CD40 Ligation Protects Bronchial Epithelium against
Oxidant-Induced Caspase-Independent Cell Death
Anna M. Merendino, Fabio Bucchieri, Rosalia Gagliardo, Arezoo Daryadel, Flora Pompeo,Giuseppina Chiappara, Roberta Santagata, Vincenzo Bellia, Sabrina David, Felicia Farina, Donna E. Davies, Hans-Uwe Simon, and Antonio M. Vignola†
Department of Medicine, Pneumology, Physiology, and Human Nutrition; Department of Experimental Medicine, Section of Human Anatomy, Universita` di Palermo, Palermo; Institute of Biomedicine and Molecular Immunology, Italian National Research Council, Palermo, Italy; Division of Infection, Inflammation, and Repair, University of Southampton, United Kingdom; and Department of Pharmacology, University of Bern, Bern, Switzerland
CD40 and its ligand regulate pleiotropic biological responses, in-cluding 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 14oⴚ cells ex-posed to oxidant stress, we found that ligation of CD40 (induced by G28-5 monoclonal antibodies) enhanced cell survival and in-creased the number of cells in G2/M (interphase between DNA synthesis and mitosis) of the cell cycle. This was associated with NF-B 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 implicat-ing CD40 ligation as a regulator of caspase-independent cell death. This was confirmed by the demonstration that CD40 ligation pre-vented 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.
Keywords: activator protein–1; apoptosis; CD40; NF-B; oxidant stress 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 inter-acting 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
(Received in original form November 25, 2005 and in final form February 28, 2006 ) †Deceased.
This work was supported by the University of Palermo, Italy, IBIM-CNR, Italy, and the Swiss National Science Foundation (grant 310000-107526).
Correspondence and requests for reprints should be addressed to Anna Maria Merendino, Ph.D., Dipartimento di Medicina, Pneumologia, Fisiologia e Nutrizi-one Umana, Universita’ di Palermo, Ospedale “V, Cervello”, Via Trabucco 180, 90146 Palermo, Italy. E-mail: annameren@yahoo.it
Am J Respir Cell Mol Biol Vol 35. pp 155–164, 2006
Originally Published in Press as DOI: 10.1165/rcmb.2005-0433OC on March 16, 2006 Internet address: www.atsjournals.org
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 microenvi-ronment, the interaction between CD40 and its ligand can modu-late several responses, including cell proliferation, differentia-tion, 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 activa-tion 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 biologi-cal effects of TRAF signaling appear to be mediated through the activation of transcription factors of NF-B and activator protein (AP)-1 family (2, 9).
CD40 has also been implicated in the regulation of the func-tional activation of structural cells, such as fibroblasts and epithe-lial 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 acti-vation of bronchial epithelial cells and to the release of inflam-matory mediators (11, 12), which is related to NF-B activation (13). In these previous studies, the proinflammatory responses triggered by CD40 ligation were evaluated alone or in the pres-ence of proinflammatory cytokines. Recognizing that the epithe-lium will be exposed to oxidants derived not only from environ-mental 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-B and AP-1 and, most significantly, that it protects epithelial cells exposed to oxidant-mediated cell death by blocking caspase-independent apoptosis.
MATERIALS AND METHODS Cell Cultures, Reagents, and Antibodies
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-cm2flasks at a
density of 5⫻ 105cells/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 indi-cated times, in the absence or presence of 200–400M hydrogen perox-ide (H2O2), and in the absence or presence of the anti-CD40 monoclonal
antibody (mAb; G28-5,g/ml). Dose–response curves have been gener-ated 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). H2O230% was purchased from
J.T. Baker (Deventer, Holland).
Protein A/G Plus-Agarose, rabbit polyclonal Abs recognizing inhibi-tory B kinase (IKK) ␣, IKK, NF-B 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 second-ary 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 cas-pase-3 and anti-human caspase-8 Abs (1:1,000) were purchased from Becton-Dickinson Biosciences, Pharmingen (Basel, Switzerland). Ago-nistic anti-Fas receptor mAb CH11 (1 g/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--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 con-trol protein loading.
Purification of the Anti-CD40 mAb G28-5
G28-5 mAb was obtained from HB 9110 hybridoma cells purchased from American Type Culture Collection (Rockville, MD). The specific-ity and functional activspecific-ity 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 Biosci-ences, 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 100l/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 ali-quots at⫺20⬚C.
Caspase-3 Enzymatic Assays
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
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 -glycero-phosphate, 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 centri-fuged at 10,000⫻ g for 5 min. The protein content of the supernatants was analyzed using a bicinchonic acid assay (Pierce); 25–30g of lysate was then denatured under reducing conditions by boiling for 3 min in 50 mM Tris-HCl (pH 6.8), 1% SDS, 2%-mercaptoethanol, and 0.01% bromophenol blue. Proteins were separated by SDS-PAGE and trans-ferred 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 appro-priate horseradish peroxidase–conjugated secondary Ab; bound Ab was detected using the ECL chemiluminescence detection system (Amersham-Pharmacia, Biotech), according to the manufacturer’s in-structions. Membranes were stripped and reprobed with housekeeping proteins-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 Plot-ting 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-actin.
Immunoprecipitation and Immunoblotting
Cells were exposed as indicated in the Results section and then har-vested into lysis buffer, as described above (19, 20); 500g of each lysate was incubated with 2.5g of specific Ab (anti-IKK␣ or anti-IKK, or anti-c-Jun N-terminal kinase-1 [JNK1]) for 2 h and then immunoprecipi-tated by incubation overnight at 4⬚C with 20 l 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). Sam-ples 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.
Cell Cycle Analysis
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 permeabiliza-tion and staining the nuclei with propidium iodide (PI); data were analyzed in the form of a DNA histogram.
Determination of Apoptosis by Cell Morphology
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 (10g/ml), and the number of cells with apoptotic nuclei was assessed as a percentage of the total cell number.
Determination of Cell Death and Apoptosis by AnnexinV
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 (1g/ml) and PI (2.5g/ml) were added to the tubes with 1 ⫻ 105cells/100l 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 detec-tor (FL) 1 (AxV-FITC) versus FL2 (PI) was performed using WinMDI 2.8 (Flow Cytometry software, University of Massachusetts). The de-gree of apoptosis was expressed as the number of AxV⫹/PI⫺cells shown as a percentage of total cells.
Quantification of Nuclear Translocation of AIF in 16HBE 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 systemati-cally 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 translo-cated compared with the total number of nuclei in each slide.
Statistical Analysis
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.
RESULTS
Ligation of CD40 Activates of NF-B and c-Jun in 16HBE Exposed to Oxidant Stress
To examine whether ligation of CD40 and/or exposure to oxi-dants affected NF-B levels, we analyzed NF-B 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-B levels, not only under basal conditions, but also in association with oxidant stress.
Both CD40 ligation and oxidant stress are known to activate NF-B in epithelial cells (13, 22). This involves a cascade in which phosphorylation of the IKK␣ and IKK serine/threonine kinases leads to phosphorylation of IB, causing its ubiquitina-tion and degradaubiquitina-tion by the 26S proteasome, allowing NF-B nuclear translocation. In bronchial epithelial cells, CD40 ligation caused a marked increase in the amount of phosphorylated IKK␣ in comparison with untreated cells or those treated with H2O2
䉴
Figure 1. CD40 ligation activates NF-B pathway in 16HBE exposed to
H2O2.16HBE cells were treated with (solid bars) or without (open bars)
G28-5 (10g/ml) for 24 h in the presence or absence of H2O2at 200,
300, and 400M. (A) Expression of p65 protein. Signals corresponding to p65 on the various Western blots were semiquantified by densitomet-ric 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-actin. (B) Expression of p-IKK␣/IKK␣: 500g of total lysate was immunoprecipitated with IKK␣ and analyzed via immunoblotting using antiphosphorylated serine/threonine mixed mouse Abs, as described in MATERIALS ANDMETHODS. Open bars,⫺G28-5; closed bars, G28-5. (C ) Expression of p-IKK/IKK: 500 g of total lysate was immunoprecipitated with IKK and analyzed as described in MATERIALS ANDMETHODS. Signals corresponding to p-IKK␣ and IKK␣ and p-IKK and IKK were semiquantified by densitometric scanning, nor-malized, and expressed as the ratio of the band intensity of the phos-phorylated 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.
alone (Figure 1B). The amount of phosphorylated IKK was also significantly increased after CD40 ligation alone and when associated with H2O2treatment at 300 and 400M (Figure 1C). These data are consistent with those of previous reports (13) showing that CD40 ligation promotes NF-B activation.
Figure 2. CD40 ligation activates
activa-tor protein (AP)-1 pathway in 16HBE ex-posed to H2O2. (A ) Expression of p-JNK1/
JNK1 on 16HBE treated with or without G28-5 (10g/ml) for 24 h in the presence or absence of H2O2 at 200, 300, and
400M; 500 g of total lysate was immu-noprecipitated with JNK1 rabbit poly-clonal Ab and analyzed via immunoblot-ting using antiphosphorylated serine/ threonine mixed mouse Abs, as described in MATERIALS ANDMETHODS. Signals corre-sponding to p-JNK1 and JNK1 on the vari-ous Western blots were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the band inten-sity of the phosphorylated protein versus the band intensity of the total protein (NIH Image/Gel Plotting analysis pro-gram). (B and C) Expression of phosphory-lated c-Jun on Ser 63 and Ser 73, respec-tively. 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-actin. Results are representative of three independent experiments. * Significant difference be-tween Ab⫺and Ab⫹groups. Open bars, ⫺G28-5; closed bars, G28-5.
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 concen-trations 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 H2O2failed to affect c-Jun phosphorylation on Ser 73 and suppressed the effect of CD40 ligation.
The Effect of CD40 Ligation on Epithelial Cell Survival In keratinocytes, CD40 signaling alters the cell cycle by decreas-ing the number of cells in the G1 (interval between the com-pletion 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, H2O2was 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 H2O2together 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
Figure 3. Flow cytometry analysis of cell cycle. 16HBE cells were treated
for 16 h with or without H2O2and G28-5 (10g/ml). DNA content
was quantified by PI staining and flow cytometry analysis, as described in MATERIALS ANDMETHODS. 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 (10g/ml) plus H2O2with H2O2alone.
H2O2alone, and a concomitant increase in the number of cells in G2/M phase (P⬍ 0.03 for all; n ⫽ 3) (Figure 3).
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 200M H2O2in 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 g/ml) was able to suppress the number of late apoptotic cells caused by H2O2treatment, with a consequent increase in viability when
compared with H2O2alone (45.7⫾ 2.2 versus 65.7 ⫾ 8.9; P ⬍ 0.05) (Figure 4A).
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 300M H2O2caused 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 10g/ml G28-5, the number of late apoptotic/necrotic cells observed after 24 h of treatment with 200M H2O2was reduced in comparison with H2O2alone (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 300M H2O2.
CD40 Induces c-IAP1
To investigate the mechanism by which CD40 ligation may pro-tect 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 400M) 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).
CD40 Ligation Suppresses Caspase-Independent Apoptosis 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 mea-sured caspase-3 and -8 activity by Western blot analysis or enzy-matic assay of epithelial cell lysates after H2O2exposure (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-H2O2-mediated cell death in 16HBE cells is not dependent on caspase- and calpain-medi-ated pathways, even though it is suppressed by CD40 ligation. CD40 Ligation Suppresses Mitochondrial Release of AIF 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 micros-copy with Abs to AIF. 16HBE were treated for 24 h with 200M H2O2plus or minus 10g/ml of G28-5. Figure 7 shows that, in basal conditions, these cells express AIF in their mito-chondria (Figure 7B); H2O2treatment 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
Figure 4. Cell death evaluation. (A )
Mor-phologic evaluation of nuclei stained with Hoechst dye. 16HBE cells (n ⫽ 3) were treated for 16 h with or without H2O2and
G28-5 (10 g/ml). Cells were stained in viable conditions with Hoechst 33342 (10g/ml), and the number of cells with apoptotic nuclei was assessed as a per-centage 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 sta-tistical 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 H2O2and
G28-5 (10g/ml) and then stained with PE-labeled AxV and PI, as described in MATERIALS ANDMETHODS. Viable (AxV⫺/PI⫺), early apoptotic (AxV⫹/PI⫺), and late apo-ptotic/necrotic (AxV⫹/PI⫹) cells were measured and results displayed as histo-grams representing the relative cell per-centage. Open circles indicate statistical significance (P ⬍ 0.001) according to ANOVA comparing treatment with un-treated control. * Statistical significance (P⬍ 0.001) according to ANOVA com-paring association of G28-5 (10g/ml) plus H2O2with H2O2alone. Black bars,
un-treated; dark gray bars, G28-5 (10g/ml);
medium dark gray bars, H2O2, (200M);
medium light gray bars, H2O2, (200M) ⫹
G28-5; light gray bars, H2O2(300M); white
bars, H2O2(300M) ⫹ G28-5.
of AIF in 16HBE cells under the described conditions are re-ported in Table 1.
DISCUSSION
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 oc-curring 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 epithe-lial 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 im-mune response by inducing an upregulation of costimulatory
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 (10g/ml) for 24 h in the presence or absence of H2O2at 200, 300, and 400M. 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-actin, according to NIH Image/Gel plot-ting 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.
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 H2O2and 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 H2O2and CD40 ligation was accompanied by a parallel decrease of G0/G1 and S cells. This cell cycle configura-tion is typical of cells that are undergoing active proliferaconfigura-tion. 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 reduc-tion 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 down-stream of the death signal: the protease pathway, involving cas-pases 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 neces-sarily 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 propa-gates to the mitochondria, triggering the release of AIF that shuttles from the mitochondria to the nucleus, inducing periph-eral 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 oxi-dant 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-B, as pre-viously reported (13), and AP-1, and that this was augmented in response to oxidant stress. Although the NF-B 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-B, 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-B activation. In our experi-ments, the CD40 ligation leads to NFB activation and induction
Figure 6. Apoptosis induced by H2O2treatment is caspase- and
calpain-independent. (A ) Effect of H2O2and 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, 0.5M) exposure of 16HBE cells for 5 h induced the enzymatically active 17-kD and 18-kD fragment of caspase-3 and caspase-8, respec-tively. 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 (10g/ml) and H2O2(200M) for 16 h. As positive control, enzymatic
activity of recombinant caspase-3 and neutrophils undergoing apoptosis by Fas receptor activation (CH11 1g/ml) were measured. Caspase-3–like activity was measured in 10 l of supernatants as enzymatic conversion of the colorimetric substrate Ac-DEVD-pNA at 405 nm, as described in MATERIALS ANDMETHODS. (C ) Pharmacological inhibition of caspase-3 (20M z-VAD,) and calpain (10 M 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 (10g/ml) plus H2O2with H2O2alone.
of at least one of the NF-B antiapoptotic target genes, c-IAP1. Recent studies have shown that IAPs directly inhibit some cas-pases, 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 phosphory-lation 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 hepato-genesis (47). In addition, using mouse embryo fibroblasts ob-tained 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). How-ever, 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 demon-strated that immunostaining for CD40L was negative in bron-chial 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 bron-chial 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 col-leagues (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 ambiva-lent 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 epi-thelial cell death could cause a persistent inflammatory response due to prolonged survival of epithelial cells. Apoptotic mecha-nisms 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 in-volves the recognition of phosphatidylserine on the outer leaflet of the plasma membrane by specific receptors on macrophages.
Figure 7. Immunofluorescent analysis of AIF
ex-pression and localization. 16HBE cells were treated for 24 h with 200 and 300M H2O2plus
or minus G28-5 (10g/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 M H2O2, (d ) 200 M H2O2 ⫹ G28-5
(10 g/ml), and (e) higher magnification of (c ). Scale bar⫽ 50 m. White arrows show trans-location of AIF into the nuclei.
In this regard, the role of CD40L-positive infiltrating lympho-cytes would be to amplify the inflammatory cascade. Blocking CD40-mediated interactions could therefore contribute to re-duce the inflammatory loop.
In summary, we have shown that CD40 ligation protects bron-chial epithelial cells from oxidant-induced cell death, thereby amplifying inflammatory responses, and have identified an inter-action between the CD40 signal transduction pathway and cas-pase-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
TABLE 1. QUANTIFICATION OF PERCENTAGE OF APOPTOSIS-INDUCING FACTOR–POSITIVE NUCLEI IN 16HBE CELLS
Treatments AIF nuclear positivity %
Untreated control 0.5⫾ 0.3
H2O2200M 17.1⫾ 4.7
H2O2200M ⫹ G28-5 3.4⫾ 2.3
H2O2300M 22.9⫾ 6.4
H2O2300M ⫹ G28-5 5.3⫾ 3.2
Definition of abbreviation: AIF, apoptosis-inducing factor.
therapeutic approaches in diseases, such as asthma and chronic obstructive pulmonary disease, where these mechanisms are known to be altered.
Conflict of Interest Statement : None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Acknowledgments : The authors dedicate this work to the memory of Professor Antonio Maurizio Vignola.
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