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Department of Medical Specialities, Southampton General Hospital, Southampton, Hampshire, United Kingdom; and
Washington University, St. Louis, MO 63110
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and 
chemokines
and, uniquely, of secretory component (SC), the extracellular
ligand-binding domain of the polymeric IgA receptor. Ig superfamily
relatives of SC, such as IgG and 2-macroglobulin, bind
IL-8. Therefore, we tested the hypothesis that SC binds IL-8, modifying
its activity as a neutrophil chemoattractant. Primary bronchial
epithelial cells were cultured under conditions to optimize SC
synthesis. The chemokines IL-8, epithelial neutrophil-activating
peptide-78, growth-related oncogene , and RANTES were released
constitutively by epithelial cells from both normal and asthmatic
donors and detected in high m.w. complexes with SC. There were no
qualitative differences in the production of SC-chemokine complexes by
epithelial cells from normal or asthmatic donors, and in all cases this
was the only form of chemokine detected. SC contains 15%
N-linked carbohydrate, and complete deglycosylation with
peptide N-glycosidase F abolished IL-8 binding. In
micro-Boyden chamber assays, no IL-8-dependent neutrophil chemotactic
responses to epithelial culture supernatants could be demonstrated. SC
dose-dependently (IC50 
0.3 nM) inhibited the neutrophil
chemotactic response to rIL-8 (10 nM) in micro-Boyden chamber assays
and also inhibited IL-8-mediated neutrophil transendothelial migration.
SC inhibited the binding of IL-8 to nonspecific binding sites on
polycarbonate filters and endothelial cell monolayers, and therefore
the formation of haptotactic gradients, without effects on IL-8 binding
to specific receptors on neutrophils. The data indicate that in the
airways IL-8 may be solubilized and inactivated by binding to
SC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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SC is a member of the Ig superfamily and a highly glycosylated protein containing 15–23% carbohydrate by weight (6, 7). SC has seven putative sites for N-linked glycosylation, and analysis of the carbohydrate composition of SC purified from human milk has revealed that glycosylated SC contains relatively high amounts of fucose (8). Although not involved in the binding of SC to IgA (9), the carbohydrate component has other important functions. In vivo studies have demonstrated the protective effects of fucosylated oligosaccharide fractions of human milk against heat stable enterotoxin of Escherichia coli in suckling mice (10). Clostridium difficile binds to glycosylated SC to a much greater extent than to the deglycosylated form (11), and the SC-dependent inhibition of adhesion of enterotoxic forms of E. coli to erythrocytes is apparently reliant on the carbohydrate portion of SC (12). Furthermore, removal of N-linked oligosaccharides from sIgA has been shown to enhance the fixation of C3b and activation of the alternative complement pathway (3). Thus it appears that fully glycosylated SC has enhanced anti-infective and decreased proinflammatory properties.
SC is synthesized uniquely by epithelial cells. Expression of SC is
increased in vitro by proinflammatory cytokines including IFN-
,
alone (13) and in synergistic combination with IL-4
(14, 15, 16), TNF- (17), IL-1, and IL-1
(18). In terms of mucosal defense, this is an important
response because infection and inflammation raise the local
concentrations of these cytokines leading to an increase in the
delivery of sIgA into the airway lumen. Many of the previous studies of
SC expression were conducted using immortalized cell lines. In this
study, our aim was to analyze constitutive release of SC from primary
human bronchial epithelial cells in culture, comparing cells from
normal subjects with those from volunteers with mild asthma.
In view of our previous observation of a complex of IL-8 with IgA
(19) and other members of the Ig superfamily such as
2-macroglobulin (20) in the
airways, we hypothesized that IL-8 may bind to SC. Therefore, we sought
evidence for modification of the form and function of IL-8 by binding
to SC released constitutively from primary bronchial epithelial cells
in culture. We provide evidence that SC forms complexes with both
and chemokines and that this is the only detectable form of the
chemokines. We report that the carbohydrate moiety of SC is critical in
binding to IL-8 and that SC inhibits neutrophil migration responses to
IL-8, both in micro-Boyden chambers and in a more physiological model
of transendothelial migration in vitro. These previously unrecognized
properties of SC indicate a potential anti-inflammatory role for SC
in the airways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bronchial brushings were obtained from volunteers undergoing bronchoscopy for research purposes. Additionally, one set of experiments was conducted using normal cells grown from an explant taken from the periphery of resected lung tumor. Donors were normal (n = 4) or atopic, mild asthmatics (n = 4). The asthmatic donors received Ventolin (Glaxo, Uxbridge, U.K.) only or were taking no medication.
Cell culture
Cells collected on one or two brushes per patient were harvested into 10 ml bronchial epithelium basal medium (Clonetics, San Diego, CA) containing 500 µg/ml each gentamicin and amphotericin-B. Cells were treated immediately with a final concentration of 6.5 mM DTT (Sputolysin; CN Biosciences, Nottingham, UK) for 30 min at room temperature, to aid dispersal of mucus and increase the yield of epithelial cells. Cells were recovered by centrifugation at 1000 x g for 5 min at room temperature before resuspension in 1 ml of bronchial epithelial growth medium. Bronchial epithelial growth medium was comprised of basal medium supplemented with the bronchial epithelium SingleQuots, as per the manufacturer’s instructions, in addition to 1% (v/v) FCS. Cells were seeded into flasks coated with 10 µg/cm2 collagen IV (Sigma, Poole, U.K.), and medium was refreshed every other day until 80% confluence was reached. Ciliary activity was observed for the first 2–3 days of culture, and it was apparent that the ciliated cells adhered to the culture flask during this time. Cells were grown in 5% CO2, at 37°C with humidity. At 80% confluence, cells were lifted with 0.025% trypsin/0.01% EDTA and reseeded into 24-well plates (Costar, Bucks, U.K.) coated with 10 µg/cm2 collagen IV. Cells were grown to 80% confluence (2–4 wk), the medium was changed, and 24-h culture supernatants were collected, centrifuged at 500 x g for 10 min at 4°C to remove any dead cells or cell debris, and the cleared supernatants were snap frozen in liquid nitrogen. Samples were stored at -20°C before analysis.
Immunoprecipitation of SC from epithelial cell supernatants
Supernatants were diluted 1:5 in immunoprecipitation buffer, comprised of 150 mM NaCl, 50 mM Tris, 5 mM EDTA, 0.1% SDS, 0.5% Tergitol Nonidet P-40, and a protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany), pH 7.6. To this was added 1 µg/ml of a polyclonal Ab to human colostral SC (Binding Site, Birmingham, U.K.) and 4 µl of protein A agarose beads (Bio-Rad, Herts, U.K.), and samples were incubated with shaking at 4°C overnight. The samples were centrifuged at 3000 x g and 4°C for 5 min, the supernatant was removed, and the pellet was washed three times with ice-cold immunoprecipitation buffer. To release the immunoprecipitated protein from the Ab-protein A agarose complex, samples were incubated with 5x Laemmli sample buffer (21) containing 100 mM DTT at room temperature for 30 min before centrifuging for 5 min at 3000 x g.
SDS-PAGE and Western blotting
Polyacrylamide mini-gels were cast in the Bio-Rad Protean II system, using Pro-Sieve 50 (Flowgen, Staffs, U.K.), a modified solution of acrylamide that forms a gradient after polymerization. Gels containing 12% Pro-Sieve 50 that have a resolvable range of 5–150 kDa and are therefore suitable for separating the chemokines (around 8 kDa) from SC (around 90 kDa) were made. Proteins were separated by electrophoresis in Tris-tricine running buffer (100 mM each Tris and tricine with 0.1% (w/v) SDS). Electrophoresis was conducted at 200 V for 30 min. Proteins were electrophoretically transferred onto a 0.45-µm nitrocellulose membrane (Bio-Rad) using semidry transfer. Transfer was conducted in 25 mM Tris, 192 mM glycine, with 0.1% (w/v) SDS containing 20% methanol for 80 min at a constant 250 mA.
Staining Western blots with Abs
The membranes were blocked in PBS with 2% (v/v) Tween 20
(blocking buffer) at 4°C overnight, then washed three times in
PBS/0.05% (v/v) Tween 20. All primary Abs were diluted in blocking
buffer, and blots were incubated for 90 min at room temperature,
without shaking. Anti-human colostral SC (Binding Site) was used at a
final concentration of 3.5 µg/ml, anti-human rIL-8 (the gift of
Dr. I. Lindley, Novartis, Vienna, Austria) at 4.1 µg/ml,
anti-human rRANTES, anti-human recombinant epithelial
neutrophil-activating peptide-78 (ENA-78), and anti-human
recombinant growth-related oncogene (GRO) (all from R&D Systems,
Minneapolis, MN) all at 0.5 µg/ml. Bound Abs were detected using a
biotinylated secondary Ab (DAKO, Cambs, U.K.) followed by a complex of
streptavidin and biotinylated HRP (DAKO StreptABComplex). Results were
visualized using a chemiluminescent substrate (SuperSignal; Pierce
Warriner, Chester, U.K.) and subsequent exposure to x-ray film (Kodak
XLS; Kodak, Rochester, NY). Control experiments (see Fig. 1
) using SC
(Binding Site) and recombinant chemokines indicated that the limits of
detection of this method were 5 ng/ml (SC), 10-9
M (IL-8 and GRO), and 10-10 M
(RANTES and ENA-78).
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Samples for analysis of the total carbohydrate content were electrophoretically separated and transferred onto nitrocellulose as above. The carbohydrate-containing components of the samples were examined using the glycoprotein detection kit from Bio-Rad. Carbohydrate residues on glycoproteins are oxidized and biotinylated, followed by detection using streptavidin alkaline phosphatase and nitroblue tetrazolium/5-bromo-4-chloro-indolyl phosphate for color development. Carbohydrate-positive bands appear blue/black on a cream background.
ELISAs for total IL-8 and total SC
Total IL-8 (i.e., free IL-8 and that in complex with SC and other binding molecules) was measured using a commercially available ELISA kit (Peli-Kine kit; Eurogenetics, Hampton, U.K.). The sensitivity of this kit is 1 pg/ml, and preliminary experiments indicated that SC, at concentrations up to 50 µg/ml, does not interfere in this assay. Unfractionated epithelial cell supernatants were diluted 1:3 in PBS/2% Tween 20 (v/v) before assaying for total SC using an in-house ELISA, as described previously (5).
Deglycosylation of SC
Standard SC, the crude preparation from human colostrum (Binding Site), was first depleted of the BSA present as a carrier protein by immunoprecipitation overnight with 10 µg/ml of a rabbit polyclonal Ab to BSA (Sigma). The BSA-depleted SC was resuspended at 8.8 µg/ml, in 100 mM sodium phosphate buffer, pH 7.5, and incubated with 10 mU recombinant peptide N-glycosidase F (PNGase F; Bio-Rad) for 3 days at 37°C. After the addition of Laemmli sample buffer (21), 2 µl of the deglycosylated SC was separated by SDS-PAGE and transferred onto nitrocellulose as described above. Blots were stained for SC, IL-8, and carbohydrate as described above.
Affinity purification of SC
Human SC was isolated from human colostrum using affinity column chromatography, then separated from sIgA by fast protein liquid chromatography. The purity of SC was established by Western blot analysis (22).
Neutrophil chemotaxis assays
Neutrophils were isolated from EDTA-anticoagulated normal venous blood. RBCs were removed by sedimentation in the presence of 6% (v/v) Dextran 70 (Macrodex; Pharmacia, Uppsala, Sweden). Leukocyte-rich supernatants were under-layered with an equal volume of Lymphoprep (Nycomed Pharma, Oslo, Norway), and tubes were centrifuged at 300 x g for 30 min at room temperature. RBCs remaining in the granulocyte pellet were subjected to hypotonic lysis. Cells were washed and resuspended at 1 x 106 cells/ml in HBSS (Life Technologies, Paisley, U.K.) buffered to pH 7.4 with 20 mM HEPES (BDH, Poole, U.K.). Neutrophils isolated in this manner were >97% pure, with eosinophils as the contaminating cells.
Neutrophil chemotaxis was assayed using 5-µm pore size polyvinylpyrrolidone-free polycarbonate filters in micro-Boyden chambers. Recombinant human IL-8 (a gift of Dr. I. Lindley) was used at 10-8 M in HBSS with 20 mM HEPES, pH 7.4, as a positive control and HBSS/HEPES alone was the negative control. A serial dilution of human colostral SC (Binding Site) was prepared (8–500 ng/ml) in HBSS/20 mM HEPES, pH 7.4, in the absence or presence of 10-8 M IL-8 and incubated for 30 min at room temperature. SC standards (with and without IL-8) were added to the lower wells of the chamber, and the upper wells were loaded with 50,000 neutrophils each. The chamber was placed in a humidity-controlled unit at 37°C for 30 min. Cells on the upper surface were removed by scraping, and cells adherent to the lower side of the filter were fixed in methanol and stained (Hema-Gurr; BDH) for counting. The number of cells in five high-power (x400) fields were counted, and the result were expressed as the percentage response to IL-8 alone (121.5 ± 25.7 neutrophils per high-power field (n = 3)), after subtraction of the value of the negative control well.
IL-8-dependent neutrophil chemotaxis in response to epithelial cell culture supernatants was tested by quantifying the neutrophil chemotactic response to undiluted culture supernatants preincubated for 1 h at 4°C in the absence and presence of 20 µg/ml polyclonal anti-IL-8 (a gift of Dr. I. Lindley).
Endothelial cell culture
Primary cultures of single donor HUVEC (Clonetics) were grown to confluence in 1% (w/v) gelatin-coated flasks in endothelial growth medium (Clonetics).
Neutrophil transendothelial migration
HUVEC, between passages 4 and 8, were seeded at 30,000 cells/well on 10 µg/cm2 collagen IV-coated Transwells (Becton Dickinson, Mountain View, CA) and grown for 1 wk. HUVEC were washed once with PBS, and IL-8 and SC, diluted in HBSS with 20 mM HEPES, added 1 h before neutrophils. Human rIL-8 was added as neutrophil chemoattractant to the lower Transwell compartment only (final concentration, 10-8 M). Crude SC was added to both the lower and upper compartments, at a final concentration of 500 ng/ml, while affinity-purified SC was added to both compartments at 100, 500, or 1000 ng/ml. Neutrophils were added to the upper chamber at 200,000 cells/well and cocultured for 3 h at 37°C. Migrated cells in the lower compartment were harvested, together with transmigrated neutrophils adhering to the lower surface of the Transwell, by adding EDTA (final concentration, 77 mM) to the lower compartment and gently agitating for 1 min. The medium in the lower well was removed, centrifuged at 200 x g for 10 min at 4°C, and the supernatant was stored at -80°C. The numbers of neutrophils in the cell pellets were counted, and the results are expressed as the percentage of cells migrated, relative to control conditions in the absence of SC. Supernatants were analyzed for the presence of IL-8 by SDS-PAGE and Western blotting, as described above, with scanning densitometry to quantify the data.
Neutrophil shape change assay
Neutrophils were incubated at 10 x 106/ml for 5 min at 37°C with IL-8 (10-8 M) that had been preincubated for 30 min at room temperature with and without SC (Binding Site) at 500 ng/ml, or with SC alone, in a final volume of 200 µl HBSS with 20 mM HEPES. Cells were mixed with an equal volume of trypan blue (0.4% in 0.85% saline; Life Technologies), and the percentage of cells that had changed shape from a round appearance were counted in 200 cells.
Binding of radiolabeled IL-8 to neutrophils
SC, at 500 ng/ml in HBSS was preincubated for 3 h at room temperature with 125I-labeled IL-8 (125I-IL-8) (0.63 nM), to allow binding. Neutrophils were isolated as described above and resuspended at 1 x 107 cells/ml in HBSS with 20 mM HEPES. Neutrophils (100 µl) were incubated with the 125I-IL-8 alone or with the 125I-IL-8-SC mixture for 1 min at room temperature before centrifugation at 200 x g and 4°C for 10 min. Cell pellets were washed twice in PBS with 1% (w/v) BSA before counting in a gamma counter (LKB Wallac, Turku, Finland) for 1 min. To test the specificity of binding, unlabeled IL-8 (1 x 10-6 M) was added to aliquots of the neutrophils immediately before the addition of 125I-IL-8. All measurements were conducted in triplicate.
Binding of radiolabeled IL-8 to polycarbonate filters
SC (500 ng/ml) and 125I-IL-8 (0.1 nM) were preincubated at room temperature for 30 min. 125I-IL-8 alone, or 125I-IL-8 in the presence of SC, was placed in the bottom of a four-well (1.5 ml) micro-Boyden chemotaxis chamber. The binding of 125I-IL-8 to a 5-µm polyvinylpyrrolidone-free polycarbonate filter was assessed after 30 min at 37°C by washing the filters twice in PBS, cutting them into four pieces, and gamma counting each.
Binding of radiolabeled IL-8 to endothelial cells
Endothelial cells, at passage 3, were cultured to confluence in
gelatin-coated, 24-well plates (Nunc, Life Technologies) in endothelial
growth medium. SC (500 ng/ml) and 125I-IL-8 (0.1
nM) were preincubated for 30 min at room temperature. HUVEC were washed
once with HBSS containing 20 mM HEPES and incubated at 37°C for
3 h with either 125I-IL-8 alone or
125I-IL-8 after preincubation with SC.
Supernatants were collected and counted in a counter. The amount of
125I-IL-8 bound to the endothelial cells was
calculated by subtracting the counts in the supernatant after 3-h
incubation from the total added.
LDH analysis
Lactate dehydrogenase activity was assayed in supernatants of neutrophils cultured for 0.5–3 h at 37°C with 500 ng/ml SC using the Roche cytotoxicity detection kit (Roche Diagnostics, Lewes, U.K.).
Data analysis
Western blots were scanned and the density of the bands measured
using QuantiScan (Biosoft, Cambridge, UK) software. All data
were statistically analyzed using Student’s t test, and
significance was assigned at p 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epithelial cells from both normal and asthmatic donors
constitutively produced SC and IL-8. No significant differences were
observed in the amounts of SC released from cells from normal and
asthmatic donors in 24-h culture, as measured by ELISA. However,
significantly more IL-8 was released from epithelial cells from
asthmatic donors, compared with that from cells from normal donors
(Table I).
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), the molar ratio of SC:IL-8 in epithelial culture
supernatants, as measured by ELISA, was calculated. For each sample the
molar concentration of SC and IL-8 was calculated, based on a molecular
mass of 90 kDa for SC and of 8 kDa for IL-8. Although significantly
more IL-8 was released by epithelial cells from asthmatic donors, the
molar ratio of SC:IL-8 in the samples (0.60 ± 0.4) was not
different to that from normal epithelial cells (0.70 ±
0.1).
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The molecular form of SC and IL-8 produced by bronchial epithelial
cells was investigated using Western blot analysis of unfractionated
supernatants (Fig. 1
, A and
B). For cells from both asthmatic and normal donors, SC was
detected with an apparent molecular mass of 130 kDa, whereas the
positive control of SC from human colostrum was, as expected, around 98
kDa. IL-8 was not detected at the predicted molecular mass of 8 kDa,
but in a high molecular mass form, also at 130 kDa. Similarly, Western
blots stained for RANTES, ENA-78, and GRO (Fig. 1, A and
B) showed that these were also present at 130 kDa and were
never observed at the molecular mass of their free form (around 8 kDa
for all). Analysis of standard human recombinant peptides in the same
way indicated that although multimers of ENA-78 and GRO formed at
the highest concentration (10-8 M) analyzed
(Fig. 1C), these did not account for the 130-kDa form
observed in the epithelial cell culture supernatants. Using this
method, the sensitivity of detection of recombinant peptides was
10-9 M for IL-8 and GRO and
10-10 M for RANTES and ENA-78.
Analysis of the high molecular mass form of IL-8
In view of our previous observation that IL-8 binds to IgA
(19) and 2-macroglobulin
(20), members of the Ig superfamily and relatives of SC,
we predicted that the high molecular mass form of the chemokines was a
complex of SC and chemokine. To confirm this, samples were
immunoprecipitated with a polyclonal Ab to SC. This resulted in the
coimmunoprecipitation of all the chemokines (Fig. 2), which were retained as part of the
130-kDa complexes. Conversely, immunoprecipitation with polyclonal Abs
to either IL-8 or RANTES resulted in the coimmunoprecipitation of SC
(data not shown). Free, i.e., 8-kDa, chemokines were not detected in
these immunoprecipitates, indicating that the majority of the
chemokines were in the SC-containing complex. Control
immunoprecipitation, conducted using protein A-agarose beads only, did
not result in immunoprecipitation of any proteins (data not shown).
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Preliminary experiments established that commercially available
human SC prepared from colostrum contained the SC-IL-8 complex (Fig. 3), and this preparation was subsequently
used to investigate the role played by carbohydrate and protein
components in chemokine binding. The material was subjected to
deglycosylation with recombinant PNGase F, following which the samples
were analyzed by Western blot and stained for SC, IL-8, and
carbohydrate (Fig. 3).
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illustrate
analysis of standard SC before treatment, and several points were
noted. First, although Coomassie Brilliant Blue-stained gels had shown
several proteins, ranging from 45 to 206 kDa, to be present in the
standard, including albumin added as a carrier protein (data not
shown), SC-specific staining indicated that SC was present in three
forms. These had molecular masses of 130, 98, and 60 kDa, and the
98-kDa form was the most abundant (lane 1, open
arrow). This is different to the published molecular mass of mature
human colostral SC of around 75.5 kDa (6) and may reflect
the binding of other components of colostrum to SC. The lower molecular
mass 60-kDa species could reflect partial degradation of SC. Second,
lane 3 (standard SC stained with polyclonal Abs to IL-8)
confirms that IL-8 is bound to 98-kDa SC. A second, unidentified IL-8
binding protein, which was not SC, was observed at 113 kDa. This
protein could be one or part of the several other molecules capable of
binding IL-8, including IgA and
2-macroglobulin (19, 20), that
are likely to be present in colostrum, in whole and degraded forms.
Third, lane 5 shows the carbohydrate profile of the SC
preparation. Glycoproteins were apparent at 158, 130, 98, 76, and 54
kDa, indicative of the relative impurity of this preparation of
SC.
Importantly, glycosylation of 98-kDa SC was confirmed
(lane 5, open arrow) and shown to be sensitive to
PNGase F treatment (lane 6). The absence of a 98-kDa
carbohydrate-positive band in lane 6 confirmed that PNGase F
treatment had effectively removed the N-linked carbohydrate
from SC. Following complete deglycosylation of SC with PNGase F, the
molecular mass of the 98-kDa SC was decreased to 74 kDa (Fig. 4, compare lanes 1 and
2, filled arrow), in good agreement with published data
regarding the percentage of carbohydrate, 15%, on SC (6, 7). Most important, however, was the observation that IL-8 was
no longer bound to SC after removal of the carbohydrate (no 74 kDa band
in lane 4). From these results it is clear that SC from
human colostrum is a glycoprotein capable of binding IL-8. Although we
have yet to confirm the precise molecular nature of this association,
the sustained presence of an SC-IL-8 complex following SDS-PAGE
suggests that covalent linkages are involved.
In contrast, the 130-kDa SC-positive band in lane 1 that was also glycosylated (lane 5) was resistant to PNGase F treatment (lane 6) and does not bind IL-8. This component is presumably comprised of nascent SC bound to another glycoprotein or fragment thereof, which contains O-linked oligosaccharides, most likely IgA (23).
The 76-kDa glycoprotein (lane 5) was confirmed by
immunoprecipitation to be the BSA added to SC as a carrier protein,
relatively insensitive to PNGase F and, importantly, incapable of
binding IL-8 (lane 3). However, IL-8 remained bound
to the nonglycosylated, and therefore PNGase F-resistant, non-SC
protein at 113 kDa. In addition, PNGase F treatment generated a
90-kDa IL-8 binding protein, which was not glycosylated and not SC
(lanes 2, 4, and 6).
SC inhibits IL-8-induced neutrophil chemotaxis
SC dose-dependently inhibited the migratory response of normal
neutrophils to an optimum concentration (10 nM) of human rIL-8 (Fig. 4). Concentrations of SC of 16 ng/ml and above significantly
(p < 0.05) inhibited the chemotactic response,
and the IC50 for this effect was 25 ± 2
ng/ml. Chemotaxis was completely inhibited by 500 ng/ml (6 nM) SC, a
molar ratio of SC:IL-8 of 0.6. Under these conditions, no evidence of
cell death as determined by increase in soluble lactate dehydrogenase
activity or decrease in trypan blue exclusion was detected.
Epithelial culture supernatants do not contain active IL-8
Greater neutrophil chemotactic responses to culture supernatants
from normal epithelial cells were observed (Table II) compared with epithelial cells from
asthmatic donors, although because of the variable responses the
difference was not significant. Polyclonal anti-IL-8 did not
significantly inhibit neutrophil chemotactic responses to supernatants
from epithelial cells from either group of donor. Conversely, this Ab
significantly (p < 0.0001) inhibited the
neutrophil response to an optimum concentration of human rIL-8 by
79.1 ± 1.9% (n = 4).
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To elucidate the mechanism by which SC inhibited IL-8-induced neutrophil chemotaxis, we investigated the effect of SC on IL-8 binding to specific receptors on neutrophils. Under the conditions used, the specific binding of 125I-IL-8 to neutrophils from normal donors was 3935 ± 280 cpm with nonspecific binding accounting for 8.5% of the total (n = 4). However, preincubation of 125I-IL-8 with 500 ng/ml of either a crude preparation of SC or affinity-purified SC did not alter the specific binding of 125I-IL-8 to neutrophils (3738 ± 136 and 3429 ± 144 cpm in the presence of crude and affinity-purified SC, respectively).
SC-bound IL-8 signals the neutrophil
To investigate whether SC bound to IL-8 inhibited the ability of soluble IL-8 to signal via the IL-8 receptor, we quantified the shape change response of neutrophils to an optimum concentration of IL-8 that had been preincubated with SC at 500 ng/ml. The shape change response of neutrophils to incubation buffer alone was 4.9 ± 2.7%. SC alone did not induce shape change and did not inhibit the shape change response to IL-8, which was 90.9 ± 2.2% in the absence of SC and 90.3 ± 3.4% in the presence of SC (n = 3).
SC inhibits IL-8 binding to polycarbonate filters
It has been demonstrated that IL-8 binds rapidly to the polycarbonate filters used in chemotaxis assays and that the haptotactic gradient so formed is responsible for directing neutrophil migration (24). Therefore, we investigated whether SC interfered with the binding of IL-8 to the polycarbonate filters. At 500 ng/ml, and under the same conditions as those employed for chemotaxis studies, SC significantly inhibited binding of IL-8 to polycarbonate. Under the conditions used, the amount of 125I-IL-8 bound to the polycarbonate filter (15,997 ± 1,480 cpm) was significantly reduced in the presence of SC (11,256 ± 431 cpm; n = 4).
SC inhibits IL-8-induced transendothelial migration
To investigate the functional effects of SC in a more
physiological model of IL-8-induced neutrophil migration, we examined
the effect of SC on the neutrophil response to a transendothelial
gradient of IL-8 in vitro. We tested the effect of both crude and
affinity-purified preparations of SC in these assays (Fig. 5). Both preparations of SC significantly
inhibited neutrophil transendothelial migration, supporting our
conclusion from the Boyden chamber assays that SC inhibits IL-8-induced
neutrophil chemotaxis. Again, we could find no evidence of SC-mediated
cytotoxicity under these conditions. The crude preparation of SC
appeared to be more efficient at inhibiting neutrophil chemotaxis,
reducing it to 9% of control, whereas the affinity-purified SC, at the
same concentration, inhibited transmigration by 40%.
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). This was
confirmed by significant inhibition of binding of
125I-IL-8 to endothelial cell layers in the
presence of SC. After a 3-h incubation, the amount of radiolabeled IL-8
in the soluble phase was significantly (p <
0.001) increased in the presence of crude SC (125 ± 4%) and
affinity-purified SC (175 ± 38%), compared with control (100%,
20,744 ± 583 cpm).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(GRO and
ENA-78) and (RANTES) subfamilies, also bind to SC. Focussing on
IL-8, we have demonstrated that the carbohydrate component of SC is
required for binding, and that binding to SC inhibits IL-8 function.
Chemokines synthesized and released constitutively by primary bronchial
epithelial cells from normal and asthmatic donors form high molecular
mass complexes with SC. Experiments using a neutralizing Ab to IL-8
indicated that IL-8 in epithelial culture supernatants is not active,
but is present as an inactive SC-IL-8 complex. The results indicate an
important role for SC in the regulation of IL-8 function in the normal
and asthmatic airways. In our experiments, SC was synthesized and released constitutively at around 30 ng/24 h from cultures of primary human bronchial epithelial cells. In cultures of primary human bronchial epithelial cells isolated from enzymatic digests of segments of bronchial tubes from patients with a history of smoking and mild chronic obstructive pulmonary disease undergoing lobectomy for lung cancer, Godding et al. (25) reported 15 times lower SC release. Because decreased Clara cell protein (CC10) levels have been measured in epithelial cells from smokers (26), it is possible that synthesis of other proteins such as SC may be similarly altered. Alternatively, the explanation may lay in the different culture conditions. The importance of the extracellular matrix and its active role in the growth and differentiation of cells is well documented (27). Godding et al. (25) used fibronectin, vitrogen, and BSA as a culture coating. However, we used collagen IV, one of the major components of basement membranes, because collagen was previously shown to promote sustained production of SC from cultures of primary human tracheal epithelial cells, compared with cells cultured on plastic (28). Similarly, collagen has been shown to strongly stimulate IL-8 synthesis by human macrophages (29).
Our primary cultures from bronchial brushings retain additional features of a valid ex vivo model, because significantly higher baseline levels of IL-8 were released over 24 h from cells from asthmatic donors than from those from normal donors, as previously described by Marini et al. (30).
All of the chemokines studied here, IL-8, RANTES, ENA-78, and GRO
were constitutively released from bronchial epithelial cells and formed
part of a 130-kDa complex. Immunoprecipitation with polyclonal Abs to
SC confirmed that SC was also a component of this complex. We could
find no evidence, by Western blotting, for the presence of the
chemokine-binding glycosaminoglycan heparan sulfate in this complex
(data not shown). This is the first report that free SC can bind one or
more chemokine(s) and suggests a role for SC other than as the receptor
for polymeric Igs. In support of previously unrecognized roles for SC,
recent evidence has demonstrated that SC produced by cultures of
primary human keratinocytes inhibits IFN- function
(31), suggesting an anti-inflammatory role for SC in
the pathogenesis of skin diseases such as psoriasis. As IFN- was
shown to increase production of SC from keratinocytes, this interaction
forms an effective homeostatic-negative feedback mechanism.
Furthermore, Moro et al. (32) reported that IL-8
stimulates SC production, and because we have shown here that SC
inhibits IL-8-mediated neutrophil migration, we propose that a similar
SC-dependent, anti-inflammatory mechanism exists in the airways to
regulate chemokine function.
This feedback model can be applied to the airways of patients with cystic fibrosis (CF), where levels of IL-8 are high (33) and the recruitment of neutrophils and the production of mediators leads to damage of airway tissue. SC isolated from the sputum of CF patients was previously reported to be more electrophoretically negative than normal (34). We have observed that the SC isolated from CF sputa is relatively degraded and incapable of binding IL-8, suggesting that in this disease SC cannot play its regulatory role in attenuating inflammation (L. J. Marshall, B. Perks, J. K. Shute, manuscript in preparation). Conversely, effective inhibition of the neutrophil-directed function of IL-8 by SC may explain the high levels of IL-8 associated with the eosinophilia of asthma and eosinophilic pneumonia (35).
Neutrophil chemotactic responses induced by epithelial cell culture supernatants were independent of IL-8, despite the presence of levels of IL-8 theoretically capable of stimulating this response (24). This indicates that the molar ratio of SC to IL-8 synthesized by epithelial cells from normal subjects (0.7) and those with mild asthma (0.6) is high enough to completely inhibit neutrophil responses to the concentrations of IL-8 present. Certainly, Boyden chamber assays demonstrated that a molar ratio of purified SC to rIL-8 of 0.6 completely inhibits the neutrophil response to an optimum concentration of IL-8. In contrast to our findings, other groups have shown IL-8-dependent neutrophil chemotactic responses to epithelial cell culture supernatants (36, 37). We suggest the difference lay in their use of cell lines or culture conditions in which SC is expressed at low levels. For example, the A549 cell line used by Smart and Casale (36) expresses SC at levels three orders of magnitude less than primary epithelial cells (25). Second, the culture of primary bronchial epithelial cells on uncoated plastic, for example by Abdelaziz et al. (37), reduces SC expression by 30-fold compared with growth on collagen-coated plastic as shown by Fiedler et al. (28). In this study, we have combined collagen coating of plastic ware with the use of primary epithelial cells to establish a relevant model of epithelial cell culture, which has demonstrated the high capacity for SC synthesis by these cells. The net neutrophil chemotactic activity we observed appears to be due to other factors produced by epithelial cells, possibly other stimulatory cytokines, complement cascade fragments or eicosanoids (38), and/or inhibitory prostanoids (39).
We have shown that SC inhibits the IL-8-induced migration of neutrophils, both in Boyden chamber assays and in a model of transendothelial migration. Neutrophils do not have a specific receptor for SC (4), and SC did not interfere with IL-8 binding to specific, CXCR1 and/or CXCR2, receptors on neutrophils. In parallel, SC did not inhibit signaling via the IL-8 receptor. However, SC did reduce the binding of IL-8 to low-affinity, nonspecific binding sites on polycarbonate filters and on endothelial cells. The SC-mediated increase in soluble forms of IL-8 we observed in our model of transendothelial migration indicates that SC exerts its effect by decreasing the gradient of immobilized chemoattractant across the endothelial cell monolayer. It has been demonstrated that an immobilized gradient of IL-8 is necessary to direct neutrophil migration, both across polycarbonate filters (24) and across endothelial cells (40). We propose that SC, synthesized exclusively by epithelial cells, may play a role in sequestering soluble IL-8 released into the tissue, preventing rebinding of IL-8 to matrix or endothelial cell surface proteoglycans (41) and effectively reducing tissue-bound gradients of chemoattractant. Because neutrophils activated by IL-8 before they contact the endothelium lose their ability to adhere to the vessel wall (40), and SC does not interfere with this binding of IL-8 to specific receptors, an anti-inflammatory function of SC is indicated.
The interaction of chemokines with carbohydrate residues in glycosaminoglycans has previously been described, and all chemokines are known to bind to heparin (42). While binding does not necessarily imply an effect on function, we showed an inhibitory effect of heparin on IL-8-induced neutrophil chemotaxis in micro-Boyden chambers (43). These observations have since been confirmed and extended to the inhibitory effect of heparin on IL-8 bound to endothelial cell surface heparan sulfate proteoglycans (44). The interaction of IL-8 with heparin involves highly sulfated residues on the glycosaminoglycan (45). However, the interaction of IL-8 with the carbohydrate residues, fucose, mannose, galactose, N-acetyl glucosamine, and neuraminic acid, on SC (8) is likely to be different and is the subject of our further investigations.
In summary, we have indicated that SC may be functionally important in down-regulating the IL-8-mediated recruitment of neutrophils to the airways. SC binds IL-8, and other chemokines, limiting the formation of haptotactic gradients of immobilized IL-8 across polycarbonate filters and endothelial cell monolayers, thereby inhibiting subsequent neutrophil responses. The carbohydrate component of SC is important in binding IL-8, and, coupled with the functional effects of SC-induced inhibition of IL-8-dependent neutrophil migration, we suggest that glycosylated SC may be part of a feedback mechanism to attenuate the inflammatory response promoted by IL-8.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Janis K. Shute, School of Pharmacy and Biomedical Sciences, St. Michael’s Building, University of Portsmouth, White Swan Road, Portsmouth PO1 2DT, U.K. E-mail address: jan.shute{at}port.ac.uk
3 Abbreviations used in this paper: SC, secretory component; pIgR, polymeric Ig receptor; sIgA, secretory IgA; ENA-78, epithelial neutrophil-activating peptide-78; GRO, growth-related oncogene-; PNGase F, peptide N-glycosidase F; CF, cystic fibrosis; 125I-IL-8, 125I-labeled IL-8.
Received for publication May 3, 2001. Accepted for publication June 1, 2001.
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16 ng/ml, SC significantly inhibited
the migratory response of neutrophils to 10-8 M IL-8. Data
are expressed as a percentage of the response in the presence of
10-8 M IL-8 alone and are shown as mean ± SEM of
three experiments using neutrophils from different normal donors. *,
p < 0.01; **, p < 0.001;
statistically significant reduction compared with control.
