCO BRL). Specific primers were designed from cDNA sequences for fractalkine, monocyte chemotactic protein (MCP)-1, ICAM-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Primers used were: fractalkine sense 5'-AACTCGAAATGGC GGCACCTT-3' and antisense 5'-ATGAATTACTACCACAG CTCCG-3'; MCP-1 sense 5'-AAACTGAAGCTCGCACTCTC GC-3' and antisense 5'-ATTCTTGGGTTGTGGAGTGAGT 3'; ICAM-1 sense 5'-CACAGTCACCTATGGCAACG-3' and antisense 5'-TTCTTGATCTTCCGCTGGC-3'; and GAPDH sense 5'-CCACCCATGGCAAATTCCATGGCA-3' and antisense 5'-AGACCACCTGGTGCTCAGTGTAGC-3'.
The amplified cDNA products for fractalkine, MCP-1, ICAM-1, and GAPDH are 887, 353, 750, and 696 base pairs (bp), respectively. Conditions for reactions were 1 × (94°C, 1 min), 30 × (94°C, 1 min, 60°C for fractalkine and GAPDH or 55°C for MCP-1 and ICAM-1, 1 min, 72°C, 1 min) and 1 × (72°C, 10 min). The products were electrophoresed on a 1.5% agarose gel containing ethidium bromide. The density of the bands was measured using a soft laser densitometer (MDA Scientific, Parkridge, IL), and the density was found to be linear against numbers of reaction cycles from 24 to 34. Specificity of PCR reactions was confirmed by sequencing the products.
For Northern blot analysis, poly(A)+RNA was isolated from
total RNA using Oligotex-dT30<Super> (Takara, Ohtsu, Japan) and subjected to electrophoresis on a 1% agarose gel containing formaldehyde. The RNA was blotted to a positively
charged nylon membrane by capillary transfer and probed with
the digoxygenin (DIG)-labeled antisense RNA for fractalkine or
-actin. A T7 promoter adapter was ligated to the 887-bp PCR
product specific for fractalkine using a Lig'nScribe kit (Ambion,
Austin, TX), and this was used as a template for the synthesis of a
DIG-labeled antisense RNA probe using a DIG RNA labeling
kit (Roche, Mannheim, Germany). The probe for -actin was
purchased from Roche. Hybridization was performed at 68°C for
16 h using a NorthernMax kit (Ambion), and the detection was
by means of a DIG-detection kit (Roche).
Enzyme-Linked Immunosorbent Assay for Fractalkine
The levels of fractalkine protein in the medium or cell lysates were determined by enzyme-linked immunosorbent assay (ELISA). For the preparation of cell lysates, the cultures were washed and incubated with the following cell lysis buffer: 20 mM phosphate-buffered saline (PBS), pH 7.4, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 0.01% protease inhibitor cocktail (Sigma, St. Louis, MO).
Noncoated 96-well plates were precoated by incubating, at 4°C for 12 h, with 4 µg/ml of an antihuman fractalkine antibody (R&D Systems, Minneapolis, MN). After incubating with a SuperBlock blocking reagent (Pierce, Rockford, IL), aliquots (100 µl/well) of the conditioned medium or cell lysates were added and incubated for 2 h at room temperature. Then 100 µl of 0.5 µg/ml biotinylated antihuman fractalkine antibody (R&D Systems) was added into each well, and the plate was further incubated for 2 h at room temperature. After washing, immunoreactive fractalkine was determined using horseradish peroxidase-labeled streptavidin and a TMB peroxidase substrate (Kirkegaard & Perry, Gaithersburg, MD). The chemokine domain of recombinant human [r(h)] fractalkine (R&D Systems) was used to generate standard curves for the measurement in culture medium and full-length r(h) fractalkine (R&D Systems) for standard curves for cell lysate. Fractalkine levels in medium or cell lysate were corrected with an average number of cells per milliliter of medium, which was obtained by repeated cell counts of similar cultures.
Adhesion of Mononuclear Cells and Polymorphonuclear Neutrophils to Bronchial Epithelial Cultures
Mononuclear cells and polymorphonuclear neutrophils (PMNs)
were separated from venous blood of healthy volunteers using
Ficoll-Paque Plus (Amersham Pharmacia Biotech, Uppsala, Sweden) (17). The cells were suspended, at a concentration of 1 × 106
cells/ml, in Dulbecco's modified Eagle's medium containing
0.5% (wt/vol) human serum albumin (DMEM-HSA). Bronchial
epithelial cells were cultured in a 12-well plate and stimulated
with 50 ng/ml IFN- for 16 h. After washing, the cultures were
further incubated for 1 h in DMEM-HSA. To assess the roles of
fractalkine or ICAM-1 in the adherence, neutralizing antibodies
against these factors or nonimmune mouse immunoglobulin (Ig)
G were added, and then 0.5 ml suspension of mononuclear cells
or PMNs was added to each well. The plate was incubated for 1 h
and nonadherent cells were removed by inverting the plate under
rolling conditions (63 rpm, 37°C, 20 min) (18). The cells were photographed under a microscope and the adherent cells counted.
Fractalkine Protein Levels in Bronchoalveolar Lavage Fluids
Bronchoalveolar lavage fluids (BALFs) obtained from 16 subjects were used for fractalkine measurement. The subjects include four patients with idiopathic pulmonary fibrosis, seven with sarcoidosis, and one each with eosinophilic pneumonia, acute respiratory distress syndrome, diffuse panbronchiolitis, hypersensitivity pneumonia, and pneumoconiosis. After a written informed consent was obtained, all subjects were premedicated with atropine sulfate (0.5 mg intramuscularly) and given topical lidocaine anesthesia. A fiberoptic bronchoscope was wedged into the right middle-lobe bronchus, and three aliquots of sterile saline solution were instilled with a total volume of 150 ml and aspirated immediately. Total cell count was obtained using a Bürker-Türk hemacytometer. Cells were isolated by centrifugation, cytologic preparations were made by cytocentrifugation and stained with Diff-Quik (Baxter, Miami, IL), and differential counts were performed by counting 300 cells per sample. About 10 ml of BALF was desalted with a Centriprep concentrator (Amicon, Beverly, MA) and dried using a vacuum evaporator. The residues were redissolved in 200 µl of PBS (pH 7.4) containing 0.5% (wt/vol) HSA and subjected to fractalkine ELISA.
Immunofluorescent Staining for Fractalkine in the Bronchial Epithelium
Biopsy specimens obtained from six subjects with suspected lung
cancer were used for immunofluorescent staining for fractalkine. After a written consent was obtained, patients were premedicated and anesthetized in a manner similar to that of BALF sampling, and biopsy was performed using a fiberoptic bronchoscope. All samples were frozen immediately and stored at 
80°C
until use. Immunofluorescent staining was performed essentially
as described previously (10). A 4-µm-thick section was mounted
on a slide glass, air-dried, and fixed with periodate-lysine-paraformaldehyde for 5 min, followed by 15% sucrose in PBS for
5 min. After blocking for 30 min with a 1:1 mixture of bovine serum and a SuperBlock blocking reagent, samples were incubated
with 2.5 µg/ml antifractalkine antibody (Santa Cruz Biotechnology, Santa Cruz, CA) in blocking solution for 2 h, washed with
HBSS (three times for 10 min each time), and incubated with a
biotinylated antigoat IgG (1:50) for 30 min. After washing, the
slides were incubated with streptavidin-fluorescein isothiocyanate (FITC) (1:50) for 1 h. The samples were examined under a LASER confocal microscope (LSM 410; Carl Zeiss, Jena, Germany).
Statistics
Data are presented in terms of means ± standard deviation (SD) and statistical significance was analyzed by Student's t test or Welch's t test.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Fractalkine Messenger RNA Expression by Bronchial
Epithelial Cells Stimulated with IFN-
IFN- stimulated the expression of fractalkine messenger
RNA (mRNA) in cultured bronchial epithelial cells as shown
in Figure 1.
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The fractalkine expression was dependent on the concentration of IFN- from 0.05 to 100 ng/ml (Figure 2A).
The IFN--induced fractalkine expression reached a maximal level after 16 h of stimulation (Figure 2B). Figure 2
also shows the concentration- and time-dependent upregulation, by IFN-, of MCP-1 and ICAM-1, which have adhesive or agonistic activities on mononuclear cells.
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Fractalkine Protein Levels in Medium and Lysates of Bronchial Epithelial Cells
A small amount of fractalkine protein was detected in the
conditioned medium and lysates of the unstimulated cells,
and IFN- clearly increased the production, as shown in
Figure 3. The IFN--induced protein production was concentration-dependent in a range similar to that for mRNA
expression (Figure 3A). Also, the time course of the IFN--induced fractalkine protein production agreed with that
of the mRNA expression and reached a maximal level after 16 h (Figure 3B). Most of the fractalkine production by
bronchial epithelial cells was cell-associated, and more
than 70% was found in cell lysates.
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P < 0.01 versus the control value
(Welch's t test).
Adhesion of Mononuclear Cells and PMNs to Bronchial Epithelial Cultures
The results of mononuclear cell adherence to bronchial
epithelial monolayers are shown in Figures 4 and 5A. The
number of adherent cells to unstimulated control monolayers was 363 ± 34 cells/field, and stimulation with 50 ng/
ml IFN- resulted in a 1.5-fold increase: 535 ± 25 cells/
field (n = 4; P < 0.01; Student's t test). Neutralizing antibodies against fractalkine or ICAM-1 significantly inhibited the
basal and IFN--induced adherence, and the treatment with
both antibodies had a slight additive effect (Figure 5A). IFN-
slightly but not significantly enhanced the adhesion of PMNs
to bronchial epithelial cells, and an anti-ICAM-1 antibody
exerted some inhibitory effect (Figure 5B). Although the effect of IFN- on PMN adhesion was not clear as compared with mononuclear cell adhesion, this may be due to the fact
that IFN--induced ICAM-1 expression reached maximum
earlier than fractalkine, and ICAM-1-dependent PMN adhesion may already have been downregulated at the time of the
experiment.
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Fractalkine Protein Levels in BALF
The average value of fractalkine concentration in BALF from 16 patients was 133 ± 193 pg/ml. Although the value in four patients with idiopathic pulmonary fibrosis was the highest, at 255 ± 299 pg/ml, there was no significant difference among patient subgroups. Linear regression analysis revealed a significant association between the fractalkine protein levels and mononuclear cell counts, as shown in Figure 6 (r = +0.633, P < 0.01).
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Immunofluorescent Staining for Fractalkine in the Bronchial Epithelium
The results of immunofluorescent staining of biopsied bronchial tissues are shown in Figure 7. The bronchial epithelium in a section with inflammation, but not the normal-appearing epithelium, was stained positively for fractalkine.
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Discussion |
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Materials and Methods
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Fractalkine is a transmembrane glycoprotein expressed on
the surface of endothelial or epithelial cells activated with
proinflammatory cytokines (2, 13, 19). In the nervous
system, fractalkine is considered as a signal molecule from
neurons to microglial cells (5). A soluble form of fractalkine is also released from membrane-bound molecules
and it exerts chemoattractant activities for monocytes and
T lymphocytes (2). In the present study we found the upregulation of fractalkine expression in bronchial epithelial
cells by the stimulation with IFN-. IFN- is a cytokine produced only by T lymphocytes and natural killer cells,
and plays a pivotal role in host defense against viral infections (20). It has beneficial effects when administered to
patients with certain tumors or viral hepatitis (21). Fractal-
kine may, in part, mediate these effects of IFN-. A recent
study demonstrated that a mutation in CX3CR1 is associated with reduced fractalkine binding and accelerated
progression of acquired immunodeficiency syndrome (22),
suggesting the importance of this chemokine in immune mechanisms.
Most of the fractalkine produced by IFN--stimulated
bronchial epithelial cells was retained within the cells and
was shown to function as a membrane adhesion molecule
for mononuclear cells. Bronchial epithelial cells also produce ICAM-1 (15, 23), which is shown to serve as a
main molecule in the adhesion of neutrophils and eosinophils to the bronchial epithelium (26). In the present study an antibody against fractalkine markedly inhibited
the adhesion of mononuclear cells, but not of PMNs, to
IFN--stimulated monolayers of bronchial epithelial cells.
An antibody against ICAM-1 effectively suppressed the
adhesion of both mononuclear cells and PMNs. Although fractalkine has a limited range of target cells as compared
with ICAM-1, it may play an important role in recruiting
monocytes and T lymphocytes to the bronchial epithelium.
Bronchial epithelial cells also produce and secrete MCP-1,
a CC chemokine with a potent chemoattractant activity
for mononuclear cells, and IFN- increases its production
(29). Increased levels of MCP-1 in the bronchial epithelium or in BALF is found in patients with asthma (30, 31).
In concert with these molecules, fractalkine derived from the bronchial epithelium may participate in the control of
airway inflammatory reactions. In fact, we detected fractal-
kine in BALF from patients with respiratory diseases, and
the levels of this chemokine correlated positively with mononuclear cell counts. In addition, immunofluorescent staining
disclosed the expression of fractalkine in the bronchial epithelium from inflammatory focus in a biopsy specimen.
We conclude that IFN- stimulates the production of
fractalkine by bronchial epithelial cells and this may play a
role in airway inflammation by eliciting leukocyte trafficking at the bronchial epithelium.
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Footnotes |
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Address correspondence to: Tadaatsu Imaizumi, Dept. of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, 5-Zaifucho, Hirosaki 036-8562, Japan. E-mail: timaizum{at}cc.hirosaki-u.ac.jp
(Received in original form June 27, 2000 and in revised form March 15, 2001).
Abbreviations: bronchoalveolar lavage fluid, BALF; digoxygenin, DIG; enzyme-linked immunosorbent assay, ELISA; glyceraldehyde-3-phosphate dehydrogenase, GAPDH, intercellular adhesion molecule, ICAM; interferon, IFN; immunoglobulin, Ig; monocyte chemotactic protein, MCP; messenger RNA, mRNA; polymerase chain reaction, PCR; polymorphonuclear neutrophil, PMN; reverse transcriptase, RT.
Acknowledgments:
The authors thank Dr. Tomoh Matsumiya for his help and
Ms. Kumiko Munakata for technical assistance.
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References |
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