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Departments of
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Internal Medicine, Division of Pulmonary and Critical Care Medicine,
Pathology and Microbiology, and
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198; and
§
Department of Immunology, Scripps Research Institute, La Jolla, CA 92037
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of C5aRs on epithelial cells may be particularly relevant
to the lung, since C5a has been strongly implicated as a mediator of
acute lung injury in animal models (5, 6) and in the adult respiratory
distress syndrome (7, 8). This C5a/C5aR connection to airway and
alveolar epithelium is further strengthened by a recent report
demonstrating that the presence of the C5aR is essential for mucosal
clearance of Pseudomonas aeruginosa infection in the lung
(9). In light of these results, we evaluated whether human bronchial
epithelial cells (HBEC)3
obtained ex vivo express C5aRs and whether these cells respond to C5a
by releasing the 
-chemokine IL-8 before and after their exposure
to cigarette smoke. HBECs were chosen for this study because their
origin in the lower respiratory tract makes them among the first cells
to encounter the irritants and toxins present in tobacco smoke.
Furthermore, HBECs are known to synthesize (10) and release IL-8 upon
exposure to cigarette smoke extract (CSE) (11). Thus, the initial HBEC
response to cigarette smoke exposure and the subsequent extent to which
these cells invoke C5a may be early and decisive events in cigarette
smoke-induced airway inflammation.
In this report we demonstrate that HBECs constitutively express C5aRs and respond to C5a by releasing small but statistically significant levels of IL-8. However, exposure of HBECs to cigarette smoke significantly enhances the C5a-mediated release of IL-8, an effect that is significantly attenuated in the presence of an anti-human C5aR Ab. These results are discussed in terms of a potential modulatory role for the C5a/C5aR system in airway inflammation induced by cigarette smoke.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Except where noted, modifying enzymes and other molecular biology reagents were purchased from Stratagene, La Jolla, CA, and used according to the manufacturer’s instructions. Anti-human C5aR Ab was generated against the N-terminal, surface-exposed linear region of the C5aR (residues 9–29) according to previously published methods (12). The conformationally biased decapeptide C5a agonist, YSFKPMPLaR, was synthesized by standard solid-phase techniques and purified in accordance with previously published methods (13). Human recombinant (hr) C5a was purchased from Sigma, St. Louis, MO.
Preparation of CSE for CSE-induced IL-8 release from HBECs
CSE was prepared as a saturated stock solution by bubbling the smoke from one research-grade 100-mm filtered cigarette (type 2R1; Tobacco-Health Research, University of Kentucky) in 25 ml of RPMI 1640 without hydrocortisone as described previously (14). A total of 0.5 ml of the CSE stock solution was then diluted with 9.5 ml of LHC9-RPMI to arrive at the 5% final concentration.
Cell retrieval and culture
Passaged HBECs were obtained from bronchial brushings of patients undergoing diagnostic bronchoscopy with lavage, or from tissue specimens obtained at autopsy as previously reported (15, 16). HBECs were also recovered by bronchoscopy from adult male and female volunteers who were either nonsmokers or had a current history of cigarette smoking as part of a clinical protocol approved by the University of Nebraska Medical Center’s Institutional Review Board. Immediately after retrieval, HBECs were washed twice and plated on collagen-coated tissue culture dishes in LHC-9/RPMI 1640 (50:50, v/v) (Life Technologies, Grand Island, NY). Cell cultures were re-fed every third day with serum-containing medium until confluent, at which time the cultures were passaged and maintained under serum-free conditions (17). Serum-free growth conditions were used to restrict contamination by fibroblasts. However, if fibroblasts persisted in cultures, they were removed by selective trypsinization (16). Second and third passage cells were cryopreserved in freezing medium supplemented with antibiotics (0.02% penicillin/streptomycin and 20% FCS in HEPES-buffered L15 medium; Biofluids, Rockville, MD) under liquid nitrogen. Anti-keratin and anti-vimentin cytochemical staining along with visual morphologic assessment was used to determine that cell cultures were >95% of epithelial phenotype (18). All subsequent culturing of HBECs was under serum-free conditions with second, third, and fourth generation passages of cells used in this study.
Flow cytometric analysis
HBECs were grown to 70 to 80% confluency in serum-free
LHC-9/RPMI 1640 medium (50:50, v/v) on collagen-coated tissue culture
dishes. Medium was removed and replaced with either LHC-9/RPMI medium
(Life Technologies) supplemented with 5% CSE, or LHC-9/RPMI alone and
incubated for various time periods. After the appropriate treatment
times, cells were rinsed once with medium, and then removed from the
dishes by treatment with 0.02% trypsin/EDTA (Life Technologies) for 5
min at 37°C followed by treatment with a 0.002% solution of soybean
trypsin inhibitor (Sigma). Cells (106) were washed,
resuspended in Dulbecco’s PBS with 1% FBS, and incubated with 5
µg/ml of anti-human C5aR9-29 Ab (12) or preimmune
rabbit 
-globulin (RG) for 20 min at 4°C. These cells were
washed and incubated in a 1:160 dilution of FITC-labeled
F(ab')2 fragment of goat anti-rabbit IgG (secondary Ab)
(Sigma) for 20 min at 4°C. The cells were then fixed in 4%
formalin/PBS, and indirect immunofluorescent-labeled cells were
analyzed on a Becton Dickinson FACStarPLUS (San Jose, CA)
flow cytometer. Forward- and side-scatter gates were set to select the
most characteristic and homogeneous population of HBECs for analysis.
In all, 104 gated events were collected for each flow
cytometric run and the data was analyzed with CELLQUEST software. The
homogeneity of the HBEC preparation used in these experiments was
verified by back gating on the C5aR-positive peak in the histogram and
comparing its scatter plot with that of the original.
Immunocytochemistry
HBECs were grown to 70 to 80% confluency as described above. Medium was removed and replaced with either LHC-9/RPMI supplemented with 5% CSE or LHC-9/RPMI alone and incubated for 1 h. Cells were rinsed once with medium and then gently removed from the dishes as described above. Cells were washed and resuspended at a concentration of 1 x 106 cell/ml in RPMI. Suspended cells were applied to individual fields of washed and hydrated 12-field adhesion slides (Bio-Rad, Hercules, CA) and allowed to sediment for 10 min. After fixing in 0.1% glutaraldehyde, cells were washed in PBS and both endogenous peroxidases and nonspecific Igs were blocked with a 30-min incubation in MAG solution (0.2% gelatin, 0.1% BSA, 0.5% H2O2 in HEPES-buffered saline, pH 7.4). Slides were then washed and incubated with the anti-human C5aR Ab (5 µg/ml in PBS containing 0.25% BSA) for 20 min at room temperature. Slides were washed again and incubated with 750 ng/ml of affinity-purified biotinylated goat anti-rabbit IgG (Sigma) or with 0.25% BSA/PBS for 20 min. Following another wash step, 500 ng/ml of horseradish peroxidase-conjugated streptavidin (Life Technologies) was applied to the cells for 20 min, washed, and incubated in chromogen solution (160 µg/ml 3,3'-diaminobenzidine, 1% H2O2, 1% NiCl2 in PBS) for 20 min. Finally, cells were washed, dehydrated in 95% ethanol, cleared with xylene, covered with a 5% glycerine solution, and sealed with Permount (Fisher, St. Louis, MO) for examination and photomicrography.
SDS-PAGE and Western blot analysis
HBECs (106) were treated with 5% CSE for 1 h as above. CSE-treated and control HBECs were rinsed with cold PBS and lysed in PBS-containing detergents (1% Triton X-100 and 0.5% sodium deoxycholate) and protease inhibitors (100 µg/ml leupeptin, 1 mM sodium orthovanadate, 34 µg/ml aprotinin, and 1 µg/ml pepstatin). Particulates were removed by centrifugation. Protein concentration was determined by the Bradford method with Bio-Rad protein reagent. Proteins were separated by SDS-PAGE under reducing conditions on a 4 to 10% discontinuous gradient gel. The resolved proteins were electroblotted to Immobilon polyvinylidene transfer membranes (Millipore, Bedford, MA). The membranes then were immunoblotted with anti-human C5aR Ab (5 µg/ml) overnight at 4°C with gentle shaking. Membranes were washed several times with buffer containing 20 mM Tris, 150 mM NaCl, 1% nonfat milk, and 0.2% Tween (pH 7.4) and incubated with 40 ng/ml anti-rabbit IgG peroxidase conjugate for 30 min at 4°C. An enhanced chemiluminescence kit (Amersham, Arlington Heights, IL) was used to visualize the blotted proteins.
RNA isolation and reverse transcriptase (RT)-PCR analysis
Total RNA was isolated from HBECs with Trizol reagent (Life Technologies) according to the manufacturer’s directions. Contaminating DNA was digested from 10 µg of total RNA mixture using 5 U of RNase-free DNase I (Promega, Madison, WI) in a 1x buffer containing 200 mM Tris-HCl, pH 8.4, 500 mM KCl, and 20 mM MgCl2. The samples were incubated at 21°C for 15 min followed by a 15-min incubation at 65°C to heat inactivate the enzyme. The RNA was reverse transcribed in a 20-µl reaction using a GeneAmp RNA PCR kit (Perkin-Elmer, Foster City, CA). cDNA was synthesized from 1 µg of RNA with murine leukemia virus RT at a final concentration of 2.5 U/µl in buffer containing 1 mM dNTPs, 5 mM MgCl2, 1.0 U/µl RNase inhibitor, and 2.5 µM random hexamers. Incubation of the reaction mixture was conducted at room temperature for 10 min, at 42°C for 15 min, at 99°C for 5 min, and at 4°C for 5 min. The PCR was performed in a total volume of 50 µl and consisted of 10 µl of the first strand reaction product, 2.5 U of Taq polymerase, 2 mM MgCl2, and primers at a concentration of 1 µM each. The 5' and 3' C5aR primers were purchased from Stratagene and were based on the human C5aR sequence to yield a 550-bp product. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were synthesized on an Applied Biosystems (Foster City, CA) model 394 DNA/RNA synthesizer in the Eppley Institute Molecular Biology Core Laboratory. The sequence of the GAPDH sense primer was 5'-CCATGGAGAAGGCTGGGG-3' and that of the antisense primer 5'-CCAAAGTTGTCATGGATGACC-3'. Both were designed using the published nucleotide sequence data from the human GAPDH gene (19). Amplification was performed in a Perkin-Elmer model 2400 thermal cycler with a 94°C hot start of 4 min followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, 72°C for 45 s, and a final extension of 7 min at 72°C.
Southern blot analysis
In all, 15 µl of the GAPDH PCR product mixture and 50 µl of
the C5aR PCR product mixture were subjected to electrophoresis on a 1%
agarose gel containing ethidium bromide and photographed under UV
light. The DNA was transferred to a Magna NT nylon membrane (Micron
Separations, Westboro, MA) using a Turboblotter (Schleicher & Schuell,
Keene, NH) rapid downward transfer system. The DNA was cross-linked by
UV light using a Stratalinker (Stratagene, San Diego, CA). The blots
were prehybridized at 42°C for 3 h in hybridization buffer
containing 50% formamide, 10x Denhardts, 6x SSPE, 1% SDS, and 50
µg/ml salmon sperm DNA (20). A 34-bp oligo-nucleotide
5'-CCGTGGCCATCGTCCGGCTGGTCCTGGGCTTCCT-3' corresponding to the 602-
to 635-bp region of the human C5aR (21) was synthesized in the Eppley
Institute Molecular Biology Core Laboratory and end labeled with
[-32P]ATP (NEN Research Products, Boston, MA).
The labeled human C5aR oligonucleotide was added to hybridization
buffer containing 50% formamide, 5x Denhardts, 6x SSPE, 0.5% SDS,
and 100 µg/ml salmon sperm DNA, and incubated for 18 h at 42°C
in rotating tubes in a hybridization oven (20). Blots then were washed
four times for 20 min each at 65°C. The first two washes contained
3x SSC and 0.1% SDS, the last two 0.1x SSC and 0.1% SDS. The blots
were visualized on a Phosphoimager (Molecular Dynamics, Sunnyvale,
CA).
Determination of IL-8 levels
Extracellular IL-8 released from HBECs was measured in culture supernatants using a sandwich ELISA according to the method of Mio et al. (11). HBECs were treated with the appropriate experimental conditions, supernatant medium was harvested and stored at -80°C, and cells were counted by a Coulter Counter (Coulter Electronics, Hialeah, FL). The levels of IL-8 released into the culture supernatants were measured and expressed as pmol of IL-8 per 106 cells.
Effect of CSE on 18-h accumulation of IL-8 in extracellular medium
HBECs were recovered as described above from adult smokers and from adults who had no history of cigarette smoking and were grown to 70 to 80% confluency as described. Cells were exposed to the following conditions to determine extracellular release of IL-8: 1) HBECs (106) were pretreated with 5% CSE for 1 h, washed, re-fed with LHC-9/RPMI medium, incubated for 2 h, washed, and incubated in the presence of 50 nM C5a for 18 h. 2) HBECs were exposed to the control vehicle for CSE (LHC-9/RPMI medium) for 1 h, washed, re-fed with medium, incubated for 2 h, washed, and incubated in the presence of 50 nM C5a for 18 h. 3) HBECS were exposed to 5% CSE for 1 h, washed, re-fed with medium, incubated for 2 h with the control vehicle for C5a (LHC-9/RPMI medium), washed, and incubated for 18 h in medium. 4) HBECs were exposed to the control vehicle for CSE (medium) for 1 h, washed, re-fed with medium, incubated for 2 h with the control vehicle for C5a (medium), washed, and incubated for 18 h in medium. A final 18-h incubation was utilized in these experiments because it is a time interval that has been used by other investigators for the assessment of C5a-mediated release of cytokines from human peripheral blood monocytes (22, 23).
Dose response of C5a and C5a peptide agonist
HBECs (106) were exposed to CSE for 1 h, washed, and then incubated in the presence of varying concentrations of hrC5a (Sigma) or a conformationally biased peptide of C5a, YSFKPMPLaR. The accumulation of IL-8 over the subsequent 18 h in extracellular medium was measured with IL-8-specific ELISA.
Inhibition of C5a-mediated IL-8 release
HBECs (106) treated with 5% CSE for 1 h
were incubated for 2 h with varying dilutions of the
anti-human C5aR Ab (500 µg/ml) or a nonspecific RG. The
Ab-treated cells were incubated for 18 h in the presence of 50 nM
C5a and the amounts of IL-8 released into the medium were determined by
ELISA.
Time course of IL-8 release from HBECs
The early time course for C5a-mediated IL-8 release in both control and CSE-treated HBECs was evaluated with the following experiment. Cells (106) were treated in growth medium containing 5% CSE for 1 h, washed, and then incubated for 2 h in medium containing 50 nM C5a. Supernatant medium was harvested and IL-8 was measured and reported as the initial or "pre" time point value for release of IL-8. The harvested HBECs were then washed and re-fed with fresh, C5a-free and CSE-free medium for 1 h, at which time extracellular IL-8 was again measured as a 1-h time point. Cells were again washed, incubated with fresh medium, and the process was repeated over the course of 2, 4, and 6 h. Extracellular IL-8 was measured at the incubation times by IL-8-specific ELISA. Three other groups of HBECs were incubated in an identical fashion except that one group was pretreated with 5% CSE for 1 h only, a second treated with 50 nM C5a for 2 h only, and a third incubated in medium only.
Statistical analysis
Statistical analysis of data was performed with the use of EXCEL, which allowed for the calculation of one- and two-tailed t tests for significant differences in IL-8 release between groups and within groups. A p value of < 0.05 was considered statistically significant. ANOVA was also performed between groups for statistical significance.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of C5aRs by HBECs was assessed by the ability of an anti-human C5aR Ab to bind to specific sites on the cell surface. This Ab was generated against the surface-exposed, N-terminal linear region of the C5aR (residues 9–29) and has been shown to bind with high affinity and specificity to C5aRs expressed on the surface of human monocytes (12), neutrophils (12), and hepatocytes (2).
Control (untreated) HBECs and HBECs treated with 5% CSE were stained
with either an FITC-labeled goat anti-rabbit IgG (secondary Ab),
preimmune RG plus secondary Ab, or the anti-human C5aR Ab plus
secondary Ab and subjected to single color flow cytometric analysis. As
shown in Figure 1
, neither the secondary
Ab (A) nor preimmune RG
(B) demonstrated any capacity to bind
specifically to either control or CSE-treated HBECs
(C). The forward and side scatter plot
(D) indicated the presence of a single
population of cells, albeit somewhat heterogeneous in terms of cell
size. All cells, regardless of size, stained positively for keratin,
but not for vimentin (data not shown), thereby supporting the
epithelial content of this preparation.
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, A–E).
Moreover, flow cytometry indicated an increase in the relative number
of HBECs that were capable of binding the anti-C5aR Ab after cell
exposure to 5% CSE for 15 min, 30 min, 1 h, and 2 h (Fig. 2, A–E, dotted lines). CSE exposure appeared to
increase the population of C5aR-bearing HBECs rather than increasing
the density of C5aRs expressed per cell. This was supported by the
increase observed in the number of C5aR-positive cells (counts)
after CSE treatment and that the resulting C5aR-positive fluorescence
peak intensity was not shifted with respect to control HBECs. After
CSE exposure for 4 h, the relative number of HBECs capable of
binding the anti-C5aR Ab returned to the levels observed in
controls (Fig. 2E).
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). Strong bands at approximately 46 kDa
were detected in lysates derived from control HBECs and HBECs treated
with 5% CSE for 1 h. The presence of the 46-kDa band is
consistent with the m.w. of the free C5aR (24). The bands at slightly
lower m.w. may reflect nonspecific binding of the anti-C5aR.
However, the faint bands at higher m.w. in both HBEC lanes may reflect
the C5aR attached to its intracellular G protein(s) (25, 26). It was
not possible to visually determine whether there were any significant
changes in the intensity of the C5aR band at 46 kDa between untreated
and CSE-treated HBECs.
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A).
Treatment of these cells with 5% CSE for 1 h resulted in an
increase in the intensity of the anti-C5aR Ab immunostain (Fig. 4B). This C5aR immunostaining pattern was observed
for all HBECs in the preparation irrespective of cell size.
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). RT-PCR analysis was performed with 5'
sense and 3' antisense primers specific for the human C5aR and GAPDH
genes. Primers were designed to yield a 550-bp PCR product for the C5aR
(A) and a 196-bp product for GAPDH
(B) as previously reported (12, 19). To
enhance visualization, the RT-PCR products were probed with a
32P-labeled oligonucleotide corresponding to the 602- to
635-bp fragment of the C5aR gene (21). The results shown in Figure 5A indicated that control HBECs expressed mRNA specific for
the C5aR. HBECs exposed to 5% CSE for 1 h also expressed
C5aR-specific mRNA, although this band appeared weaker than that
observed with the control HBECs.
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Since the synthesis and release of IL-8 is a typical C5a-mediated response in C5aR-bearing cells of myeloid origin such as monocytes (22), a first step in assessing the functional role of C5aRs expressed on HBECs was to examine the ability of control and CSE-treated HBECs to respond to C5a by measuring the release of IL-8.
Figure 6 shows the results of the 18-h
accumulation of IL-8 released into the medium from control HBECs,
control HBECs in the presence of C5a, CSE-treated HBECs, and
CSE-treated HBECs in the presence of C5a. Panels A,
B, and C represent IL-8 released from passaged
cells obtained from a patient (nonsmoker) with idiopathic pulmonary
fibrosis at autopsy, a healthy nonsmoker, and an asymptomatic smoker,
respectively. Control HBECs incubated in the presence of 50 nM C5a for
18 h (A) did not result in a significant
accumulation of IL-8 beyond that observed with control HBECs in the
absence of C5a. However, the control HBECs depicted in B and
C did release significantly more IL-8 in the presence of 50
nM C5a than did control cells in the absence of C5a. CSE-treated HBECs
incubated for 18 h in medium only resulted in a significant
accumulation of IL-8 relative to the untreated controls, results that
are consistent with earlier observations of CSE-induced release of IL-8
from HBECs (11). However, the most significant accumulation of IL-8 was
observed with CSE-treated HBECs incubated for 18 h in growth
medium containing 50 nM C5a.
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). A similar dose-response pattern
was observed with CSE-treated HBECs challenged with a conformationally
biased peptide agonist of C5a, YSFKPMPLaR. In this case, however, the
YSFKPMPLaR agonist was significantly less potent in stimulating the
release of IL-8 than the natural factor, a result consistent with other
C5a agonist-mediated responses (13, 22).
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, the
release of IL-8 from HBECs exposed to CSE for 1 h and subsequently
challenged with C5a was significantly attenuated in the presence of the
anti-C5aR Ab. Moreover, this inhibition of IL-8 was incrementally
recovered with increasing dilutions of the C5aR Ab. In contrast to the
C5aR Ab, the use of a nonspecific RG did not significantly block
IL-8 release from HBECs. These results support the release of IL-8
being mediated through the binding of C5a with its specific C5aR
expressed on the surface of the HBEC.
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shows
that a single challenge of control HBECs with 50 nM C5a alone for
2 h (circles) resulted in a small but significant increase in the
release of IL-8 relative to control cells in the absence of C5a
challenge (squares) over the course of 6 h. A 1-h exposure of
HBECs to 5% CSE (diamonds) resulted in the release of greater amounts
of IL-8 over the course of 6 h than control cells over the same
time interval. IL-8 levels for cells treated with CSE alone reached
significance only immediately after CSE exposure as well as 2 h
later, but did not differ significantly from HBECs challenged with C5a
alone over the same 6-h time course. Although the mean 1-h IL-8 level
obtained from HBECs exposed to CSE alone was higher than the
corresponding mean level obtained from cells treated with a single
challenge of C5a, this difference was not significant (Fig. 9). In
contrast, a single exposure of HBECs to CSE for 1 h followed by a
2-h challenge with 50 nM C5a (triangles) resulted in a significantly
greater release of IL-8 over the next 6 h relative to control
HBECs. Furthermore, levels of IL-8 released from HBECs pretreated with
CSE and followed by a 2-h challenge with C5a were significantly greater
than that from cells treated with a single challenge of C5a alone at
the 1-, 2-, and 4-h time points and at the 2-, 4-, and 6-h time points
for HBECs treated with CSE alone. Thus, these results indicate that a
sustained release of IL-8 was achieved for at least 6 h from HBECs
treated to a 1-h exposure to 5% CSE followed by a 2-h challenge
with C5a.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This enhanced functional responsiveness toward C5a could be the result
of a CSE-induced increase in the population of HBECs bearing the same
relative number of C5aRs, an up-regulation (increase) in the number of
C5aRs per HBEC, or other possible mechanisms. Flow cytometric data
suggest that CSE exposure increases the number of cells that express
the C5aR rather than increases the number of C5aRs expressed per cell.
This is based on the increase in the number of C5aR-positive cells
(counts) observed after CSE exposure, but not a rightward shift in the
flow cytometric peak that would be indicative of enhanced mean
fluorescence intensity. However, in the absence of radioligand binding
or cross-linking studies, we cannot exclude the possibility that CSE
exposure concomitantly increases C5aR density or receptor expression
within individual cells. The immunostaining results shown in Figure 4
are consistent with the flow cytometric results in that CSE appears to
result in an increase in the population of cells bearing the C5aR and
possibly greater C5aR expression per cell.
Results from Western blot analysis demonstrated the presence of C5aR in both control and CSE-treated HBECs, but offered no evidence to suggest any significant changes in the amount of C5aR protein expressed in the control or CSE-treated cells. Evidence for C5aR expression was also supported by the detection of C5aR-specific mRNA by RT-PCR and Southern blot analysis. In this case, however, there was an apparent decrease in the amount of C5aR-specific mRNA in the CSE-treated HBECs. The difference between the degree of mRNA and protein expression for the C5aR in the CSE-treated cells cannot be readily explained on the basis of these results. However, the RT-PCR findings are still consistent with other data supporting the presence of C5aR expression in HBECs. Thus, our results are consistent with the notion that exposure of HBECs to CSE results in increased numbers of cells bearing the C5aR, while some cells that constitutively express the C5aR may also increase receptor expression/density after exposure to CSE.
There are several possible explanations that could account for the enhanced C5a-mediated release of IL-8 from HBECs exposed to CSE. Again, one is based simply on an increase in the number of C5aRs per cell or, more likely, an increase in the number of cells expressing the C5aR as was observed in flow cytometry and immunohistochemical staining. However, flow cytometric results indicated changes in the population of C5aR-bearing HBECs within 15 min of CSE treatment and that the increase in C5a-mediated release of IL-8 was obtained within 1 h of CSE exposure. Thus, the time course of these observations argues against an increase in C5aRs via de novo synthesis as an explanation of the enhanced release of IL-8 by CSE-treated cells.
In addition, despite the fact that control HBECs constitutively express C5aRs, these cells were less responsive to the C5a-mediated release of IL-8 than their CSE-treated counterparts. These observations tend to favor another posttranslational mechanism(s) as the principal event that renders the C5aR more functionally responsive in CSE-treated HBECs than untreated control cells.
It may be that exposure of HBECs to CSE somehow makes existing pools of C5aRs more available on the cell surface for binding with the C5a ligand. Studies on other G protein receptors, such as the type 1 angiotensin II, ß2-adrenergic, and muscarinic receptors suggest that G protein receptors are capable of intermolecular "cross-talk" and dimerization, which can influence the affinity of agonist binding and biologic response (27, 28, 29). It is possible that an inflammatory stimulus such as cigarette smoke promotes intermolecular interactions between transmembrane components of the C5aR that result in receptor dimerization. In turn, the dimerized receptor may bind C5a with higher affinity and effect a more potent biologic response. Alternatively, exposure to cigarette smoke may induce more effective coupling of the intracellular signaling pathway(s) to the C5aR resulting in an enhanced biologic response. Whatever mechanism(s) is responsible for the enhanced C5a-mediated release of IL-8, our findings suggest that cigarette smoke modulates the C5a-mediated release of this chemokine from airway epithelial cells in vitro.
This modulation of C5aR expression in airway epithelial tissues has potentially important implications in describing the pathogenesis of smoking-induced airway inflammation in vivo. The ability of these cells to invoke C5a for the local release of IL-8 would afford these cells another mechanism by which they could recruit the necessary neutrophils and possibly lymphocytes in response to cigarette smoke exposure. However, in contrast to myeloid cells, limited information is available as to what regulates expression and functional responsiveness of the C5aR on airway epithelial cells (30). Recently, Haviland et al. (4) demonstrated that mice injected with LPS expressed more C5aR-specific mRNA than untreated controls. Although their results suggested that a stimulus such as LPS could alter the expression of C5aR-specific mRNA, the study did not attempt to determine whether the observed increases in mRNA were derived specifically from airway or alveolar epithelial cells or from inflammatory cells such as neutrophils and macrophages present in the lung tissue.
Perhaps it would make sense for airway epithelial cells to exhibit a gated responsiveness to C5a. Since these cells are in frequent contact with inhaled irritants and toxins, it may be advantageous for the epithelial mucosa to mount a local inflammatory response that is commensurate with the extent of its exposure and injury. Airway epithelial cells that are relatively unresponsive to C5a under normal conditions could then modulate their overall responsiveness to C5a by modulating C5aR number or expression, C5aR sensitivity to local inflammatory stimuli or cytokine/chemokine exposure, or coupling of intracellular signaling events to the C5aR. Thus, the C5aR expressed on airway epithelial cells may represent more of a molecular rheostat than an all-or-nothing molecular switch when engaged by the C5a ligand. Our results are consistent with such a concept of C5a and C5aR-mediated events.
The results of this study raise the question of whether the C5aRs
expressed on epithelial cells are in any way different from the C5aRs
expressed on myeloid cells. Also unclear is the precise cellular
location of these C5aRs on airway epithelium in vivo, though apical
immunostaining for the C5aR has been previously shown in
paraffin-embedded sections of human bronchial epithelium (4). In
addition, the source of C5a for interaction with C5aR-bearing
epithelial cells is unknown. Prior reports have indicated that the
human type II epithelial cell line A549 and rat alveolar type II
epithelial cells can generate complement components C3 and C5 and that
their synthesis can be modulated by IFN-, IFN-, IL-1, IL-2, and
IL-4 (31, 32, 33). Since C5a is a cleavage product of C5, one potential
source of C5a in the lung could be from airway or alveolar epithelial
cells themselves and/or from extravasation of serum sources by local
blood vessels. In addition, since the epithelial tissue layer is one of
the first to encounter airborne Ag, interesting questions arise about
the types of stimuli that might influence the functional expression
of C5aRs on airway epithelial cells and how the C5a/C5aR system might
contribute to airway mucosal defense against infectious pathogens.
In this light, Höpken et al. (9) have recently demonstrated that
C5aR-deficient mice were incapable of clearing intratracheally
instilled Pseudomonas aeruginosa. In contrast to the
wild-type mice, the C5aR-deficient mice succumbed to this infection
despite a vigorous airway and alveolar influx of neutrophils that were
capable of killing the bacteria in vitro. The results of this study
suggest that normal mucosal defense is established by C5a-mediated
responses other than the chemotaxis of neutrophils to the lung and that
C5aR-bearing cells other than neutrophils are likely involved. In
conjunction with our present data, obvious implications exist for the
C5a/C5aR system and exposure of the bronchial mucosa to environmental
toxins, bacteria, viruses, or carcinogens.
In summary, the results of this study demonstrate that HBECs obtained ex vivo constitutively express the C5aR. These C5aRs appear to be functionally more responsive to C5a after cell exposure to cigarette smoke. Upon exposure, HBECs respond to C5a in dose-dependent manner by releasing IL-8. The mechanism by which cigarette smoke modulates the C5aR is unclear, but may suggest that C5a-mediated release of IL-8 by airway epithelial cells in vivo could contribute to local cytokine responses important to airway epithelial cell function.
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Acknowledgments |
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2 Address correspondence and reprint requests to Dr. Sam D. Sanderson, Eppley Institute for Research in Cancer and Allied Diseases, 600 South 42nd Street, Omaha, NE 68198-6805.
3 Abbreviations used in this paper: HBEC, human bronchial epithelial cells; CSE, cigarette smoke extract; hrC5a, human recombinant C5a; RG, rabbit -globulin; RT, reverse transcriptase; uppercase single letters, the L stereoisomeric of the amino acid; lowercase letters, the D stereoisomeric form.
Received for publication July 28, 1997. Accepted for publication January 21, 1998.
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