The human bronchial epithelial cell is one of the first cell types to be exposed to the irritants and toxins present in inhaled cigarette smoke. The ability of the bronchial epithelium to modulate inflammatory and immune events in response to cigarette smoke is important in the pathogenesis of smoke-induced airway injury. We have shown that cigarette smoke extract and the complement anaphylatoxin C5a both independently induce increased expression of intercellular adhesion molecule (ICAM)-1 on airway epithelial monolayers compared with unstimulated cells in vitro. This enhanced ICAM-1 expression is associated with a greater capacity of the airway epithelial cells to bind mononuclear cells, a process that appears to require the proinflammatory cytokine tumor necrosis factor-α and protein kinase C intracellular signaling. Exposure of epithelial monolayers to the combination of cigarette smoke followed by C5a results in an additive response for ICAM-1 expression and mononuclear cell adhesion compared with smoke or C5a challenge alone. Inhibiting C5a receptor expression can attenuate these responses. These findings suggest that smoke exposure in some way enhances the functional responsiveness of the C5a receptor expressed on these airway epithelial cells for subsequent C5a-mediated increases in ICAM-1 expression and mononuclear cell adhesion. Our results may help explain the initiation and propagation of inflammatory events in vivo induced by chronic airway exposure to cigarette smoke.
The human bronchial epithelium is exposed to a variety of inhaled ambient pollutants. In habitual smokers, this includes those toxins suspended in the vapor phase of cigarette smoke. Increases in lung neutrophils (1–4), as well as the numbers of macrophages and CD8+ T-lymphocytes, have been described in the airways of smokers and those with chronic obstructive pulmonary disease (COPD) (5, 6). In addition, increases in chemotactic cytokines responsible for the recruitment and activation of these inflammatory cells have been reported in smokers and those with COPD, including increased interleukin (IL)-6 and IL-8 expression in sputum and bronchoalveolar lavage (BAL) fluid (7–11). Although the causative association between tobacco smoke and the development of this airway inflammation is well established, the precise molecular factors and mechanisms responsible for the initiation of these inflammatory events following chronic smoke exposure remain unknown. However, as there is increasing evidence that the human bronchial epithelial cell (HBEC) helps regulate immune and inflammatory responses (12–14), it is likely that these cells play a pivotal role in the development of chronic airway inflammation and injury induced by tobacco smoke. It would also appear likely that the interactions between airway epithelial cells and local mononuclear cells would have important consequences in the pathogenesis of COPD, as monocytes and alveolar macrophages are potent immune effector cells in the lung (15–17).
We have previously shown that HBECs constitutively express the high-affinity receptor (C5aR/CD88) for the anaphylatoxin C5a (18). HBECs initially exposed to cigarette smoke extract (CSE) followed by challenge with C5a released significantly more IL-6 and IL-8 compared with cells treated with either alone (18, 19). We have reported that this process is both protein kinase C (PKC)-and C5aR-dependent, and our results suggest that exposure of HBECs to cigarette smoke somehow “primes” the functional responsiveness of the C5aR expressed on these HBECs for their increased release of IL-6 and IL-8 (19). One mechanism that could explain the increased presence of IL-6 and IL-8 in the respiratory secretions of smokers may be the C5a-mediated production of these cytokines from smoke-exposed airway epithelium.
Because the recruitment of inflammatory cells to sites of airway injury is an early hallmark of inflammation, factors involved in the recognition and binding of these inflammatory cells to injured airway epithelium are likely to be important. It is known that airway epithelial cell expression of intercellular adhesion molecule-1 (ICAM-1) can be increased by several stimuli, a phenomenon that could potentially augment leukocyte function–associated antigen-1 (LFA-1)–bearing inflammatory cell binding to ICAM-activated airway epithelium (20, 21). Gonzalez Rodriguez and coworkers have recently shown that the corticosteroid budesonide inhibits C5a-induced upregulation of ICAM-1 expression on HBECs (22). The authors however, did not examine the role of cigarette smoke on airway epithelial ICAM-1 expression, or how such alterations in expression may modulate the adhesion of mononuclear cells to airway epithelium. Indeed, little is known about the direct effects of cigarette smoke on airway epithelial cells in terms of modulating ICAM-1 and epithelial–inflammatory cell adhesion events. Furthermore, because C5a is a potent mediator of neutrophil and eosinophil transendothelial migration and eosinophil adhesion to epithelial monolayers, the potential interactions between C5a and smoke in cell adhesion would seem important (23–25).
We present evidence to indicate that exposure of primary HBECs to either cigarette smoke or C5a results in an increase in mononuclear cell adhesion to these cells, and that this increased cell adhesion is associated with increased ICAM-1 expression. These results are similar to those we found in the human bronchial cell line BEAS-2B, though these transformed cells exhibit an overall decreased capacity to bind mononuclear cells compared with primary HBECs obtained ex vivo. It is possible that the in vivo recruitment and activation of mononuclear cells to sites of smoke-induced airway injury may enhance C5a-mediated inflammatory cell adhesion to the bronchial epithelium. This process could then amplify the local inflammatory milieu through autocrine and paracrine interactions between these effector cells. Taken together, such events may provide an important link between the chronic exposure of the respiratory mucosa to tobacco smoke and the subsequent development of chronic airway inflammation and bronchoalveoar damage characteristic of COPD.
Human recombinant (hr) C5a was obtained from Sigma, St. Louis, MO. Anti-C5a receptor antibody (anti-C5aR ab) was a gift of Dr. Edward Morgan (PharMingen; San Diego, CA). The anti-C5aR antibody (ab) was a monoclonal antibody (C85–41124 antibody) produced in rabbits immunized against the N-terminal tail region (residues 9–29) of the human CD88 C5aR (26–29). Previous studies have demonstrated that this antibody is highly specific for the human CD88 C5aR in terms of receptor binding and inhibition of C5a-mediated cytokine production from mononuclear and epithelial cells (18, 30–32). A neutralizing anti–tumor necrosis factor (TNF)-α ab (R&D Systems, Minneapolis, MN) was used to inhibit ICAM-1 expression and mononuclear cell–epithelial adhesion events. The PKC inhibitor calphostin C was obtained from Calbiochem (San Diego, CA), and an anti–ICAM-1 ab (CD54) was purchased from PharMingen. Soluble ICAM-1 (sICAM-1) was obtained from Endogen (Cambridge, MA) and was used for mononuclear cell LFA-1 blockade in adhesion experiments.
Primary HBECs were obtained from bronchial brushings or endobrochial biopsies of patients and volunteer subjects undergoing diagnostic bronchoscopy and BAL, or from tissue specimens obtained at autopsy following a modification of the method of Kelsen and colleagues (33). The Institutional Review Board for the Protection of Human Subjects at the University of Nebraska Medical Center has approved this protocol. Visual assessment and anti-keratin and anti-vimentin cytochemical staining confirmed the epithelial morphology of all cells. Only HBEC cultures that were found to be > 95% epithelial phenotype were used. Cells cryopreserved under liquid nitrogen were thawed, plated on collagen-coated dishes (Vitrogen-100; Cohesion, Palo Alto, CA), maintained in serum-free medium (LHC9/RPMI 50:50), and serially passaged not more than six times before use in these experiments. This technique permits the routine replication of experiments using identical cells. Some experiments were repeated using strains of cells isolated from various donors. In addition, parallel experiments using the human bronchial epithelial cell line BEAS-2B (CRL-9609; ATCC, Manassas, VA) were conducted.
THP-1 monocytes used for adhesion assays were obtained from ATCC (TIB-202). Peripheral blood mononuclear cells (PBMCs) were freshly isolated by density gradient elutriation from peripheral blood of healthy donors. Suspensions of cells were maintained in culture and fed with RPMI medium containing 10% fetal calf serum, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol until used in adhesion assays. Macrophages were either obtained by terminally differentiating THP-1 monocytes in the presence of serum-free RPMI supplemented with 10 nM phorbol myristate acetate, or were isolated from BAL fluid of asymptomatic subjects and cultured until use. Greater than 90% of these cells became adherent, and the macrophage phenotype was confirmed visually and by immunostaining for CD68-positive cells.
Inflammatory cell adhesion to epithelial cell monolayers was analyzed using a modification of the method of Striz and coworkers (34). Briefly, primary HBECs or transformed epithelial cells (BEAS-2B) were grown to confluence on collagen-coated 24-well tissue culture plates in serum-free growth medium (LHC9/RPMI). Epithelial cells were then incubated in the presence of various stimuli and/or inhibitors for selected periods. Mouse monoclonal anti-human ICAM-1 (anti-CD54 ab) or preimmune mouse serum (nonspecific ab control) were used to block epithelial ICAM-1. In some adhesion experiments, preparations of PBMCs and alveolar macrophages were loaded with soluble (s) ICAM-1 or bovine serum albumin (BSA) (control) before adding the mononuclear cells to the confluent epithelial monolayers. The use of sICAM-1 was intended to bind LFA-1 (CD11a) sites on monocytes/macrophages and prevent LFA-1 binding to epithelial ICAM-1. Inflammatory cells (THP-1, PBMC, Macrophages; 0.6 × 106/ml) grown in suspension or freshly isolated were labeled with fluorescent dye (BCECF/AM, Calbiochem, San Diego, CA) for 30 min, washed, then allowed to attach to epithelial monolayers for 20 min at 37°C. At no time were the mononuclear cell preparations exposed to any of the activators or inhibitors except sICAM-1 protein or its control, BSA. Nonadherent cells were removed by serial washes, and the cell co-culture was solubilized with 1% Triton for 20 min. Lysed cell emulsions were transferred to black 96-well microfluorimeter plates (Costar, Corning, NY) and read on a Fluorlite 1000 microplate reader at 490/530 nm excitation/emission wavelengths (Dynex Technologies, UK, Chantilly, VA or Sussex, UK). Mean fluorescence intensities were calculated from six replicate determinations and number of adherent cells calculated from a standard curve consisting of fluorescence labeled inflammatory cells.
HBEC or BEAS-2B cell monolayers (106 cells per condition) were variously treated under the conditions described below, serially washed, and gently detached from the plate with 0.25% trypsin/EDTA. Epithelial cell suspensions then were washed and incubated with phycoerythrin (PE)-conjugated monoclonal ab against human CD54 (1:50; PharMingen) or with control rabbit γ-globulin for 30 min. Some HBECs and BEAS-2B were treated initially with CSE followed by C5a, but instead of the PE-conjugated anti-CD54 as the primary ab, a PE-conjugated mouse monoclonal ab against human CD45RA (PharMingen) was used as a primary antibody control. Labeled epithelial cell suspensions were then fixed with 1% paraformaldehyde and resuspended at 0.8 × 106 cells/ml in phosphate-buffered saline. Flow cytometric analysis was performed on a FACScan cell sorter (Becton Dickinson, San Jose, CA) using CELLQUEST software. Mean fluorescence intensity was reported for 10,000 ungated events for each experimental condition.
Fresh CSE was prepared by using unfiltered, 85-mm Code 2RI cigarettes (University of Kentucky Tobacco Health Research Division) connected to a peristaltic pump apparatus. The cigarette was lit and the smoke produced was then bubbled through 25 ml of sterile RPMI medium. The peristaltic pump was equilibrated at a rate of one cigarette per 10 min pump time to produce 160 cm3 of tobacco smoke. The smoke-saturated medium was then sterile filtered and diluted into LHC9/RPMI medium as 5% of total volume. For all of the following experiments freshly made 5% CSE was used, as this concentration has been consistently shown to stimulate HBEC biological responses without displaying evidence of epithelial cell cytotoxicity (7).
ANOVA models were used to compare the average number of adherent cells among the treatment groups. The treatment factor was considered a fixed effect, and a random technician effect was included in the ANOVA model as well as an interaction between technician and treatment effects for those experiments replicated by different technicians. A natural log transformation of the response was used when the variability of the residuals was not constant over the range of the predicted response value. Post hoc comparisons between multiple pairs of treatment groups were conducted using the Tukey's multiple comparison procedure. A two-sided 0.05 significance level was used for analysis of significance. For all of the cell adhesion results described below, the reported P values were based on the natural log transformed number of adherent cells.
Because C5a stimulates the transepithelial migration of eosinophils and neutrophils (23, 25), we assessed the possible effects of C5a and cigarette smoke on the ability of primary HBECs to adhere the monocyte cell line THP-1. HBECs were pretreated for 2 h with either the anti-C5aR ab or with nonspecific rabbit IgG before exposure to CSE or C5a. HBECs then were treated with either 5% CSE in LHC9/RPMI medium or with 50 nM C5a for 1 h, washed, and then incubated again with 50 nM C5a or medium alone for 18 h. Control HBECs were treated with LHC9/RPMI medium alone for 18 h. Separate HBECs were also treated with an anti–ICAM-1 ab (anti-CD54 mab; PharMingen) or with mouse preimmune serum for the last hour of the 18-h incubation to block ICAM-1–associated attachment. The anti-CD54 ab or preimmune serum were added at the end of this 18 h interval to prevent possible washing out of the abs before application of the THP-1 cells. Thus, in some cases HBECs were treated with both the anti-C5aR ab and the anti-CD54 ab. After BEAS-2B were treated and incubated as described above, they were washed and refed with fresh medium alone. Thus, at no time in these experiments were the THP-1 monocytes exposed to CSE, C5a, anti-C5aR ab, anti-CD54 ab, or controls such as nonspecific rabbit IgG or mouse preimmune serum. THP-1 cells were added to the HBEC wells as described above for 20 min, followed by determination of THP-1 cellular adhesion to HBECs.
As shown in Figure 1, the adhesion of THP-1 cells to HBECs was increased over 5-fold when the HBECs were stimulated with 5% CSE alone or C5a alone (P < 0.001, P < 0.001, respectively, relative to control). The ability of HBECs to adhere THP-1 cells was further enhanced to a small, but statistically significant degree when HBECs were incubated in the presence of both CSE and C5a compared with CSE or C5a alone (P < 0.001, P < 0.001, respectively). Pretreatment of HBECs with the anti-C5aR ab decreased binding of THP-1 cells to HBECs exposed to C5a, though this effect was not seen in HBECs treated with CSE alone (P < 0.001, P = 0.78, respectively). When HBECs were pretreated with the anti-CD54 ab, the ability of smoke-exposed HBECs to adhere THP-1 cells was significantly reduced (P < 0.001) and C5a-stimulated adhesion was reduced by nearly 50% (P < 0.001). When both the anti-CD54 and anti-C5aR abs were applied, THP-1/HBEC adhesion decreased ∼ 90% relative to treatment with both CSE and C5a (P < 0.001; Figure 1). However, pretreatment of the HBECs with the mouse preimmune serum or rabbit IgG did not significantly diminish THP-1 attachment to epithelial monolayers exposed to both CSE and C5a, suggesting that the observed differences in adhesion were not due to nonspecific ab interactions at the cell surface. These results suggest that the binding of THP-1 to primary HBECs induced by cigarette smoke is precipitated largely through an ICAM-1–mediated mechanism and appears not to involve the C5aR, whereas C5a-stimulated cell adhesion is a process that requires both ICAM-1 and the C5aR.
Because transformed epithelial cell lines are frequently used as in vitro cell models, we wished to determine whether mononuclear cell binding to the transformed epithelial cell line BEAS-2B differed from our findings with primary HBECs. In addition, elutriated PBMCs and human alveolar macrophages were compared with THP-1 cells in terms of CSE- and C5a-stimulated adhesion to epithelial monolayers. BEAS-2B cells were incubated in medium alone, or were treated with 5% CSE alone, 50 nM C5a alone, or CSE followed by C5a. In some cases, BEAS-2B were pretreated with the anti-C5aR ab or cells were treated with the anti-CD54 ab at the end of the 18-h incubation, as was described in the above experiments. Separate preparations of PBMCs and alveolar macrophages also were loaded with sICAM-1 or an equimolar concentration of BSA as a nonspecific ab control before adding these cells to the confluent BEAS-2B. This was done to determine if saturation of inflammatory cell LFA-1, a ligand for epithelial ICAM-1, would inhibit or abolish cell adhesion. Other than sICAM-1 and BSA, the monocytes were not exposed to CSE, C5a, or inhibitory abs.
BEAS-2B treated with either CSE or C5a demonstrated significantly greater binding of THP-1cells (Figure 2A), alveolar macrophages (Figure 2B), and PBMCs (Figure 2C) compared with unstimulated control BEAS-2B monolayers (P < 0.001 for each comparison). BEAS-2B treated with the combination of CSE and C5a exhibited more than a 2-fold increase in binding of all the mononuclear cell types relative to BEAS-2B treated with CSE or C5a alone (P < 0.001 for each comparison). Incubation of the CSE- and C5a- treated epithelial monolayers with either the anti-C5aR or anti-CD54 abs reduced by > 50% THP-1, alveolar macrophage, and PBMC adhesion (Figure 2) (P < 0.001 for each comparison). This also was the case when both of these abs were simultaneously applied to the cell system relative to treatment with CSE and C5a (P < 0.001). Furthermore, blocking the LFA-1 integrin on mononuclear cells with sICAM-1 reduced mononuclear cell -BEAS-2B adhesion to a degree comparable to that of anti-C5aR and anti-CD54 abs. In contrast, mononuclear LFA-1 blockade with BSA had no significant effect on BEAS-2B adhering mononuclear cells (data not shown). The additive effect of CSE followed by C5a for cell adhesion appeared to be proportionally greater for BEAS-2B/mononuclear cell adhesion than it was for HBECs binding THP-1 cells. This is seen by comparison of Figure 1 (HBECs), which indicates a < 1.5-fold increase in the number of THP-1–adherent cells, with Figure 2A (BEAS-2B), which shows a > 2.4-fold increase in cell adhesion. However, the maximal adhesion response of BEAS-2B is less than the respective adhesion of THP-1 cells to primary HBECs treated with CSE and/or C5a.
Collectively, these results suggest that BEAS-2B exposed to CSE or C5a demonstrate a significant augmentation in their ability to bind PBMCs and alveolar macrophages. Second, these other mononuclear cells behave in a fashion very similar to THP-1 cells in terms of their adhesion to airway epithelium. It is also apparent that mononuclear cell/epithelial cell binding is further increased in the presence of both CSE and C5a, though overall cell adhesion is less when BEAS-2B are used in the assay in place of primary HBECs. Again, these adhesion events involve the C5aR on BEAS-2B cells and appear to be mediated through epithelial ICAM-1 interactions with its corresponding LFA-1 integrin on mononuclear cells.
Because cigarette smoke activates bronchial epithelial PKC in vitro (19, 35), we determined to what extent THP-1/BEAS-2B adhesion was dependent upon PKC signal transduction. We also examined whether the ability of BEAS-2B to adhere THP-1 cells was dependent on TNF-α in our in vitro model. BEAS-2 were incubated in the presence or absence of the PKC inhibitor calphostin C or nonspecific rabbit IgG. In separate BEAS-2B cultures, goat anti-human TNF-α neutralizing abs, or control goat preimmune serum, was added to the LHC9/RPMI medium. Only BEAS-2B were exposed to smoke/C5a, calphostin C, the anti–TNF-α abs, or controls. After BEAS-2B were treated with CSE and/ or C5a as described earlier, they were washed and refed with fresh medium containing none of the above reagents. The reported treatment differences were averaged across technicians.
As shown in Figure 3, attachment of THP-1 cells to BEAS-2B was increased 3.3-fold after BEAS-2B exposure to CSE alone, and was increased 3-fold when challenged with C5a alone (P < 0.001 for each comparison). Cells exposed to the combination of CSE followed by C5a, however, exhibited a 7.9-fold enhancement of adhesion (P < 0.001). The presence of anti–TNF-α abs in the growth medium decreased cell adhesion by 67% in CSE- or C5a-stimulated BEAS-2Bcells (P < 0.001 for each comparison). Likewise, when BEAS-2B were exposed to both CSE and C5a in the presence of anti–TNF-α abs, over 68% of THP-1 adhesion events were inhibited (Figure 3) (P < 0.001). It also can be seen that inhibiting PKC with calphostin C decreased THP-1/epithelial adhesion ∼ 60% or more in BEAS-2B treated with CSE alone or in those cells stimulated with both CSE and C5a, whereas blocking PKC in C5a-treated BEAS-2B resulted in a mere 7% reduction in cell binding (P < 0.001, P < 0.001, and P = 0.99, respectively). Indeed, it can be seen from Figure 3 that inhibition of PKC in cells treated with both CSE and C5a essentially drives cell adhesion down to the level observed with C5a alone; i.e., it has removed the stimulus from smoke for THP-1 binding to epithelium. That preimmune goat serum or rabbit IgG had no significant effect on THP-1 binding to BEAS-2B indicates that decreases in cell adhesion were not due to nonspecific properties hindering adhesion events. These results suggest that smoke invokes the activation of PKC in BEAS-2B to increase the ability of these epithelial cells to adhere THP-1 cells. In addition, airway epithelial/THP-1 binding induced by smoke or C5a appear to operate through the local, cellular effects of TNF-α.
Because CSE and C5a modulated the capacity of airway epithelial cells to adhere mononuclear cells, we then assessed whether these cell adhesion events might be associated with altered ICAM-1 surface protein expression in primary cultured HBECs after exposure to CSE, C5a, or their combination. HBECs were treated with either 5% CSE in LHC9/RPMI medium for 1 h, washed, and then incubated with medium alone or with 50 nM C5a for 18 h. Control HBECs were treated with medium alone for 18 h. In separate cultures, HBECs were pretreated for 2 h with either the anti-C5aR ab, calphostin C, or neutralizing anti–TNF-α ab and then exposed to CSE followed by C5a. The rationale for using calphostin C was to determine whether possible changes in ICAM-1 expression induced by CSE or C5a entail a PKC-dependent mechanism. In addition, as TNF-α is a potent activator of ICAM-1 gene and protein expression both in vivo and in vitro (36, 37), we wanted to assess whether the expected increase in observed ICAM-1 expression was dependent on the presence of TNF-α in the culture microenvironment. A PE-conjugated mouse monoclonal ab against human CD45RA (PharMingen) was used as a primary ab control in some HBECs stimulated with CSE and C5a.Figure 4B), and indicate that both C5a and CSE can independently stimulate ICAM-1–specific immunofluoresence on HBECs. It is further apparent that in those HBECs stimulated by the combination of CSE followed by C5a, a 2- to 6-fold greater effect on ICAM-1 expression is observed compared with the treatment of cells with either CSE or C5a alone (Figure 4B).
Pretreatment of cells with the anti-C5aR ab or anti–TNF-α ab resulted in a 50% attenuation of mean ICAM-1 immunofluoresence in cells exposed to CSE and C5a. A more dramatic effect, roughly an 80% inhibition of mean ICAM-1 immunofluoresence, occurred when HBECs were pretreated with the PKC inhibitor calphostin C and then exposed to both CSE and C5a. These results suggest that the modulation of ICAM-1 expressed on HBECs appears to be at least in part C5aR-dependent, with alterations in the expression of this adhesion molecule mediated through the PKC intracellular signaling pathway and the proinflammatory cytokine TNF-α.
We then compared ICAM-1 activation between primary HBECs and the transformed HBEC BEAS-2B. BEAS-2B monolayers were incubated in the presence or absence of either calphostin C, anti-C5aR ab, or anti–TNF-α ab in LHC9/RPMI medium. Mouse monoclonal PE-conjugated anti-CD45RA was used as a primary ab control in some BEAS-2B treated with CSE and C5a. Cells then were exposed to CSE and/or C5a as previously described and analyzed for alterations in ICAM-1 by flow cytometry.Figures 5A, 5B, and 5C). Inhibiting the effects of TNF-α in cell culture attenuates mean immunofluoresence for ICAM-1 by ∼ 50%, but only in response to the combination of CSE followed by C5a (Figure 5A). Treatment of cells with the anti-C5aR ab reduced ICAM-1 expression in BEAS-2B challenged with C5a alone and C5a combined with CSE, but not in cells exposed to only CSE (Figure 5B). As expected, this latter result indicates that C5a alters ICAM-1 expression through binding to its receptor. It is also evident that as in HBECs, calphostin C significantly reduced ICAM-1 expression in BEAS-2B treated with CSE alone or CSE followed by C5a, although this was not the case for cells treated only with C5a (Figure 5C). Results of these parallel experiments suggest that the smoke-induced increase in BEAS-2B ICAM-1 expression involves activation of PKC as was observed in primary HBECs. Second, stimulating cells with the combination of smoke followed by C5a has an additive effect on ICAM-1 surface expression that in part involves a TNF-α–dependent mechanism not present when the cells are challenged with CSE or C5a alone. Interestingly, when comparing the response shifts of CSE/C5a-challenged BEAS-2B versus primary HBECs the trends are similar, though the magnitude of the mean fluorescent signal is reliably 2-fold higher in the primary HBECs.
We have presented evidence to indicate that the binding of mononuclear cells to airway epithelium in vitro is increased by prior exposure of the epithelial monolayers to CSE or C5a. Our findings suggest that this augmented adhesion between epithelial cells and mononuclear cells is due at least in part to the enhanced expression of epithelial ICAM-1 induced by smoke or C5a. In this in vitro model, it appears that increases in ICAM-1 expression and epithelial/mononuclear cell adhesion induced by cigarette smoke also involve the activation of the PKC signaling pathway and require the local presence of TNF-α. However, C5a-induced increases in ICAM-1 expression and cell adhesion are not dependent on epithelial PKC activity, though such binding is mediated through the C5aR and again involves TNF-α. In addition, these events appear to require ICAM-1 interacting with its corresponding LFA-1 integrin on mononuclear cells. These findings are consistent with previously published results in which we reported that cigarette smoke activates PKC in airway epithelium and that both PKC and the C5a/C5aR complex are needed for maximal release of IL-6 and IL-8 from these cells in vitro (18, 19). The additive effect seen for ICAM-1 expression and cell adhesion when airway epithelial cells are treated with the combination of CSE and C5a is also consistent with the additive effect we reported for C5a-mediated release of these cytokines in smoke-exposed airway epithelium.
Our findings imply that epithelial/mononuclear cell binding was not solely mediated through ICAM-1 since blocking ICAM-1 with an anti-CD54 antibody did not completely abrogate cell adhesion events. Other adhesion molecules have been identified on airway epithelial cells, including CD44, LFA-3 (38, 39) and the β6 integrin subunit on tracheal epithelial cells exposed to ozone (40). It is possible that adhesion events mediated by smoke and C5a function through these other adhesion molecules in addition to ICAM-1.
In addition, we observed an ∼ 50% inhibition of epithelial/mononuclear cell adherence when epithelial cells were pretreated with the anti-C5aR ab. This degree of inhibition may reflect cell binding independent of the C5a/C5aR system, but it may also be due to other factors. In myeloid cells, the C5aR appears to cycle between the surface membrane and internalization into the cytoplasm before it is metabolized (41, 42). Thus, at any given moment after our epithelial cells were exposed to smoke or C5a, an unknown number of epithelial C5aRs may still have been internalized and therefore unavailable for binding to its C5a ligand or an anti-C5aR antibody. Furthermore, recent reports indicate that another subtype of the C5aR or “orphan C5L2” receptor has been identified and cloned (43, 44). Possibly this “orphan C5L2” receptor is also expressed on airway epithelium, with the anti-C5aR ab used in our model binding less avidly to this other C5aR. However, the anti-C5aR ab we used has been shown to be highly specific and have a high affinity for the CD88 C5aR (27, 28, 31, 32). The use of this anti-C5aR ab has also been shown to decrease in a dose-dependent manner the C5a/C5aR-mediated cytokine release from HBECs and mononuclear cells (18, 30).
Constitutive ICAM-1 expression on BEAS-2B was slightly greater than that observed with primary HBECs at baseline. Both HBECs and BEAS-2B cells displayed a greater increase in ICAM-1–specific immunofluoresence and ability to bind mononuclear cells in response to CSE followed by C5a. After such stimulation, the effect on ICAM-1 expression and cell adhesion was proportionately greater for the BEAS-2B cell line than primary HBECs. Overall, however, these transformed cells adhered THP-1 cells less well in terms of absolute or total adhesion events and expressed less total ICAM-1 immunofluoresence than did HBECs. It is unknown why there were differences in functional responsiveness at baseline and upon stimulation between primary HBECs and a related cell line, though such differences may be an artifact of using the SV-40 transformants. However, in HBECs stimulated with smoke and C5a both ICAM-1 and the C5aR were important to mononuclear cell adhesion, as blocking both ICAM-1 and the C5aR reduced adhesion events to control levels (Figure 1).
Our current findings indicate that ICAM-1 immunofluoresence was affected by the presence of TNF-α only when epithelial cells were treated with the combination of CSE and C5a. This was in contrast to our cell adhesion data, in which BEAS-2B cells stimulated with either CSE or C5a exhibited increased binding to THP-1 cells through a TNF-α–dependent mechanism. Inhibiting TNF-α with neutralizing abs in the cell medium implies that this cytokine generates its effects on cell adhesion by local autocrine and paracrine mechanisms that are independent of mononuclear cell production and release of TNF-α. Based on our results, we cannot specify whether CSE and C5a independently generate the release of TNF-α from airway epithelial cells into the cell medium, though it has been shown that airway epithelial cells are capable of releasing TNF-α (14, 45).
It is unknown why there is a greater response to the combination of CSE and C5a in both BEAS-2B and HBECs, but our findings suggest that smoke somehow enhances the functional responsiveness of the C5aR for increases in ICAM-1 expression and cell adhesion. We previously have described increased C5aR-specific immunofluoresence in HBECs stimulated with CSE (18), though it is not clear how CSE alters C5aR surface expression in these cells. One explanation for the greater effect of CSE and C5a in combination could involve the direct release of TNF-α from epithelial cells in response to CSE, with an ensuing activation of epithelial PKC by the presence of the locally active TNF-α. Wyatt and colleagues have described TNF-α−induced activation of airway epithelial PKC (37), and recently Krunkosky and coworkers reported that TNF-α increased epithelial cell ICAM-1 gene and protein expression through its stimulatory effect on epithelial PKC (36). Results of the present study suggest a scenario in which TNF-α generated by smoke-exposed epithelial cells could trigger PKC activation that could then lead to enhanced C5aR availability and/or affinity for C5a. Alternatively, it may be that smoke directly modifies the C5aR or directly activates PKC in addition to stimulating epithelial production of TNF-α. Collectively, these events could render the C5aR more available to, and/or enhance its affinity for, C5a, thereby leading to greater increases in ICAM-1 expression and cell adhesion than is possible with cell exposure to smoke or C5a alone.
Cell-to-cell interactions such as the adhesion of inflammatory effector cells to the airway epithelium are likely to be important events in the pathogenesis of chronic airway inflammation. This process involves epithelial ICAM-1 binding to its counterpart β1 (α4β1) (24) or β2 integrins (αMβ2, CD11b/18) (23, 25, 46, 47) on inflammatory cells, and a variety of factors have been shown to increase ICAM-1 expression on airway epithelium both in vivo and in vitro. These include viral and bacterial infection (20, 48, 49), LPS (21), diesel exhaust particles (50), as well as the cytokines TNF-α and interferon-γ (23–25, 51, 52). ICAM-1 expression by immunohistochemistry has been demonstrated in the airways and lung parenchyma of cigarette smokers with or without airflow obstruction (53) and a greater release of sICAM-1 has been described following smoke exposure of HBEC explants in vitro (54). Cigarette smoke condensate has been reported to increase ICAM-1 immunofluoresence in human endothelial cells, the surface expression of CD11b on monocytes, and monocyte adhesion to endothelial monolayers (55). In addition, the binding of monocytes to endothelial cells is partially inhibited by PKC inhibitors as well as abs to ICAM-1 and the β2 integrin CD11b (55). Thus, our findings in airway epithelium are consistent with earlier results reported for the effects of smoke condensate on monocyte adhesion to human endothelial cells.
C5a mediates eosinophil and neutrophil adhesion to epithelial monolayers (24, 25). This process was found to be ICAM-1– and CD18-dependent in eosinophils when the epithelial cells were previously stimulated with TNF-α and interferon-γ (25), and required β2 integrins for neutrophil attachment regardless of the activation state of the epithelium. More recently, it has been reported that TNF-α facilitates or “primes” C5a-induced eosinophil adhesion to airway epithelial cells, a process that requires fibronectin in association with α5β1 integrins (24). The focus of these other investigations have been primarily on the role of C5a in terms of eosinophil and neutrophil function rather than on the epithelial monolayers to which those inflammatory cells adhered. Our study differed from a recent report by Burke-Gaffney and colleagues (24) in that we exposed only airway epithelial cells to C5a and/or cigarette smoke, whereas in their study both inflammatory cells (eosinophils) and HBECs were incubated in the presence of C5a and TNF-α. We examined whether CSE “primed” airway epithelium, whereas in their study TNF-α was the “priming” agent for C5a-mediated eosinophil adhesion to HBECs. Because little is known about the direct effects of CSE or C5a on airway epithelium, we chose to isolate the effects of C5a and CSE on airway epithelial ICAM-1 expression and to determine whether such alterations in ICAM-1 expression could modulate the capacity of airway epithelial cells to adhere inflammatory cells. Our data are consistent with the observations of Burke-Gaffney and coworkers (24) in that we have shown that smoke- or C5a-stimulated airway epithelium adhere mononuclear cells through a TNF-α–dependent mechanism. Furthermore, as our results implicate a role for ICAM-1 and LFA-1 (CD11a) as integrins involved in the adhesion of mononuclear cells to airway epithelium, these data are consistent with other studies indicating the involvement of β1 integrins on epithelial adhesion events (23, 25, 46, 47). Our results are also similar to a recent report describing eosinophil transepithelial migration in response to C5a, in which C5a was found to increase ICAM-1 expression on HBEC monolayers (22). Again, in that study, however, the focus was primarily on the inhibition of eosinophil migration and ICAM-1 expression by the topical corticosteroid budesonide. In addition, the additive effects of cigarette smoke on epithelial ICAM-1 expression were not examined, nor were the effects of PKC signaling or TNF-α on the capacity of airway epithelium to adhere inflammatory mononuclear cells.
In summary, we have shown that exposure of airway epithelial cells to cigarette smoke or C5a results in enhanced expression of ICAM-1 on these epithelial cells compared with unstimulated epithelial monolayers. This augmented ICAM-1 expression is reflected in a greater ability of the airway epithelial cells to bind mononuclear cells, a process that appears to require the proinflammatory cytokine TNF-α and PKC intracellular signaling. Exposure of these airway cells to the combination of cigarette smoke and C5a results in an additive response for ICAM-1 expression and cell adhesion compared with smoke or C5a challenge alone, suggesting that smoke exposure in some way enhances the functional responsiveness of the C5aR for ensuing C5a-mediated responses. Thus, our findings may provide another mechanism in vivo for the initiation and propagation of inflammatory events induced by chronic airway exposure to cigarette smoke.
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