Abstract
CD40 and its ligand regulate pleiotropic biological responses, including cell proliferation, differentiation, and apoptosis. In many inflammatory lung diseases, tissue damage by environmental or endogenous oxidants plays a major role in disease pathogenesis. As the epithelial barrier is a major target for these oxidants, we postulated that CD40, the expression of which is increased in asthma, plays a role in the regulation of apoptosis of bronchial epithelial cells exposed to oxidants. Using 16HBE 14o− cells exposed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and increased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-κB and activator protein–1 activation and increased expression of the inhibitor of apoptosis, c-IAP1. However, oxidant stress–induced apoptosis was found to be caspase- and calpain-independent implicating CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation prevented mitochondrial release and nuclear translocation of apoptosis inducing factor. In conclusion, we demonstrate a novel role for CD40 as a regulator of epithelial cell survival against oxidant stress. Furthermore, we have identified, for the first time, an endogenous inhibitory pathway of caspase-independent cell death.
The normal bronchial epithelium acts as a physical barrier to protect the internal milieu of the lung by secreting mucus and cytoprotective molecules and displaying ciliary activity. It also responds to environmental stimuli by signaling to, and interacting with, cells of the innate and adaptive immune systems through secretion of cytokines and chemokines and expression of adhesion molecules such as intercellular adhesion molecule-1 and CD40 (1).
CD40 belongs to the TNF receptor family, which includes the TNF receptors (TNFRI and TNFRII), low-affinity nerve growth factor receptor, Fas, and CD30 (2). It is a 50-kD integral membrane glycoprotein that was independently identified as a surface marker on bladder carcinomas and on B cells (2, 3). Many studies have shown that CD40 plays a key role in the regulation of humoral cell–mediated immunity. Its natural ligand is a type II, 39-kD membrane glycoprotein, known either as CD40L or CD154, which was originally identified on activated T cells (4). Depending on the cell type and the local microenvironment, the interaction between CD40 and its ligand can modulate several responses, including cell proliferation, differentiation, apoptosis, isotype switching, and inflammatory mediator production (5).
CD40 expression and function has been studied extensively in B lymphocytes and other antigen-presenting cells (monocytes and dendritic cells). In these cells, CD40 plays an important role as a costimulatory molecule and regulates cell activation and proliferation. CD40 can also modulate apoptosis of lymphoid cells by different mechanisms, such as the inhibition of Fas-dependent apoptosis or by inducing the expression and/or activation of caspase family members, such as CPP-32 (6, 7). The signal transduction events leading to activation of cytokine gene transcription by CD40 ligation have also been studied mainly in B cells. Like other members of the TNFR family, CD40 has no intrinsic catalytic activity, but interacts with “signaling adapter proteins” termed TNFR-associated factors (TRAFs). Several studies have demonstrated that the cytoplasmic domain of CD40 has two binding sites for TRAF proteins (8). Many of the biological effects of TRAF signaling appear to be mediated through the activation of transcription factors of NF-κB and activator protein (AP)-1 family (2, 9).
CD40 has also been implicated in the regulation of the functional activation of structural cells, such as fibroblasts and epithelial cells. We previously reported that CD40 and CD40L are expressed in bronchial epithelial cells of normal subjects and, to a greater extent, subjects with asthma (10). It has also been demonstrated that CD40 ligation can lead to the functional activation of bronchial epithelial cells and to the release of inflammatory mediators (11, 12), which is related to NF-κB activation (13). In these previous studies, the proinflammatory responses triggered by CD40 ligation were evaluated alone or in the presence of proinflammatory cytokines. Recognizing that the epithelium will be exposed to oxidants derived not only from environmental sources (e.g., cigarette smoke and air pollutants, such as ozone and diesel exhaust fumes), but also from endogenous inflammatory cell products, especially in chronic inflammatory lung diseases, we investigated the interaction between oxidant stress and CD40 ligation on the intracellular signal transduction and epithelial survival. Using the human bronchial epithelial (HBE) cell line 16HBE 14o− (subsequently referred to as 16HBE), we show that CD40 ligation in the presence of oxidants activates NF-κB and AP-1 and, most significantly, that it protects epithelial cells exposed to oxidant-mediated cell death by blocking caspase-independent apoptosis.
The SV40 large T antigen–transformed 16HBE cell line is an HBE cell line that retains the differentiated morphology and function of normal human airway epithelia (14). Peripheral blood neutrophils were purified from healthy normal individuals by Ficoll-Hypaque centrifugation, as previously described (15, 16), and cultured in RPMI 1640 with 10% heat-inactivated FCS (complete culture medium).
For experiments, 16HBE cells were plated in 25-cm2 flasks at a density of 5 × 105 cells/ml in 5 ml Dulbecco's modified Eagle's medium/FBS). When cells were at 70–80% confluent, the medium was replaced with Dulbecco's modified Eagle's medium plus 1% FBS for the indicated times, in the absence or presence of 200–400 μM hydrogen peroxide (H2O2), and in the absence or presence of the anti-CD40 monoclonal antibody (mAb; G28-5, μg/ml). Dose–response curves have been generated previously to define the best concentrations of G28-5 mAb and of H2O2 in bronchial epithelial cells. Irrelevant mouse IgG1 isotype control Ab (clone MOPC-21) was purchased from Sigma (Buchs, Switzerland).Unless otherwise stated, cell culture reagents were from GIBCO BRL Life Technologies (Milan, Italy).
The general calpain inhibitor (L)-3-carboxy-trans-2,3-epoxypropionyl-Leu-amino-(4-guanidino) butane ethylester (E64-d) and staurosporin were purchased from Sigma. The caspase inhibitor N-benzyloxycarbonyl (z)1-Val-Ala-Asp (VAD)-fluoromethylketone (fmk) was purchased from Alexis Corporation (Laufelfingen, Switzerland). H2O2 30% was purchased from J.T. Baker (Deventer, Holland).
Protein A/G Plus-Agarose, rabbit polyclonal Abs recognizing inhibitory κB kinase (IKK) α, IKKβ, NF-κB p65 subunit, Bfl1/A1, goat polyclonal anti-c-IAP1 (D19), c-IAP2 (H-85), anti-apoptosis-inducing factor (AIF) rabbit polyclonal Ab (clone H-300) (working dilutions, 1:200, 1:100, 1:100, 1:200, 1:200, 1:200, and 1:100, respectively) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A secondary Alexa Fluor546–conjugated goat anti-rabbit Ab (1:500; Molecular Probes, Eugene, OR) was used to reveal AIF positivity.
Phospho c-Jun (Ser 63) and phospho c-Jun (Ser73) rabbit polyclonal Abs (used at 1:1,000) were from Cell Signaling (Beverly, MA) and anti-phospho-serine/threonine (mixed mouse monoclonal IgGs used at 1:500) were from Upstate Biotechnology (Lake Placid, NY). Anti-human caspase-3 and anti-human caspase-8 Abs (1:1,000) were purchased from Becton-Dickinson Biosciences, Pharmingen (Basel, Switzerland). Agonistic anti-Fas receptor mAb CH11 (1 μg/ml) was purchased from Beckman Coulter International (Nyon, Switzerland).
Horseradish peroxidase–conjugated secondary Abs goat anti-rabbit IgG (whole molecule; 1:12,000), rabbit anti-goat IgG (whole molecule; 1:8,000), and anti-β-actin (1:5,000; clone AC-15), were from Sigma; rabbit anti-mouse IgG (1:1,000) was from DAKO (Copenhagen, Denmark). Anti-glyceraldehyde-3-phosphate dehydrogenase mAb (1:200; Chemicon International, Inc., Temecula, CA) was used to control protein loading.
G28-5 mAb was obtained from HB 9110 hybridoma cells purchased from American Type Culture Collection (Rockville, MD). The specificity and functional activity of G28-5 mAb were tested by Clark (17).
HB 9110 cells were cultured in RPMI with 10% FBS, and G28-5 mAb (IgG1) released in the supernatant was purified by protein G affinity chromatography (HiTrap Protein G HP; Amersham Biosciences, Milan, Italy) according to the manufacturer's instructions.
Briefly, the supernatant was adjusted to pH 7.0 with binding buffer (20 mM sodium phosphate, pH 7) and then applied to a protein G column equilibrated with PBS. After washing, bound Ab was eluted with 2–5 vols of elution buffer (0.1 M glycine HCl, pH 2.5) and collected into 100 μl/ml of 1 M Tris-HCl (pH 9) to neutralize the Ab-containing elution buffer. The sample was then dialyzed overnight against 20 mM sodium phosphate buffer (pH 7.0), and the G28-5 mAb was quantified using the bicinchonic acid method according to the manufacturer's instructions (Pierce, Rockford, IL) before being stored frozen in aliquots at −20°C.
16HBE cells were cultured under the conditions indicated, then washed with cold PBS and lysed as previously described (18), using a QuantiZyme caspase-3 cellular activity assay kit (Biomol, Plymouth Meeting, PA). Caspase-3–like activity of recombinant caspase-3 (Calbiochem, San Diego, CA) was also analyzed as positive control for the experiments.
Western blot analysis was performed as previously described (19, 20). Briefly, 16HBE were lysed into ice-cold lysis buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM NaCl, 5 mM ethylendiaminetetraacetic acid, 1% Nonidet P-40; phosphatase inhibitors consisted of 20 mM β-glycerophosphate, 0.3 mM Na3VO4, 1 mM Benzamidine (ICN Biochemicals Inc, Aurora, OH); protease inhibitors consisted of complete protease inhibitors cocktail (Roche, Milan, Italy); the 16HBE lysates were centrifuged at 10,000 × g for 5 min. The protein content of the supernatants was analyzed using a bicinchonic acid assay (Pierce); 25–30 μg of lysate was then denatured under reducing conditions by boiling for 3 min in 50 mM Tris-HCl (pH 6.8), 1% SDS, 2% β-mercaptoethanol, and 0.01% bromophenol blue. Proteins were separated by SDS-PAGE and transferred by electrophoresis onto Immobilon-P membranes (Millipore, Bedford, MA). After transfer, the membranes were blocked overnight at room temperature in PBS containing 3% BSA and 0.5% Tween 20 before being incubated for 1 h at room temperature with the primary Abs. After washing, the blot was incubated for 45 min with the appropriate horseradish peroxidase–conjugated secondary Ab; bound Ab was detected using the ECL chemiluminescence detection system (Amersham-Pharmacia, Biotech), according to the manufacturer's instructions. Membranes were stripped and reprobed with housekeeping proteins β-actin or glyceraldehyde-3-phosphate dehydrogenase Abs to normalize differences in protein loading. Autoradiographic films were scanned by densitometry and analyzed using the NIH Image/Gel Plotting analysis program (National Institutes of Health, Bethesda, MD). Results were normalized and expressed as the ratio of the quantification of the band intensity of protein tested after correction with the band intensity obtained for the β-actin.
Cells were exposed as indicated in the Results section and then harvested into lysis buffer, as described above (19, 20); 500 μg of each lysate was incubated with 2.5 μg of specific Ab (anti-IKKα or anti-IKKβ, or anti-c-Jun N-terminal kinase-1 [JNK1]) for 2 h and then immunoprecipitated by incubation overnight at 4°C with 20 μl protein A/G Plus-Agarose. Precipitates were washed three times with lysis buffer and then solubilized by boiling into 2× sample buffer (2% SDS, 10% glycerol, 100 mM DTT, 60 mM Tris-HCl [pH 6.8], and 0.001% bromophenol blue). Samples were separated by SDS-PAGE using 10% polyacrylamide gels and then transferred onto nitrocellulose membranes. Membranes were probed with an anti–phosphoserine/threonine mixed mouse monoclonal IgG at 1:500 dilution (Upstate Biotechnology) with ECL detection, as described above. Autoradiographic films were scanned by densitometry using the NIH Image/Gel Plotting analysis program, and data were expressed as the ratio of the band intensity of the phosphorylated protein versus the band intensity of the total protein.
After treatment, cells were harvested by trypsinization, pelleted, and gently resuspended in PBS. The percentage of cells in different phases of the cell cycle was determined by flow cytometry after permeabilization and staining the nuclei with propidium iodide (PI); data were analyzed in the form of a DNA histogram.
Morphologic evaluation of nuclei was accomplished by fluorescent staining with Hoechst dye. 16HBE cells (n = 3) were cultured for 16 h, and, where indicated, treated with H2O2, G28-5, z-VAD-fmk, and E64-d. Cells were stained in viable conditions with Hoechst 33342 (10 μg/ml), and the number of cells with apoptotic nuclei was assessed as a percentage of the total cell number.
Apoptosis was also measured according to the technique of Vermes and coworkers (21), in which binding of AnnexinV (AxV) was used to detect phosphatidylserine, which is externalized on the outer leaflet of the plasma membrane of apoptotic cells. AxV-FITC (1 μg/ml) and PI (2.5 μg/ml) were added to the tubes with 1 × 105 cells/100 μl binding buffer. The cells were incubated in the dark for 15 min, and then analyzed using a FACScan flow cytometer (Becton Dickinson, Oxford, UK). Control tubes lacking either AxV-FITC or PI, or both, were included for the acquisition. Analysis of dot plots of fluorescence detector (FL) 1 (AxV-FITC) versus FL2 (PI) was performed using WinMDI 2.8 (Flow Cytometry software, University of Massachusetts). The degree of apoptosis was expressed as the number of AxV+/PI− cells shown as a percentage of total cells.
The percentage of nuclei positive for AIF in 16HBE cells was quantified by computer-assisted image analysis (Coolorvision 1.7.6; Improvision, Coventry, UK). For each slide, the total number of nuclei was systematically assessed based on color balance. At the beginning of each session, the image analysis system was standardized using the same control slide to ensure reproducibility of analysis.
Each slide was coded, and measurement of AIF-positive nuclei expression was performed by an independent observer. Results were expressed as a percentage of the number of nuclei in wich AIF translocated compared with the total number of nuclei in each slide.
Data are expressed as the mean ± SD of replicate determinations as indicated. Unpaired t tests were used for Western blot analyses. ANOVA was used for all the other analyses. P < 0.05 was considered significant.
To examine whether ligation of CD40 and/or exposure to oxidants affected NF-κB levels, we analyzed NF-κB p65 subunit protein levels by Western blots. As shown in Figure 1A, CD40 ligation for 24 h induced a significant increase in p65 subunit expression, and this showed a further increase in the presence of H2O2. These findings suggest that, in bronchial epithelial cells, CD40 engagement affected NF-κB levels, not only under basal conditions, but also in association with oxidant stress.
Figure 1. CD40 ligation activates NF-κB pathway in 16HBE exposed to H2O2.16HBE cells were treated with (solid bars) or without (open bars) G28-5 (10 μg/ml) for 24 h in the presence or absence of H2O2 at 200, 300, and 400 μM. (A) Expression of p65 protein. Signals corresponding to p65 on the various Western blots were semiquantified by densitometric scanning and analyzed using the NIH Image/Gel Plotting analysis program. Results were normalized and expressed as the ratio of the quantification of the band intensity of p65 after correction with the band intensity obtained for the β-actin. (B) Expression of p-IKKα/IKKα: 500 μg of total lysate was immunoprecipitated with IKKα and analyzed via immunoblotting using antiphosphorylated serine/threonine mixed mouse Abs, as described in Materials and Methods. Open bars, −G28-5; closed bars, G28-5. (C) Expression of p-IKKβ/IKKβ: 500 μg of total lysate was immunoprecipitated with IKKβ and analyzed as described in Materials and Methods. Signals corresponding to p-IKKα and IKKα and p-IKKβ and IKKβ were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the band intensity of the phosphorylated protein versus the band intensity of the total protein (NIH Image/Gel Plotting analysis program). The results are representative of three independent experiments. * Significant difference between Ab− and Ab+ groups.
[More] [Minimize]Both CD40 ligation and oxidant stress are known to activate NF-κB in epithelial cells (13, 22). This involves a cascade in which phosphorylation of the IKKα and IKKβ serine/threonine kinases leads to phosphorylation of IκB, causing its ubiquitination and degradation by the 26S proteasome, allowing NF-κB nuclear translocation. In bronchial epithelial cells, CD40 ligation caused a marked increase in the amount of phosphorylated IKKα in comparison with untreated cells or those treated with H2O2 alone (Figure 1B). The amount of phosphorylated IKKβ was also significantly increased after CD40 ligation alone and when associated with H2O2 treatment at 300 and 400 μM (Figure 1C). These data are consistent with those of previous reports (13) showing that CD40 ligation promotes NF-κB activation.
Cytokines and various cellular stresses are known to activate JNK1, which phosphorylates c-Jun, resulting in its activation and stabilization. Thus, the involvement of JNK1 and c-Jun in the response to CD40 ligation and oxidant stress was also examined. After CD40 ligation, the phosphorylation of JNK1 was increased (Figure 2A), and the phosphorylation was further increased when the cells were exposed, concomitantly, to different concentrations of H2O2. Consistent with their ability to activate JNK1, CD40 ligation, or exposure to different concentrations of H2O2 enhanced the relative amount of phosphorylation of c-Jun on Ser 63 (Figure 2B) at all the doses tested, whereas higher doses of H2O2 failed to affect c-Jun phosphorylation on Ser 73 and suppressed the effect of CD40 ligation.
Figure 2. CD40 ligation activates activator protein (AP)-1 pathway in 16HBE exposed to H2O2. (A) Expression of p-JNK1/JNK1 on 16HBE treated with or without G28-5 (10 μg/ml) for 24 h in the presence or absence of H2O2 at 200, 300, and 400 μM; 500 μg of total lysate was immunoprecipitated with JNK1 rabbit polyclonal Ab and analyzed via immunoblotting using antiphosphorylated serine/threonine mixed mouse Abs, as described in Materials and Methods. Signals corresponding to p-JNK1 and JNK1 on the various Western blots were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the band intensity of the phosphorylated protein versus the band intensity of the total protein (NIH Image/Gel Plotting analysis program). (B and C) Expression of phosphorylated c-Jun on Ser 63 and Ser 73, respectively. Signals corresponding to c-Jun on the Western blots were semiquantified by densitometric scanning and results were normalized and expressed as the ratio of the quantification of the band intensity of c-Jun after correction with the band intensity obtained for the β-actin. Results are representative of three independent experiments. * Significant difference between Ab− and Ab+ groups. Open bars, −G28-5; closed bars, G28-5.
[More] [Minimize]In keratinocytes, CD40 signaling alters the cell cycle by decreasing the number of cells in the G1 (interval between the completion of mitosis [M] phase and the beginning of synthesis [S] phase) and S phases and causing an accumulation in G2/M phase of the cell cycle (23). In the case of bronchial epithelial cells, CD40 ligation alone had no effect on cell cycle progression. However, H2O2 was found to suppress the proportion of cells in G0/G1, and there was an increase in the number of cells in S phase (Figure 3). In contrast, the presence of H2O2 together with G28-5 caused a significant reduction in the number of cells in the G0/G1 and S phases of the cell cycle in comparison with H2O2 alone, and a concomitant increase in the number of cells in G2/M phase (P < 0.03 for all; n = 3) (Figure 3).
Figure 3. Flow cytometry analysis of cell cycle. 16HBE cells were treated for 16 h with or without H2O2 and G28-5 (10 μg/ml). DNA content was quantified by PI staining and flow cytometry analysis, as described in Materials and Methods. Results are displayed as histograms representing the percentage of cells in the different phases of cell cycle (G0/G1, S, G2/M). Open circles indicate statistical significance (P < 0.03) according to ANOVA comparing treatment with untreated control. * Statistical significance (P < 0.03) according to ANOVA comparing association of G28-5 (10 μg/ml) plus H2O2 with H2O2 alone.
[More] [Minimize]The effects of CD40 ligation on cell cycle progression led us to consider its effect on cell survival. To determine whether signals transduced by CD40 ligation in bronchial epithelial cells were able to affect oxidant-induced cell death, 16HBE cells were treated for 16 h with 200 μM H2O2 in the absence or presence of G28-5. Morphologic evaluation of nuclei stained with Hoechst dye showed that H2O2 alone caused a significant increase in the number of late apoptotic cells; concomitantly, viability was significantly decreased from 89.7 ± 2.4% to 45.7 ± 2.2% (P < 0.001; n = 3) (Figure 4A). The presence of G28-5 (10 μg/ml) was able to suppress the number of late apoptotic cells caused by H2O2 treatment, with a consequent increase in viability when compared with H2O2 alone (45.7 ± 2.2 versus 65.7 ± 8.9; P < 0.05) (Figure 4A).
Figure 4. Cell death evaluation. (A) Morphologic evaluation of nuclei stained with Hoechst dye. 16HBE cells (n = 3) were treated for 16 h with or without H2O2 and G28-5 (10 μg/ml). Cells were stained in viable conditions with Hoechst 33342 (10 μg/ml), and the number of cells with apoptotic nuclei was assessed as a percentage of the total cell number. Results are displayed as histograms representing the percentage of cells whose nuclei are not apoptotic (viable cells). * Statistical significance (P < 0.001) according to ANOVA comparing H2O2 treatment with untreated control. Open circles indicate statistical significance (P < 0.05) according to ANOVA comparing association of G28-5 plus H2O2 with H2O2 alone. (B) Flow cytometric analysis of AnnexinV-PE and PI staining. 16HBE cells (n = 6) were treated for 24 h with or without H2O2 and G28-5 (10 μg/ml) and then stained with PE-labeled AxV and PI, as described in Materials and Methods. Viable (AxV−/PI−), early apoptotic (AxV+/PI−), and late apoptotic/necrotic (AxV+/PI+) cells were measured and results displayed as histograms representing the relative cell percentage. Open circles indicate statistical significance (P < 0.001) according to ANOVA comparing treatment with untreated control. * Statistical significance (P < 0.001) according to ANOVA comparing association of G28-5 (10 μg/ml) plus H2O2 with H2O2 alone. Black bars, untreated; dark gray bars, G28-5 (10 μg/ml); medium dark gray bars, H2O2, (200 μM); medium light gray bars, H2O2, (200 μM) + G28-5; light gray bars, H2O2 (300 μM); white bars, H2O2 (300 μM) + G28-5.
[More] [Minimize]To confirm the morphologic observations based on nuclear staining of treated cells, FACS analysis was undertaken using AnnexinV and PI to characterize apoptotic and necrotic cells. As expected, 200 and 300 μM H2O2 caused a significant reduction in cell viability by 24 h (Figure 4B), and this was accompanied by a concomitant increase in the number of early, late apoptotic, and necrotic cells (Figure 4B). In the presence of 10 μg/ml G28-5, the number of late apoptotic/necrotic cells observed after 24 h of treatment with 200 μM H2O2 was reduced in comparison with H2O2 alone (P < 0.01; n = 6) (Figure 4B). In contrast, G28-5 was not able to significantly suppress the induction of cell death by concomitant exposure to 300 μM H2O2.
To investigate the mechanism by which CD40 ligation may protect against oxidant-induced apoptosis, we examined expression levels of the inhibitors of apoptosis, c-IAP1 and c-IAP2, by Western blot analysis. This showed that c-IAP1 expression was significantly higher in cells treated with G28-5 and H2O2 (200, 300, or 400 μM) at 24 h, either alone or in combination when compared with untreated cells (Figure 5A). In contrast, the levels of c-IAP2 or the BCL2-related gene, Bfl1/A1, at 24 h were not significantly different in any of the samples studied (Figures 5B and 5C).
Figure 5. Expression of c-IAP1, c-IAP2, Bfl1/A1 proteins. 16HBE cells were treated as described in Materials and Methods. (A) Expression of c-IAP1 protein. Representative Western blot analysis of c-IAP1 in 16HBE cells treated with or without G28-5 (10 μg/ml) for 24 h in the presence or absence of H2O2 at 200, 300, and 400 μM. Open bars, −G28-5; closed bars, G28-5. Signals corresponding to c-IAP1 on the various Western blots were semiquantified by densitometric scanning and results were normalized and expressed as the ratio of the quantification of the band intensity of c-IAP1 after correction with the band intensity obtained for the β-actin, according to NIH Image/Gel plotting analysis program. (B and C) Representative Western blot analysis of c-IAP2 and Bfl1/A1 in 16HBE cells treated as described above. The results are representative of three independent experiments. * Statistical significance (p < 0.001) between Ab− (−G28-5 treatment) and Ab+ (+G28-5 treatment) groups.
[More] [Minimize]The ability of CD40 ligation to induce c-IAP1 and to suppress oxidant-induced apoptosis led us to investigate the effect of CD40 ligation on caspase activation. However, when we measured caspase-3 and -8 activity by Western blot analysis or enzymatic assay of epithelial cell lysates after H2O2 exposure (Figure 6A), we found that the caspases were not activated by H2O2 treatment either in the absence or presence of CD40 ligation; in contrast, treatment with staurosporin induced cleavage of caspase-3 and -8 to generate active fragments of 17 kD and 18 kD, respectively. Similarly, we failed to detect any increase in caspase-3–like activity in H2O2-treated cells, contrasting with high caspase-3–like activity of recombinant caspase-3, and with caspase-3–like activity of Fas-activated neutrophils that were used as positive controls for the experiment (Figure 6B). To confirm the absence of caspase activation in response to oxidant stress, we compared the effect of a combination of caspase (Z-VAD-fmk) and calpain (E64-d) inhibitors on induction of apoptosis by Hoechst staining. Figure 6C shows that treatment with Z-VAD and E64-d failed to rescue 16HBE from H2O2-mediated cell death, suggesting that oxidant-mediated cell death in 16HBE cells is not dependent on caspase- and calpain-mediated pathways, even though it is suppressed by CD40 ligation.
Figure 6. Apoptosis induced by H2O2 treatment is caspase- and calpain-independent. (A) Effect of H2O2 and CD40 ligation on caspase-3 and caspase-8 activation. Representative Western blot analysis of caspase-3 and caspase-8 in 16HBE cells. Lane 1: staurosporine (positive control, 0.5 μM) exposure of 16HBE cells for 5 h induced the enzymatically active 17-kD and 18-kD fragment of caspase-3 and caspase-8, respectively. Filters were reprobed with an anti–glyceraldehyde-3-phosphate dehydrogenase Ab to ensure equal loading of the gels. The results are representative of three independent experiments. (B) Caspase-3 activity assay in 16HBE cells cultured in the presence or absence of G28–5 (10 μg/ml) and H2O2 (200 μM) for 16 h. As positive control, enzymatic activity of recombinant caspase-3 and neutrophils undergoing apoptosis by Fas receptor activation (CH11 1 μg/ml) were measured. Caspase-3–like activity was measured in 10 μl of supernatants as enzymatic conversion of the colorimetric substrate Ac-DEVD-pNA at 405 nm, as described in Materials and Methods. (C) Pharmacological inhibition of caspase-3 (20 μM z-VAD,) and calpain (10 μM E 64-d) failed to rescue 16HBE cells from H2O2-mediated cell death. Viability was measured with Hoechst staining. Values are means ± SEM of three independent experiments. Open circles indicate statistical significance (P < 0.001) according to ANOVA comparing treatment with untreated control. * Statistical significance (P < 0.001) according to ANOVA comparing association of G28-5 (10 μg/ml) plus H2O2 with H2O2 alone.
[More] [Minimize]Recent studies have shown that caspase-independent apoptosis can be mediated by mitochondrial membrane depolarization and translocation of AIF from mitochondria into the nucleus (24). To determine whether oxidant-mediated apoptosis of 16HBE cells and its suppression by CD40 involved effects on nuclear translocation of AIF, we performed immunofluorescent microscopy with Abs to AIF. 16HBE were treated for 24 h with 200 μM H2O2 plus or minus 10 μg/ml of G28-5. Figure 7 shows that, in basal conditions, these cells express AIF in their mitochondria (Figure 7B); H2O2 treatment determined translocation of AIF from the mitochondria to the nuclei (Figure 7C). The translocation of AIF into the nuclei was blocked by G28-5 (Figure 7D). The results of quantification of nuclear translocation of AIF in 16HBE cells under the described conditions are reported in Table 1.
Figure 7. Immunofluorescent analysis of AIF expression and localization. 16HBE cells were treated for 24 h with 200 and 300 μM H2O2 plus or minus G28-5 (10 μg/ml). Cells were then fixed, permeabilized, and stained with an anti-AIF rabbit polyclonal Ab, followed by a secondary Alexa Fluor546–conjugated goat anti-rabbit Ab. (a) Negative control, (b) untreated control, (c) 200 μM H2O2, (d) 200 μM H2O2 + G28-5 (10 μg/ml), and (e) higher magnification of (c). Scale bar = 50 μm. White arrows show translocation of AIF into the nuclei.
[More] [Minimize]Treatments | AIF nuclear positivity % |
|---|---|
| Untreated control | 0.5 ± 0.3 |
| H2O2 200 μM | 17.1 ± 4.7 |
| H2O2 200 μM + G28-5 | 3.4 ± 2.3 |
| H2O2 300 μM | 22.9 ± 6.4 |
| H2O2 300 μM + G28-5 | 5.3 ± 3.2 |
CD40 ligation has been reported to play an important role in the immune and inflammatory responses mediated by bronchial epithelial cells, inducing the release of inflammatory mediators, such as IL-8, RANTES (regulated upon activation, normal T-cell expressed and secreted), and monocyte chemotactic protein-1, and modifying the expression of important adhesion molecules (i.e., intercellular adhesion molecule-1) (11, 25). Moreover, CD40 ligation can regulate cell survival in many different cell types, including B cells (26, 27), keratinocytes (23), monocytes (28), and dendritic cells (29). We now report for the first time that CD40 ligation can suppress oxidant-induced apoptosis in bronchial epithelial cells. Furthermore, our findings that CD40 signaling suppresses caspase-independent apoptosis by blocking AIF release represents the first identification of a naturally occurring inhibitory pathway for this process.
Oxidant damage, either from environmental (cigarette smoke, ozone exposure, etc.) or endogenous oxidants (catabolic cell products, neutrophil-derived reactive oxygen, etc.), is one of the first and most important stimuli to stress bronchial epithelial cells. It is well known that oxidant stress plays a key role in the pathogenesis of many inflammatory lung diseases (30). Several studies have shown that oxidant stress may also affect the immune response by inducing an upregulation of costimulatory molecules, such as CD40 and CD86, as well as the expression of human leukocyte antigen-DR, determining a persistent state of immune activation (31). Consistent with this observation, we have previously found that CD40 is increased in asthmatic bronchial epithelium (10).
To understand whether CD40 functions as a regulator of bronchial epithelial cell survival in response to oxidant stress, we exposed cells to H2O2 and analyzed cell cycle progression and induction of apoptosis. CD40 ligation was found to increase the number of cells in G2/M, and concomitantly reduced the number of dead cells. The increase of G2/M cells observed after treatment with H2O2 and CD40 ligation was accompanied by a parallel decrease of G0/G1 and S cells. This cell cycle configuration is typical of cells that are undergoing active proliferation. These data are compatible with those of previous reports on the effects of CD40 in other cell types (26, 27, 32).
Together with increased proliferation, we observed a reduction in cell death associated with CD40 signaling. In particular, there was a significant reduction of late apoptotic/necrotic cells, as shown with both Hoechst and AnnexinV staining when 16HBE cells were exposed to oxidant stress with CD40 ligation. In vertebrates, there are two major execution programs downstream of the death signal: the protease pathway, involving caspases and calpain and the organelle dysfunction pathway, of which mitochondrial dysfunction is the best characterized (33). Our data show that in oxidant stress–induced epithelial cell death, apoptosis occurs even in the presence of caspase and calpain inhibition. Thus, although 16HBE cells upregulated c-IAP1 in response to CD40 ligation and oxidant stress, this did not seem to explain the mechanism of cell survival induced by CD40 signaling.
There is now increasing evidence that caspases are not necessarily sufficient for apoptosis, and that complex interactions of death signaling pathways are required for commitment to, and execution of, apoptosis (34). This led us to examine whether CD40 ligation affected AIF, which is released from mitochondria in response to activation of poly (ADP-ribose) polymerase-1 (PARP-1), an important activator of caspase-independent cell death. Activation of PARP-1 initiates a nuclear signal that propagates to the mitochondria, triggering the release of AIF that shuttles from the mitochondria to the nucleus, inducing peripheral chromatin condensation and large-scale fragmentation of DNA (24, 35). These are the characteristic hallmarks of late apoptosis. In contrast, intramitochondrial AIF acts as a free radical scavenger, decreasing H2O2-mediated cell death (36). Consistent with the ability of CD40 ligation to reduce the number of late apoptotic/necrotic cells observed in cells exposed to oxidant stress, we found that it also suppressed mitochondrial AIF release, identifying the CD40 signal transduction pathway as an inhibitor of caspases-independent apoptosis.
In our studies, we demonstrated that engagement of CD40 triggers different signaling pathways, including NF-κB, as previously reported (13), and AP-1, and that this was augmented in response to oxidant stress. Although the NF-κB pathway is important for regulating the expression of cellular genes that are involved in the control of the immune and inflammatory response (37, 38), it can also prevent cellular apoptosis (39, 40). Several genes, the expression of which is regulated by NF-κB, may play a role in blocking apoptosis. These include cellular inhibitors of apoptosis c-IAP1, c-IAP2, TRAF1, and TRAF2 (41–43). The c-IAPs and TRAF1 are known to bind to TRAF2, and TRAF2 is required for NF-κB activation. In our experiments, the CD40 ligation leads to NFκB activation and induction of at least one of the NF-κB antiapoptotic target genes, c-IAP1. Recent studies have shown that IAPs directly inhibit some caspases, such as caspase-3 (44, 45), thus arresting the proteolytic cascade and providing protection from Fas/caspase-8–induced apoptosis. In the mitochondrial pathway, c-IAP1 and c-IAP2 bind directly to the primary caspase, pro-caspase-9, and prevent its processing and activation induced by cytochrome c (46). In our model of bronchial epithelial cells exposed to oxidant stress, we demonstrate that the caspases are not activated in stress-induced cell death. Thus, if c-IAP1 is involved in this system, its mechanism of action would be different from that described for other systems.
We also demonstrated that CD40 ligation induces phosphorylation of c-Jun, suggesting activation of AP-1, a transcription factor that also regulates expression of genes, such as Fas-L or BIM, the products of which are regulators of apoptosis. In a murine model of kainate-induced neuronal apoptosis, Behrens and colleagues showed that c-Jun N-terminal phosphorylation is required for the antiapoptotic function of c-Jun during hepatogenesis (47). In addition, using mouse embryo fibroblasts obtained from E11.5 murine embryos, it has been demonstrated that protection from apoptosis in response to UV irradiation requires Serine 63 and 73 phosphorylation of c-Jun (48). However, no studies have specifically investigated the role of c-Jun as a regulator of caspases-independent apoptosis. Thus, it would be of interest to investigate the effects of inhibitors of JNK on CD40-dependent suppression of oxidant-induced apoptosis in bronchial epithelial cells.
In our model, CD40 ligation was achieved by means of an agonist Ab. In preliminary studies, we had already demonstrated that 16HBE cells do not express CD40L, either under basal conditions or after oxidative stress (data not shown), and these data are supported by other evidence produced by Gormand and colleagues in 1999 (12). In this work, the authors demonstrated that immunostaining for CD40L was negative in bronchial epithelial cells. These data are apparently in contrast with what was demonstrated by Vignola and colleagues in 2001, even though in that case the CD40L positivity reported by the authors could have been related to infiltrating lymphocytes. In the bronchial airways, in fact, the main source of the natural ligand is represented by infiltrating activated lymphocytes.
CD40/CD40L interaction is of fundamental importance in a number of cellular processes that give rise to inflammatory responses. It is not yet clear the role that these two related molecules have in the pathogenesis of chronic inflammatory diseases. On the one side it would seem that CD40L expressed on T cells, but not CD40, is required for bronchial hyperreactivity in a murine model (49), suggesting a minor role for CD40 in asthma pathogenesis. On the other hand, Takahashi and colleagues (50) in 2003 demonstrated an increased susceptibility of CD40-deficient mice to airway responsiveness, suggesting a clear protective role of CD40 in this model. This contrasting ambivalent function of these two molecules could reflect differences in methodologies, but more likely seems to suggest that these two related molecules have different roles in the development of chronic inflammatory diseases.
In our model, CD40 ligation determined a protective effect on oxidative stress–induced cell death, via both inhibition of AIF and increased expression of c-IAP1. The inhibition of epithelial cell death could cause a persistent inflammatory response due to prolonged survival of epithelial cells. Apoptotic mechanisms play a key role after tissue injury by enabling disposal of a dying cell without induction of a proinflammatory response, as occurs after necrosis. One of these disposal mechanisms involves the recognition of phosphatidylserine on the outer leaflet of the plasma membrane by specific receptors on macrophages. In this regard, the role of CD40L-positive infiltrating lymphocytes would be to amplify the inflammatory cascade. Blocking CD40-mediated interactions could therefore contribute to reduce the inflammatory loop.
In summary, we have shown that CD40 ligation protects bronchial epithelial cells from oxidant-induced cell death, thereby amplifying inflammatory responses, and have identified an interaction between the CD40 signal transduction pathway and caspase-independent apoptosis. Knowledge of the key intracellular signal activated by CD40 ligation to regulate this process is of great importance, as it may eventually allow us to find new therapeutic approaches in diseases, such as asthma and chronic obstructive pulmonary disease, where these mechanisms are known to be altered.
The authors dedicate this work to the memory of Professor Antonio Maurizio Vignola.
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