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B activation in pancreatic and tracheal cystic fibrosis cells
,1
* INSERM, U770, Le Kremlin-Bicêtre, France; Université Paris-Sud, Faculté de Médecine, Hôpital de Bicêtre, le Kremlin-Bicêtre, France;
Université Louis Pasteur, Faculté de Médecine, Institut d'Hématologie et d'Immunologie, Strasbourg, France;
INSERM, U680, Paris, France; and Université Pierre et Marie Curie, Site Saint-Antoine, Paris, France
1Correspondence: CNRS UMR 6214-INSERM U771, Faculté de Médecine, rue Haute de Reculée, 49045 Angers, France. E-mail: carmen.martinez{at}univ-angers.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508 mutation (CFPAC-1 and CFT-2, respectively) were assessed. An increased susceptibility to apoptosis was observed in CFPAC-1 and CFT-2 cells. Apoptosis was reduced by treatment with a pan-caspase inhibitor and by incubation at 27°C, allowing recruitment of CFTRF508 at the plasma membrane. Inhibition of CFTR function in wild-type cells induced an increase of apoptosis. Apoptosis in CFPAC-1, but not in CFT-2 cells, was associated with overexpression of the proinflammatory mediators interleukin-6 and interleukin-8. In CF cells, apoptosis was linked to NF-
B pathway activation. Conditioned medium from actinomycin D-treated CFPAC-1 cells produced an increase in apoptosis of wild-type cells, suggesting that proinflammatory mediators secreted by mutant cells promote apoptosis. This was confirmed through the induction of apoptosis in wild-type cells by exogenous interleukin-6 and interleukin-8. These results suggest that CFTRF508 mutation, apoptosis, and activation of the NF-B pathway contribute to the self-perpetuating inflammatory cycle, at least in pancreatic cells, and provide evidence that excessive apoptosis may account for the exaggerated proinflammatory response observed in CF patients.—Rottner, R., Kunzelmann, C., Mergey, M., Freyssinet, J-M., Martínez, M. C. Exaggerated apoptosis and NF-B activation in pancreatic and tracheal cystic fibrosis cells.
Key Words: CFTR • phosphatidylserine • inflammation • cytokines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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that functions as part of a cyclic AMP-regulated chloride channel in epithelial cells of various organs such as lung, pancreas, liver, gastrointestinal tract, and sweat glands (2)
. Mutations of this gene result in the most common autosomal recessive and lethal disease in the Caucasian population (2)
: cystic fibrosis (CF). The largely prevalent mutation [i.e., the deletion of residue Phe-508 (F508)] results in a partially functional trafficking mutant that is capable of conducting chloride but is prematurely degraded from the endoplasmic reticulum (ER) (3
, 4)
.
CF is the most common hereditary disease involving the exocrine pancreas. More than 80% of patients with CF have pancreatic insufficiency at the time of diagnosis, and even more have partial impairment in enzyme secretion. Pancreatic secretions have a reduced water content and are viscous (5)
. Thus, in CF patients the obstruction of pancreatic ducts by hyperconcentrated secretions results in destruction of the pancreas epithelia, which could promote inflammation (6)
. Even though chronic pulmonary disease is the major cause of mortality in CF, gastrointestinal manifestations also lead to significant morbidity, such as recurrent abdominal pain and recurrent acute pancreatitis, which can turn into chronic pancreatitis (5)
. Airway obstruction, chronic infection, and inflammation are hallmarks of CF lung disease (7)
. The origin of airway inflammation in CF has been a matter of debate following the findings of neutrophil-dominated inflammation in the absence of bacterial or viral pathogens in bronchial alveolar lavage liquid in CF infants (8
9
10)
. In these studies, airway fluids of patients with CF showed increased levels of the proinflammatory cytokines IL-6 and IL-8 but decreased levels of anti-inflammatory IL-10 (11)
. One possible explanation is that inflammation precedes infection by some direct contribution of the defective CFTR (12)
. Furthermore, epithelial cells are involved in cytokine release and play a major role in local inflammation, which leads to further speculation that defective CFTR function may be directly related to excessive inflammation.
Expression of many genes encoding proinflammatory cytokines requires activation of NF-B (13)
. This transcription factor is complexed in the cell cytoplasm with IB-
and IB-ß, which are selectively phophorylated, ubiquitinated, and degraded in the proteasome in response to stimuli. The lack of functional CFTR, the abnormal trafficking of CFTRF508, and its accumulation in the ER trigger intracellular stress, resulting in NF-B activation (14)
. Furthermore, NF-B can be activated by proinflammatory cytokines (15)
, suggesting a double-sense interaction between the former and the latter.
Contradictory data on the sensitivity of CF cells to apoptosis have been reported. Whereas intestinal epithelial CF cells show a higher fragmentation of DNA (4)
, suggesting elevated susceptibility to programmed cell death, respiratory epithelial CF cells undergo delayed Pseudomonas aeruginosa-induced apoptosis (16)
. Independent of the susceptibility to apoptosis of CF cells, it has been shown in CF that clearance of apoptotic cells is defective and that accumulation of such cells could contribute to ongoing inflammation (17)
.
In the present study, we investigated whether CFTR mutations in pancreatic and tracheal cells could lead to activation of apoptosis, which favors inflammation. We have used two pancreatic and tracheal cell lines expressing the wild-type CFTR (PANC-1 and NT-1, respectively) or CFTRF508 protein (CFPAC-1 and CFT-2, respectively). The results reveal that cells presenting the CFTR mutation are more sensitive to apoptosis than those expressing wild-type CFTR. Furthermore, recruitment of CFTRF508 at the plasma membrane (18)
is associated with a decrease in apoptosis. In addition, apoptosis is dependent on caspases. However, in pancreatic cells apoptosis is accompanied by an increase in the expression of proinflammatory cytokines due to NF-B activation; in tracheal cells, activation of NF-B was not associated with cytokine production.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B- was from U.S. Biological (Swampscott, MA, USA). Mouse anti-CFTR IgM was from Affinity BioReagents (Golden, CO, USA). RayBioHuman cytokine antibody array, the enzyme-linked immunosorbent assays (ELISA) for IL-6, IL-8, and MCP-1, were obtained from Bender Medsystems (Burlingame, CA, USA), ELISA kit for TIMP-2 was from RayBioTech, Inc. (Norcross, GA, USA). Inhibitor of IB- phosphorylation (Bay 11–7082) was purchased from BioMol Research Labs, Inc. (Exeter, UK). Annexin V-biotin was the same as that used before in our laboratory (19)
. The In Situ Cell Death detection kit was from Roche Diagnostics (Meylan, France).
Cell culture and induction of apoptosis
The pancreatic cancer cell line PANC-1 that expresses endogenous CFTR and CFPAC-1, showing the CFTRF508 mutation, was purchased from the American Type Culture Collection (Rockville, MD, USA). PANC-1 is a human epithelioid pancreas carcinoma cell line and was grown in DMEM. CFPAC-1 is a human pancreatic adenocarcinoma cell line and was grown in IMDM. The tracheal cell line NT-1, derived from non-CF human fetus, and the CFT-2 cell line, homozygous for the F508 mutation (20)
, were grown in DMEM/F12. All media were supplemented with 10% heat-inactivated FCS, 100 µg/ml streptomycin, and 100 U/ml penicillin. CFPAC-1, NT-1, and CFT-2 cells were incubated in humidified 5% CO2 atmosphere at 37°C. PANC-1 cells were cultured at 37°C in a humidified atmosphere of 7.5% CO2. Cell viability was checked by Trypan blue exclusion. Cells were seeded at 7.5 x 104 cells in T75 flasks. All experiments were carried out when the cells were 80–90% confluent. They were incubated in the presence or absence of actinomycin D (0.5 µg/ml), St (0.33 nM), z-vad.fmk (50 µM), Bay 11–7082 (7.5 µM) (21
, 22)
, or CFTR-Inh 172 (10 µM) for 24 h. All agents were used at concentrations at which no cytotoxicity was observed, as deduced from Trypan blue exclusion.
Correction of CFTR defect in CFPAC-1 and CFT-2 cell lines
All cell lines were grown as previously described up to 80–90% confluence at 27°C prior exposure to Act D or St for 24 h.
Determination of hypodiploid DNA
After the various treatments, culture medium was removed from cells growing in monolayers; adherent cells were trypsinized, detached, combined with floating cells from the original culture medium, and centrifuged. Cells were then fixed in 70% ethanol for at least 4 h at 4°C and washed once in 1 mM HBSS Ca2+ before resuspension for 10 min in a solution containing type I-A RNase A (0.05 mg/ml) in HBSS containing 1 mM Ca2+ at 37°C. PI was then added at a final concentration of 0.1 mg/ml. After 15 min in the dark at room temperature, samples were analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA). Data acquisition (10,000 events in each case) and analysis were conducted using CELLQuest software (Becton Dickinson). The forward light scatter setting was E-01.
Determination of apoptosis and necrosis
Culture medium was removed from pancreatic cells growing in monolayers; adherent cells were trypsinized, detached, combined with floating cells from the original culture medium, and centrifuged. Cells were placed in HBSS containing 1 mM Ca2+ and exposed for 10 min to annexin V-biotin (17.5 µg/ml) and PI (6.66 µg/ml) at room temperature. Cells were stained for 5 min with streptavidin-FITC (14 µg/ml) at room temperature in the dark. Samples were analyzed by flow cytometry as described above.
Cell death was detected in situ in cells by enzymatic labeling of DNA strand breaks using TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) according to the manufacturer's instructions.
Assay of multiple proinflammatory mediators by microarrays and ELISA
Cells were cultured for 24 h at 37°C or 27°C, then supernatants were harvested and prepared according to the manufacturer's instructions. Cells were cultured in the presence or absence of Act D (0.3 µg/ml), St (0.33 nM), and z-vad.fmk (50 µM) for 24 h at 37°C. ELISA for IL-6, IL-8, TIMP-2, and MCP-1 was performed according to the manufacturer's instructions. Data were expressed as ng/ml per 1 x 105 cells.
Evaluation of the response of pancreatic cell lines to cytokine stimulation
To determine whether mediators secreted by the cell lines are able to induce apoptosis, CFPAC-1 and PANC-1 cells were grown as described and culture media were removed. PANC-1 cells were treated with the conditioned medium of Act D-treated CFPAC-1 cells for 24 h. Culture medium was removed from the cells growing in monolayers; adherent cells were briefly trypsinized, detached, combined with floating cells from the original culture medium, and centrifuged. To determine the effect of endogenous proinflammatory mediators on apoptosis, PANC-1 cells were grown as described previously. At confluence, PANC-1 were treated or not with Act D (0.5 µg/ml) for 24 h in the absence or presence of a cocktail of cytokines (IL-8 12 µg/ml, IL-6 1.4 µg/ml, and TIMP-2, 600 ng/ml). Determination of hypodipoid DNA was performed as described above.
Cell extracts and Western blot analysis
After incubation with apoptosis-inducing agents for 12 h, cells were scrapped in the presence of 1 ml of Rippa buffer with 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 1 mM PMSF. Samples containing 20–30 µg protein (Bio-Rad protein assay kit) were separated on 10% SDS-PAGE. Separated proteins were then blotted onto Hybond-ECL nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK). Blots were probed with mAb to phosphorylated IB- and developed with the anti-mouse HRP-conjugated secondary Ab. Bound Ab were revealed by chemiluminescence (Pierce, Rockford, IL, USA); ß-actin staining was used as control. IB- phosphorylation levels were determined by densitometry analysis.
Immunofluorescence analysis of CFTR protein expression
PANC-1 and CFPAC-1 cells were analyzed for CFTR expression by immunohistochemical analysis. The cells were grown to 
80% confluence in 35 x 10 mm tissue culture dishes, rinsed briefly with PBS, and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. Samples were then rinsed in PBS; after fixation, nonspecific binding sites on PANC-1 and CFPAC-1 cells were blocked with PBS containing 10% bovine serum albumin (BSA) for 20 min at room temperature. The cells were then stained with the CFTR-specific antibody at 1 µg/ml in PBS-1.5% (w/v) BSA for 60 min at room temperature and rinsed in PBS. Samples were next stained with goat anti-mouse IgM-FITC-conjugated secondary antibody at 1 µg/ml diluted in PBS-1.5% BSA for 45 min in the dark at room temperature. Samples were then rinsed twice with PBS. Samples were analyzed by fluorescence laser scanning microscopy. Fluorescence intensity was recorded using Spot Advance software from Diagnostic Instruments, Inc. (Sterling Heights, MI, USA). To enable comparison, all images were recorded using the same parameters of laser power and photomultiplier sensitivity. Images shown are representative of at least three separate experiments for each condition and were processed using identical values for contrast and brightness.
Statistical analysis
The results of multiple experiments (n=number of experiments) are expressed as mean ± SE. Statistical analysis was carried out using Student's t test. Differences were considered statistically significant at a value of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A shows that basal apoptosis (e.g., in the absence of treatment) was not significantly different among normal cells or cells with CFTR dysfunction. However, at 24 h of incubation with Act D, a 7-fold increase of hypodiploid DNA was observed in CFPAC-1 cells; in control cells the increase was only 2-fold. In addition, St induced an increase of 2-fold and 3-fold of hypodiploid DNA in PANC-1 and in CFPAC-1 cells, respectively.
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To correlate the increase in hypodiploid DNA with an increase of apoptotic cells, double labeling with annexin V and PI was performed in nonpermeabilized cells. Figure 1B
shows that treatment of CFPAC-1 cells with Act D induced an increase (4.5-fold) in the percentage of annexin V+/PI– reflecting the apoptotic population, while the necrotic (annexin V+/PI+) population was not affected. When exposed to Act D, PANC-1 cells also underwent apoptosis, but to a much lesser extent and still without a significant change in the necrotic population. The apoptotic character of Act D- and St-treated cells was confirmed by TUNEL assay. TUNEL-positive staining was observed in Act D- (201±20) and St- (217±22 fluorescence arbitrary units) treated CFPAC-1 cells vs. untreated cells (84±17 fluorescence arbitrary units). No change in TUNEL labeling was observed in PANC-1 cells (77±8, 82±6, and 79±5 fluorescence arbitrary units for untreated, Act D-, and St-treated PANC-1 cells, respectively). These results suggest that the increase in the hypodiploid population is correlated with an increase in the apoptotic population and that CFPAC-1 cells are more sensitive to the apoptotic agents than PANC-1 cells.
To evaluate whether the difference in the degree of apoptosis in both types of cells was not due to a delayed PANC-1 response, hypodiploid DNA was measured in PANC-1 and CFPAC-1 cells after stimulation with Act D for 12 h, 24 h, and 48 h. Act D induced DNA fragmentation in a time-dependent manner in both cell lines, but at all time points CFPAC-1 cells were more sensitive to apoptosis than were PANC-1 cells (Fig. 2
A).
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Apoptosis is caspase dependent in CFPAC-1 cells
To investigate the implication of caspases in Act D-induced apoptosis, the effect of the nonselective caspase inhibitor, z-vad.fmk, was tested in CFPAC-1 cells. As shown in Fig. 2B
, this inhibitor significantly reduced the degree of apoptosis evoked by Act D in a concentration-dependent manner. At 50 µM, z-vad.fmk inhibited apoptosis induced by Act D by 60%; at 100 µM, the effect of Act D was abolished. These results clearly indicate that caspases play a critical role in apoptosis induced by Act D in CFPAC-1 cells.
Recruitment of CFTRF508 at the plasma membrane is associated with a reduction of apoptosis in CFPAC-1 cells
To further confirm that the increase of apoptosis in CFPAC-1 cells is related to the lack of CFTR at the plasma membrane, CFPAC-1 cells were incubated at 27°C. This condition allows the translocation of CFTR at the plasma membrane (18)
. At 37°C, CFTR immunostaining was detected in PANC-1 but not in CFPAC-1 cells (Fig. 3
A). By contrast, after incubation of CFPAC-1 cells at 27°C, CFTR immunostaining was observed at the membrane surface, suggesting that this treatment induces recruitment of CFTR at the plasma membrane. As shown in Fig. 3B
, incubation at 27°C resulted in the abolition of apoptosis in CFPAC-1 cells, and thus the levels of apoptotic cells were similar in the two cell lines. Abolition of apoptosis at 27°C was reversed in the presence of the specific inhibitor, CFTR-Inh 172.
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In addition, inhibition of CFTR function in PANC-1 cells with CFTR-Inh 172 (23)
resulted in an increase of apoptosis in PANC-1 cells (Fig. 3C
). This suggests that CFTR dysfunction plays a crucial role in apoptosis of mutant cells.
Cytokine production in response to apoptogenic agents
Because cytokines and others proinflammatory mediators promote inflammation in patients with CF, we also examined whether the apoptogenic agents Act D and St induced the expression of proinflammatory mediators. By using microarrays, we have identified the different mediators secreted by the two cells lines.
As shown in Fig. 4
A, B, D, CFPAC-1 cells secreted constitutive amounts of IL-8, TIMP-2, and IL-6 significantly higher (3-, 2.5-, and 10-fold for IL-8, TIMP-2, and IL-6, respectively) than PANC-1 cells. Treatment with proapoptotic agents resulted in increased levels of these proinflammatory mediators in CFPAC-1 cells. Furthermore, incubation of CFPAC-1 cells at 27°C decreased IL-8 and IL-6 production to levels similar to those in PANC-1 cells. As described (24)
, PANC-1, but not CFPAC-1 cell, secreted MCP-1 (Fig. 4C
).
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To establish whether apoptosis induction is involved in the generation of proinflammatory mediators, we measured IL-6 secretion in the presence of the caspase inhibitor, z-vad.fmk. After incubation, the increase of IL-6 production induced by Act D or St was abolished or considerably reduced, respectively (Fig. 4D
). These results strongly suggest that the caspase pathway plays a major role in the production of IL-6 in CF cells.
Activation of NF-B is associated with apoptosis in epithelial cells with CFTR dysfunction
Because NF-B activation can be presumed to precede cytokine production, we studied this pathway at 12 h. PANC-1 and CFPAC-1 cells were treated with Act D, as described previously, for 12 h and phosphorylation of IB- was monitored by Western blot. As shown in Fig. 5
, basal phosphorylation of IB- was higher in CFPAC-1 than in PANC-1 cells. In addition, a weak signal was detected in PANC-1 cells after incubation with Act D, whereas Act D treatment caused a substantial increase in phosphorylation of IB- in CFPAC-1 cells. These observations suggest that the NF-B pathway is indeed mobilized in CFPAC-1 cells under basal as well as apoptotic conditions.
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We investigated the effect of inhibition of IB- phosphorylation on apoptosis. As shown in Fig. 5C
, the presence of Bay 11–7082, an inhibitor of IB- phosphorylation, reduced apoptosis induced by Act D or St. These results implicate the NF-B pathway in the apoptotic process induced by Act D or St.
Effect of conditioned medium from CFPAC-1 cells on apoptosis in PANC-1 cells
The foregoing results suggest that the NF-B pathway is implicated in both the production of proinflammatory mediators and apoptosis. To assess whether proinflammatory mediators secreted by CFPAC-1 cells are involved in apoptosis, PANC-1 cells were exposed to conditioned medium from CFPAC-1 cells treated with apoptogenic agents. As shown in Fig. 6
A, conditioned medium obtained from Act D-treated CFPAC-1 cells induced apoptosis in PANC-1 cells, and this effect was abolished by the caspase inhibitor z-vad.fmk. The effect of conditioned medium from St-treated CFPAC-1 cells on PANC-1 cells apoptosis was weaker. In addition, we determined the role of exogenous proinflammatory mediators on apoptosis. For this, we treated PANC-1 cells with a cocktail of IL-8, IL-6, and TIMP-2 for 24 h in the absence or presence of Act D. As shown in Fig. 6B
, purified cytokines induced apoptosis to a degree comparable to that elicited by Act D, with a statistically significant additive effect when combining both treatments.
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Effect of apoptotic agents in normal and CFTRF508 tracheal cells
To determine whether the effects observed in pancreatic cell lines expressing CFTRF508 mutation are tissue specific, tracheal NT-1 (wild-type) and CFT-2 (CFTRF508 mutation) cells were used. After treatment with proapoptotic agents for 24 h, basal apoptosis (e.g., in the absence of treatment) was higher in NT-1 cells than in cells with CFTR dysfunction. By contrast, incubation with either Act D or St induced an increase of 3- and 4-fold of hypodiploid DNA in CFT-2 cells; in control cells the increase was only 1.3- and 1.8-fold, respectively (Fig. 7
A). These results were confirmed by TUNEL assay (not shown). Moreover, z-vad.fmk inhibited apoptosis induced by Act D in CFT-2 cells. These results suggest an increased sensitivity to apoptogenic agents in tracheal cells expressing CFTRF508 mutation, as observed in pancreatic CF cells.
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When tracheal cells were incubated at 27°C so as to allow CFTR translocation to the plasma membrane, the degree of apoptosis was similar to that at 37°C in wild-type cells, but an increase was abolished in CFT-2 cells (Fig. 7A
).
Regarding the effect of apoptotic agents in IL-8 and IL-6 production (Fig. 7B, C
), NT-1 and CFT-2 cells secreted lower basal levels of both cytokines than did pancreatic cells. Treatment with proapoptotic agents resulted in weak changes in the levels of these proinflammatory mediators in both cell types. Surprisingly,incubation at 27°C of NT-1 and CFT-2 cells induced an enhancement of basal cytokine production that was not affected by treatment with Act D or St. By contrast, apoptotic agents caused a significant increase in phosphorylation of IB- in CFT-2 (18% and 32% for Act D and St, P<0.05 and P<0.01, respectively), but not in NT-1 cells (10% and 14% for Act D and St, respectively).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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F508 at the plasma membrane is associated with a reduction of apoptosis. Furthermore, apoptosis in CF cells involved caspase activation and is associated with increased production of proinflammatory mediators through the NF-B pathway. In addition, these mediators are able to promote apoptosis by themselves. Tracheal cells presenting the CFTRF508 mutation were also more susceptible to caspase-dependent apoptosis than tracheal wild-type cells. As observed in pancreatic cells, apoptosis was directly related to the CFTR mutation and linked to activation of the NF-B cascade. By contrast, tracheal cells did not produce large amounts of cytokines. These results provide valuable evidence to help explain that an excess of apoptosis in pancreatic CF cells may account for the sustained proinflammatory response observed in CF patients.
Some studies have reported defective apoptosis of cells expressing mutant forms of CFTR (16
, 21)
. However, other authors have observed excessive apoptosis in CF cells (4
, 22)
. These contradictory observations might be related to the different cell types or apoptogenic agents used. In the present study we found that, under basal conditions, apoptosis is similar in pancreatic wild-type and CF cells but higher in tracheal wild-type than CF cells. By contrast, caspase-dependent apoptosis measured by DNA fragmentation (propidium iodide and TUNEL) and annexin V-positive cells was higher in CF cells after Act D or St treatment than in wild-type cells, suggesting that CF cells are more sensitive to apoptosis, as has been described for tracheal cells (25)
. The fact that recruitment of CFTR508 at the plasma membrane at 27°C (18)
abolished apoptosis in CF cells independent of their tissue origin and that inhibition of CFTR function by the selective inhibitor CFTR-Inh 172 (23)
induced apoptosis in wild-type cells or in CF cells after incubation at 27°C indicates that programmed cell death is directly related to CFTR mutation.
It has been shown that CF cells may exhibit an exaggerated inflammatory response even in the absence of bacterial infection, suggesting a constitutive dysfunction in the regulation of cytokine production (9)
. Several studies have noted elevated concentrations of proinflammatory mediators such as IL-6 and IL-8 in CF cells (for a review, see ref. 26
). In the present investigation, basal levels of proinflammatory mediators are higher in CFTRF508 pancreatic cells than in wild-type cells, except for MCP-1, confirming earlier reports (24)
. In tracheal cells, by contrast, basal levels of IL-6 and IL-8 were lower, as was shown in other tracheal cells by Vilela et al. (27)
. Among the proinflammatory mediators detected here, IL-6 and IL-8 play a respective role in the resolution of acute and chronic inflammatory processes and participate in the chemotaxis of neutrophils to the site of inflammation. TIMP-2, a specific tissue inhibitor of matrix metalloproteinases, has also been found to be elevated in CF patients (28)
. In conjunction with a similar basal degree of apoptosis and an enhanced basal level of phosphorylation of IB-, these results indicate that mutant cells present an exaggerated intrinsic activation of the NF-B pathway, as reported by others in airway epithelial CF cells (14)
, and support the hypothesis that CF cells display an exacerbated inflammatory profile. In addition, we show that in the presence of Act D or St, proinflammatory mediators secreted by pancreatic CF cells have a tendency to increase concomitant with the enhancement of phosphorylation of IB- and that susceptibility to apoptosis decreases in the presence of the NF-B pathway inhibitor, indicating a link between apoptosis, CFTR mutation, and inflammatory response. In addition, secretion of proinflammatory mediators by pancreatic CF cells was abolished after incubation at 27°C, allowing recruitment of CFTRF508 at the plasma membrane. In the presence of a nonspecific caspase inhibitor, proinflammatory IL-6 production was also abolished or reduced in either Act D- or St-treated cells, respectively. Regarding cytokine production in tracheal cells, we have observed that proapoptotic agents did not modify proinflammatory production at either 37°C or 27°C, whereas phosphorylation of IB- was increased. One possible explanation is that basal production of IL-6 and IL-8 in tracheal cells is not related to apoptosis but to CFTR mutation and the NF-B pathway, as described previously (14)
. Additional studies are needed to establish whether other proinflammatory mediators are secreted by tracheal CF cells. These results suggest a double sense regulation between apoptosis and the NF-B pathway.
Finally, we have assessed whether the mediators secreted by CFTRF508 cells could affect apoptosis in pancreatic wild-type cells. Thus, the effect of generated conditioned medium from apoptotic mutant cells was examined with respect to the apoptotic response in the latter. Under these conditions, PANC-1 cells underwent apoptosis when treated with the conditioned medium obtained from apoptotic CFTRF508 cells. The conditioned medium generated in the presence of the caspase inhibitor did not induce apoptosis. The results suggest that paracrine regulation via the mediators secreted by CF cells may account for their apoptotic response. This was confirmed by the fact that proinflammatory mediators induced apoptosis in wild-type cells. Taken together, we can propose that apoptosis of pancreatic cells induces release of proinflammatory mediators, which in turn are able to evoke apoptosis. Thus, all these processes would contribute to the self-perpetuating inflammatory cycle.
In conclusion, the present study highlights the role of the CFTRF508 mutation in the apoptotic process and initiation of the proinflammatory response in pancreatic CF cells. Although in tracheal cells the link between CFTRF508 mutation, apoptosis, and the NF-B pathway activation is clearly demonstrated, the nature of the proinflammatory response remains to be further elucidated. Hence, it appears reasonable to assume that the accumulation of senescent cells in CF patients would not only be related to a defect of elimination of apoptotic cells, as described by Vandivier et al. (17)
, but could also result from an excess of apoptosis, which could worsen the clinical situation through the development of inflammation, possibly depending on the nature of the tissue.
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ACKNOWLEDGMENTS |
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Received for publication October 27, 2006. Accepted for publication March 26, 2007.
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P < 0.01, significantly different from their respective controls in the absence of zvad.fmk.
