Materials and methods

Cell Culture

The human insulinoma cell line CM was obtained from Professor P Pozzilli of St Bartholomew's Hospital, London, UK. The cells were grown in Iscove's modified Dulbecco's medium/RPMI 1640 (4:1) supplemented with 10% fetal bovine serum, 5 mmol/l L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were maintained at 37°C in 5% CO₂.

Assessment of TRAIL-Induced Apoptosis

Sensitivity of CM cells to TRAIL-mediated apoptosis was determined using 100 ng/ml of human recombinant Killer^TM TRAIL (Alexis, San Diego, CA, USA) for 6 h. To test for cytokine-enhanced sensitivity of CM cells towards TRAIL-mediated apoptosis, cells were pretreated with 1 ng/ml TGF (Roche) for 24 h followed by 500 U/ml IFN (Roche) for 18 h. Apoptotic death rates were determined by annexin V binding using an FITC-labeled mouse anti-human annexin V monoclonal antibody (mAb) (IgG1 isotype; PharMingen, San Diego, CA, USA) and 2 l propidium iodide (50 mg/ml). After gating for propidium iodide negativity to exclude dead cells, 10⁵ events were examined for each determination. Flow cytometry was performed on a FACScalibur with CellQuest software (Becton Dickinson, Heidelberg, Germany).

Flow Cytometry

Surface expression of TRAIL-R1, -R2 and -R4 on CM cells was determined by flow cytometric analysis. A total of 10⁶ cells/sample were suspended in 50 l FACS medium (phosphate-buffered saline containing 2% fetal bovine serum, 0.01 mol/l HEPES and 200 ng/ml sodium azide) and incubated for 1 h in an equal volume of the appropriate dilution of the mouse mAbs to TRAIL-R1 (clone M272, IgG1 isotype), TRAIL-R2 (clone M412, IgG1 isotype) and TRAIL-R4 (clone M440, IgG2a isotype) kindly provided by Amgen (Thousand Oaks, CA, USA). The mouse anti-human CD3 (clone Leu4, IgG1 isotype) (DAKO, Copenhagen, Denmark) was used as an irrelevant isotype-matched negative control. Cells were washed twice and incubated with 2 ng of FITC-labeled F(ab')₂ goat anti-mouse immunoglobulins (DAKO) for 30 min on ice. Finally, cells were washed twice and resuspended in 500 l cold FACS medium containing 1 g/ml propidium iodide (Sigma). Analysis was restricted to propidium-negative (viable) cells and was performed on a FACSCalibur cytometer with CellQuestPro software.

Pancreatic Tissues

In all, 18 patients, whose informed consent was obtained prior to surgery, were chosen for this study. A total of 14 patients underwent partial pancreatectomy for CP: 10 were suffering from alcoholic pancreatitis, two had chronic nonalcoholic pancreatitis associated with pancreas divisum, one developed a cyst after initial surgery and one had cryptogenic CP. Sections were subjected to morphometric assessment for the grading of fibrosis (see below). Four patients underwent partial pancreatectomy for pancreas divisum. The specimens of these patients were histopathologically diagnosed as NP and included in the study as normal controls. All pancreatectomy specimens were immediately transferred to the laboratory where representative tissue samples were snap frozen in liquid nitrogen and kept there until further processing. Other parts of the tissues were stored at -80°C, for later use in frozen section immunohistochemistry and laser capture microdissection (LCM).

Grading of Fibrosis

NP and CP cases for this study were selected and graded independently by two pathologists (UKB and CH) on hematoxylin and eosin (H&E)-stained cryosections based on the relative proportions of parenchyma and stromal fibrosis. There was generally good agreement in NP and CP cases with severe fibrosis. However, there was only partial agreement in grading of CP with mild and moderate fibrosis. Total agreement was achieved, however, on reviewing these cases together on a multiheaded discussion microscope. Further, stromal tissue in NP and the proportion of fibrosis in different cases of CP was accurately quantitated on H&E-stained cryosections using a light microscope with a video camera (Sony 3CCD) attached to an image capture card in a standard desktop computer with image analysis software (ImageTool, San Antonio, TX, USA) to calculate the volume fraction of fibrosis. Per slide 10 different areas were assessed and the extent of fibrosis was then calculated as percentage of fibrosis in mild, 12.41.8%, moderate, 41.20.6%%, severe fibrosis, 73.96%, and that of normal stromal tissue in NP, as 6.31.9%.

Tissue Processing for LCM

Frozen tissue specimens were cut at 8 m thickness and serial sections transferred to PALM^® MembraneSlides (PALM, Bernried, Germany). Four to 10 tissue sections were required to obtain sufficient number of cells from various cellular compartment of NP and CP with varying grades of fibrosis. Sections were stained with H&E as follows: slides were immersed in 70% ethanol for about 10 min followed by sequential dips in sterile deionized water, Mayer's hematoxylin solution, sterile deionized water, diluted alcoholic eosin, 95% ethanol and finally dehydrated with three changes of absolute ethanol for 3 min each. Slides were air-dried for 5 min before proceeding for LCM.

LCM

PALM^® Laser Microbeam Microdissection System with Laser Pressure Catapulting and Robocut Software (PALM, Germany) was used for microdissection. Approximately 2000 cells from each epithelial compartments of NP and CP with various grades of fibrosis were microdissected and catapulted into LCM caps with their inner surface coated with a thin layer of inert mineral oil.

RNA Isolation from Microdissected Tissue

The material from two to three caps was pooled in a single purification column. Total RNA was isolated using Picopure^TM RNA isolation Kit (Arcturus, Mountain View, CA, USA) according to the manufacturer's instructions. Total RNA was qualitatively and quantitatively assessed on RNA LabChip (Agilent, Wilmington, DE, USA) following the manufacturer's instructions. Only those samples with clearly visible ribosomal peaks and OD_280/260 ratios between 1.6 and 1.8 were further processed.

RNA Isolation from CM Cells

Human insulinoma cell line CM was used for stimulation experiments. CM (2 10⁵) were stimulated with TRAIL, IFN and TRAIL+IFN in ideal conditions for 48 h. Exactly the same number of cells kept under identical culture conditions without addition of any substance was used as controls. Total cellular RNA was isolated using the RNeasy RNA isolation system (Qiagen, Valencia, CA, USA) following the manufacturer's recommendations. RNA was assessed by agarose gel electrophoresis and quantitated by UV absorbance.

RNA Amplification and Semiquantitative Real-Time PCR

The RiboAmp^TM RNA Amlification Kit was used for amplifying the total RNA from microdissected tissues. The manufacturer's protocol for performing one round of amplification was used. The amount of RNA obtained after amplification was qualitatively and quantitatively assessed using the RNA LabChip (Agilent). RNA (2.5 g) was reversely transcribed into cDNA using Superscript Reverse Transcriptase (Invitrogen), random hexamer primer and addition of an RNase inhibitor following the manufacturer's protocol for expression analysis of various NFB subunits in microdissected tissues. Primers were designed using Primer Express Software (Perkin-Elmer, Foster City, CA, USA) to meet the following criteria: nonoverlapping primers as close as possible to each other, avoidance of sequences with propensity for dimer formation, low G–C content, avoiding run of nucleotides and a T_m at around 60°C. The sequences of the primer pairs are shown in Table 1. Primer pairs were purchased from MWG Biotech (Ebersberg, Germany). Cyclophilin expression was chosen as the endogenous control (forward primer: 5'-ATGGTCAACC CCACCGTGT-3'; reverse primer: 5'-TCTGCTGTCTTTGGGACCTTGTC-3'). First, the absence of nonspecific amplification was confirmed by analyzing the PCR amplification products by agarose gel electrophoresis. Amplicons generated from cDNA were also tested against no template control and RNA. In a further step, optimal primer concentrations were selected by mixing primer concentrations from 50 to 900 nmol of each forward and reverse primer. The curves were checked for low C_T, fast rising and for confirmation analyzed by agarose gel electrophoresis. Real-time PCR was performed using 4 l cDNA (12.5 ng/l), 4 l primer mix (forward/reverse) at optimal concentrations, 12 l sterile distilled water and 20 l SYBR Green PCR Master Mix (PE Biosystems, Foster City, CA, USA) per reaction. The following cycling conditions were set: 50°C for 2 min, denaturation at 95°C for 10 min, followed by 45 cycles at 95°C for 20 s each, 60°C for 20 s and 72°C for 20 s. Gene expression of NFB subunits of tissue affected by CP was measured relative to whole NP tissue as the calibrator sample. All quantitations were also normalized to cyclophilin as an endogenous control to account for variability in the initial concentration of total RNA. Analysis of quantitation was carried out by calculating as follows: 1. mean C_T value of three replicates per sample; 2. difference between mean C_T values of samples for each target (CP) and those of the endogenous controls (C_T); 3. difference between mean C_T values of the samples for each target (CP) and the mean C_T value of the corresponding calibrator (NP) (C_T). The quantitation is expressed as 2^-C_T to allow graphical presentation, and is shown as x-fold expression of the target gene in CP compared to NP set as 1.

Table 1 - Sequence and concentration of primers.

Full table

Immunohistochemistry

Immunohistochemistry was performed on four NP and 14 pancreatic tissues affected by CP with different extents of fibrosis. Serial, 2-m-thick cryosections were immediately fixed in ice-cold acetone for 10 min, air-dried and incubated for 1 h with the following mouse anti-human mAbs: anti-RelA (IgG1 isotype), IB/MAD-3 (IgG1 isotype), IKK/NEMO (IgG1 isotype) and cIAP1 (clone B75-1, anti-IgG1 isotype), all purchased from BD Biosciences (PharMingen, Belgium). For detection of survivin, a polyclonal rabbit anti-human antibody (R&D Systems, Germany) was used. Bound primary antibody was detected via goat anti-mouse and swine anti-rabbit immunoglobulins, respectively, conjugated to peroxidase-labeled dextran polymer in Tris-HCl buffer containing carrier protein (EnVision^TM, Dako). 3-Amino-9-ethyl-carbazole (Sigma, St Louis, MA, USA) was used as the substrate. Hematoxylin was used as a counterstain. The monoclonal mouse anti-human TTF1 (clone 8G7G3/1, anti-IgG1 isotype) purchased from Zymed Laboratories (South San Francisco, USA) and rabbit anti-human thyroglobulin obtained from DAKO (Copenhagen, Denmark) were used as irrelevant isotype-matched negative controls.

RelA Transcription Factor Assay

DNA-binding activity of RelA of the NFB transcription factor family in CM treated with 500 U/ml IFN (Roche) and 100 ng/ml of human recombinant Killer^TM TRAIL (Alexis, San Diego, CA, USA) separately and together for 48 h was determined using an ELISA-based assay (TransAM Kit^TM) obtained from Active Motif (Rixensart, Belgium). CM (2 10⁵/well) in a six-well plate were used and each stimulation was performed in triplicate. An equal number of cells kept under identical conditions without the addition of any substance were used as controls. In brief, the nuclear extracts were prepared following the manufacturer's instructions and 5 g of nuclear extract was added to microwells coated with a cold oligonucleotide containing the consensus-binding site for NFB. After 1 h incubation at room temperature, the microwells were washed thrice with washing solution. Antibody directed against phosphorylated RelA was used to label the RelA bound to the oligonucleotide and this was followed by an incubation with a secondary antibody conjugated to horseradish peroxidase. Finally, the results were quantified by a chromogenic reaction. Results were analyzed using the Revelation software (Dynatech) and are given in pg/ml as means with standard deviations.

Results

TRAIL-R Surface Profile of Stimulated CM Cells Corresponds to that of Islets in CP

Against the background that islet cells are TRAIL-R1^-, TRAIL-R2^- and TRAIL-R4⁺,⁴ we tested human insulinoma cell line CM for TRAIL receptor surface expression. CM cells were constitutively devoid of TRAIL-R1, -R2, -R3 (not shown) and -R4, which is a phenotype corresponding to that of islet cells of NP.⁴ The TRAIL-R profile of CM upon stimulation with TGF (24 h), IFN (18 h) and TRAIL (6 h) mimicked that of islets in CP,⁴ in being TRAIL-R1^-, TRAIL-R2^-, TRAIL-R3^- (not shown) and TRAIL-R4⁺ in a major subset of cells (Figure 1a).

Figure 1.

(a) FACS histograms showing surface expression of TRAIL-R1 (left panel), -R2 (middle panel), and -R4 (right panel) in black lines. M1 marks the subset of CM cells positive for TRAIL-Rs. Isotype-matched negative controls are shown as gray lines. (b) Semiquantitative real-time PCR for NFB subunits in CM cells stimulated for 48 h with TRAIL, IFN and TRAIL+IFN compared with untreated cells. (c) Semiquantitative real-time PCR for NFB targets IB and survivin in CM cells stimulated with cytokines for 48 h. IB mRNA levels increased with IFN, while a decrease was noted with TRAIL and TRAIL+IFN. Survivin mRNA increased considerably with TRAIL, IFN and TRAIL+IFN. (d) RelA transcriptional activity in CM activated by TRAIL, IFN and IFN+TRAIL determined by ELISA compared with untreated control. The bars indicating standard deviation are not always visible due to the minimal variations of values compared to the scale used.

Full figure and legend (143K)

TRAIL Modulates NFB Subunit Expression in CM

Next, CM cells were stimulated with TRAIL, IFN and TRAIL+IFN for 48 h to determine the mRNA expression of NFB subunits and NFB targets IB and survivin.

The combination of TRAIL and IFN brought about a four-fold rise in NFB1 mRNA levels. On the other hand, mRNAs of transcriptionally active NFB subunits RelA, RelB and c-Rel were between two- to 9.5-fold increased compared to untreated cells on TRAIL+IFN stimulation (Figure 1b). Additionally, mRNA of NFB target IB was downmodulated, while survivin message increased considerably (Figure 1c).

Thus, TRAIL-R4 acts as a signal transducer in modulating NFB subunits in CM. Moreover, CM did not show sensitivity to TRAIL-mediated apoptosis, as determined by quantifying the sub-1 fraction in FACS, nor did the amount of insulin secretion change significantly under TRAIL treatment (data not shown).

RelA Transcriptional Activity in CM

The DNA-binding activity of RelA of NFB family of transcription factors in CM cells as determined by an ELISA-based assay increased significantly following 48 h stimulation with IFN (20-fold rise) and TRAIL+IFN (10-fold) in comparison with untreated control (Figure 1d).

General Aspects in Epithelia of NP and CP

Turning to tissues, NP had low mRNA levels of NFB subunits. These were increased in CP in parenchymal areas with fibrosis and most intensely in islets. The expression of NFB-regulated proteins IB, survivin and cIAP1 was found in corresponding sites, again at their highest levels in islets surrounded by fibrosis. Specimens from patients with CP were markedly microheterogeneous with respect to each aspect aimed at in this study. As observed in the pilot phase of this study, this heterogeneity seemed to be paralleled not by the degree of inflammatory lymphohistiocytic infiltration but by the degree of fibrosis. Even within the same section, the relative amount of antigen detected was most pronounced in fibrotic areas, the intensity being markedly lower in relatively normal unaffected areas. Therefore, we morphometrically estimated the relative proportion of the stromal and the epithelial compartment in each specimen and defined three grades of fibrosis, that is, mild fibrosis, 12.41.8%, moderate, 41.20.6%, and severe fibrosis, 73.96%; stroma constituted 6.31.9% of NP (Figure 2).

Figure 2.

Morphometric analysis of the relative proportion of stromal and epithelial tissue in NP and CP. Three grades of fibrosis, that is, mild fibrosis, 12.41.8%, moderate, 41.20.6%, and severe fibrosis, 73.96%, and NP, 6.31.9% were calculated by binary analysis of H&E-stained cryosection (left panel). White areas depict the epithelial compartment, gray areas empty spaces (eg ductal and vessel lumina) and the black areas represent stromal tissue (right panel).

Full figure and legend (636K)

Cryosections were subjected to LCM to obtain cells from each epithelial compartment of NP and CP in order to analyze the mRNA expression levels of NFB subunits and stained for proteins for which reliable antibodies were available. Next, we correlated the mRNA levels (data given in Figure 3) and the staining intensities in IHC with the extent of fibrosis (data given in Table 2 and paradigmatic situations depicted in Figure 4). To summarize, NP was largely devoid of expression of NFB constituents, survivin and cIAP1. Message and/or protein levels in pancreatic epithelial compartments increased with the degree of fibrosis. At the extreme, that is, in severe fibrosis, mRNA and/or protein was detected in high amounts, especially in islets spared in the midst of fibrosis.

Figure 3.

Semiquantitative real-time PCR for NFB subunits. Cyclophilin was used as the internal standard. The diagram depicts the x-fold expression of NFB1, NFB2, RelA, RelB and c-Rel in different microdissected epithelial compartments of CP with mild, moderate and severe fibrosis compared with corresponding epithelial compartments of NP, set as 1, after normalization of the C_T values of each sample to its internal standard. The bars indicating standard deviation are not always visible due to the minimal variations of values compared to the scale used.

Full figure and legend (130K)

Figure 4.

Immunohistochemistry for IKK, IB, RelA, survivin and cIAP1. IKK: NP tissues show no or only a very weak expression of IKK. Weak expression emerges in islets of mild fibrosis, whereas acini and ducts remain largely negative or exhibit only a weak staining. In CP with moderate fibrosis, there is a marked enhancement in staining intensity in the islets, acini and focally in the ducts; the islets are slightly positive in CP with severe fibrosis. IB: NP, CP with mild and moderate fibrosis are largely devoid of IB. In CP with severe fibrosis, there is a moderate expression in the islets and weakly so in the acini, but none in the ducts. RelA: normal acini, ductal epithelium and islets are devoid of RelA; in CP with mild fibrosis, staining is appreciable in acinar cells and much more intense in islets, while ducts show a very weak or no expression. In CP with moderate fibrosis, the acini and islets exhibit a stronger cytoplasmic positivity. In CP with severe fibrosis, the islets cells show a very strong cytoplasmic and nuclear staining; the remaining acini are also strongly positive. Survivin: acini, islets and ducts in NP express survivin only very weakly; staining is conspicuous in acini, islets and ducts in CP with mild fibrosis. With moderate fibrosis, there is an intense staining of all the epithelial compartments. In severe fibrosis, the islets exhibit further accentuation in the staining intensity in the cytoplasm and the nucleus. cIAP1: acini and ducts in NP do not stain for cIAP1. Staining emerges in the cytoplasm of acinar and islets cells with mild fibrosis; ducts are negative. In moderate fibrosis, there is further accentuation of staining in the acini, islets and staining emerges in ducts. In severe fibrosis, islets exhibit an even stronger reaction; acini and ducts are also stained intensely; staining is cytoplasmic as well as nuclear (^*islets).

Full figure and legend (1,031K)

Table 2 - RelA, survivin and cIAP1 expression in NP and CP.

Full table

RelA

In severe fibrosis, there was a sharp increase in RelA transcripts in islet cells but not in acini and ducts (Figure 3). Normal acini, ductal epithelium and islets showed no expression of RelA protein. In CP, RelA protein was detectable and the intensity of staining increased with progressive severity of fibrosis. Staining was most pronounced in the acini and islets in areas of severe fibrosis. In both, the acinar and islet cells, the staining was cytoplasmic and nuclear. The ductal epithelium showed either no or, focally, very low expression of RelA. In CP with severe fibrosis associated with exocrine parenchymal regression, the acini in fibrotic areas featuring a tubular morphology exhibited strong cytoplasmic and nuclear positivity. Pancreatic islets that were intact within severely fibrotic areas stained most intensely for RelA, staining being cytoplasmic as well as nuclear (Figure 4).

NFB1 (p105)

There was a sharp, about 10-fold, increase in NFB1 (p105) transcript numbers in islet cells in areas of severe fibrosis (Figure 3). As was the case for NFB1 (p100), no protein data are available for NFB2 (P100), RelB and c-Rel due to the lack of a reliable antibody.

NFB2 (p100)

A similar pattern was observed in NFB2 (p100) mRNA levels with the exception of acinar cells showing 4.6- and 69-fold increase in moderate and severe fibrosis, respectively (Figure 3). Islets showed nearly 24-fold amount of message in areas of severe fibrosis.

RelB

RelB mRNA was overexpressed in islet cells in mild and even more so in severe fibrosis (Figure 3).

c-Rel

In acini and ducts, there was a slight increase in c-Rel mRNA with higher degrees of fibrosis. This enhancement was by far exceeded by the extensive, 58-fold overexpression of c-Rel in islets, occurring in conjunction with severe fibrosis (Figure 3).

IKK

In NP, IKK protein expression was restricted to some scattered acinar cells.

In CP, an induction of IKK was observed in the exocrine and endocrine epithelium paralleled by the degree of fibrosis. In specimens showing only mild fibrosis, IKK expression was restricted to islets; all pancreatic epithelia showed moderate to high immunoreactivity for IKK in areas with moderate fibrosis. In severe fibrosis, within the remaining endocrine epithelium, IKK expression was at mild to moderate levels with lower levels in ducts and acini (Figure 4).

IB

The IKK target, IB, was undetectable in NP and in CP with mild and moderate fibrosis. In contrast, tissues affected with severe fibrosis showed expression of IB, this being more pronounced in islets and the surrounding acinar epithelium (Figure 4).

Against the constitutive paucity of RelA transcripts and low activators IKK and IB protein levels in NP, CP was characterized by fibrosis-related neoexpression and/or partial increase of mainly IKK and RelA, and less so of IB. The question thus arose as to whether RelA immunohistochemically shown to be translocated to the nucleus (Figure 4) contributes to the remarkably prolonged survival in pancreatic islet cells in the context of CP by inducing antiapoptotic factors, for example, IAPs such as survivin and cIAP1.

Survivin

In NP, survivin protein expression was absent or at low levels in ductal, acinar and islet cells. In CP, there was a survivin expression, the intensity of which increased with the extent of fibrosis. In CP cases with severe fibrosis, the intact islets exhibited intense cytoplasmic and nuclear staining. The remaining acini and the tubular acini also stained strongly (Figure 4). This pattern closely resembles those of IKK, IB and RelA immunoreactivity.

cIAP1

The staining pattern mostly parallels the ones described above for RelA and survivin. While NP did not stain for cIAP1, the staining could be discerned in acini, ducts and pancreatic islets in a perinuclear cytoplasmic location in CP with mild fibrosis, becoming more pronounced with increasing fibrosis. In CP with severe fibrosis, the intact islets exhibited an intense cytoplasmic and nuclear pattern. The acini spared by the fibrotic process and the tubular acini also stained strongly (Figure 4).

Discussion

TRAIL-R4 does not transmit a direct death signal but increasing evidence suggests that it activates NFB transcription factors, first demonstrated in T cells and transfectants.⁵ We show here for the first time that this is also true for a human insulinoma cell line, CM. CM cells essentially lack surface expression of functional death receptors, TRAIL-R1 and -R2, and constitutively express TRAILR4 in a minority of cells. Pretreatment of CM cells with TGF and IFN upregulated R4, while R1 and R2 remained unchanged. Treatment of these in vitro-activated cells with TRAIL led to an increase in transcripts for transcriptionally active RelA, RelB and c-Rel subunits together with a slight decrease in mRNA of the generally transcriptionally inactive molecules NFB1 (p105) and NFB2 (p100). Furthermore, this resulted in a downmodulation of IB and remarkable increase in survivin transcripts. Since TRAIL-R4 is the only functionally expressed TRAIL receptor in CM cells, this TRAIL effect most probably is transmitted via TRAIL-R4. Untreated and IFN/TGF-stimulated CM cells were resistant to TRAIL-mediated apoptosis. Moreover, TRAIL modulated their NFB repertoire in a way that activated antiapoptotic programs. Further, we found elevations in DNA-binding activity of RelA upon treatment with IFN and IFN+TRAIL.

With this, we turned to the in situ analysis of NP and CP. We show that NP is characterized by low expression levels of NFB subunit transcripts in both exocrine and endocrine compartments. In CP, there is a massive induction of IKK and RelA protein, which is at its maximum in islets. Immunohistochemistry further suggested that this extent of enhancement might be associated with the degree of fibrosis. Since commercially available antibodies to other NFB subunits failed to meet our quality standards, we had to rely solely on the mRNA levels. Therefore, we first morphometrically assessed the degree of fibrosis and isolated mRNA of microdissected islets, acinar and ductal epithelial cells from corresponding serial sections. The results are clearcut and reflect the pattern observed at the protein level. The second important and unprecedented finding is the fibrosis-associated increase in mRNA for NFB1, NFB2, RelA, RelB and c-Rel in islet cells. Together with our findings that transcriptionally active NFB subunits are upregulated in CM cells upon TRAIL treatment, this interaction may also play a role in CP. Thus, TRAIL, via TRAIL-R4 broadly expressed in CP, may be one, if not a major, factor bringing about the above scenario.¹⁰

At present, there is a wealth of data on the defensive and protective role NFB plays in the acute phase response in inflammation.^{11, 12} It was suggested that RelA and c-Rel are functional antagonists with respect to apoptosis, RelA overexpession inhibiting apoptosis, while c-Rel acts in a proapototic way by enhancing TRAIL-R1, -R2 or suppressing cIAP1, cIAP2 and survivin.¹³ It was, however, also suggested that NFB2 (p100) might be a direct activator of programmed cell death.^{14, 15} NFB was shown to be involved in acute pancreatitis.¹⁶ Activation of NFB was detected as early as 10 min after supramaximal stimulation in the cerulein model of acute pancreatitis and seemed to limit the damage induced, while blocking NFB activation had an adverse effect.¹⁶ Conversely, intraductally administered in vivo gene transfer of RelA led to NFB activation, acinar damage and acute pancreatitis in rats.¹⁷ Thus, it remains unclear whether NFB activation in pancreas is protective or deleterious.¹⁸ Remarkably, in the acini of CP with severe fibrosis, we found a 69-fold NFB2 mRNA expression, eventually tilting the balance in favor of apoptotic cell death in the exocrine compartment.

Our new findings collectively suggest that in CP, a progressive disease smoldering for years, there is a reprogramming of endocrine and, less so, exocrine cells for an altered state of NFB-regulated transcriptional activity, the functional consequences of which, at the single-cell level, are largely unclear. One family of genes regulated by NFB are IAPs, acting as suppressors of apoptosis by inhibiting caspase-3, -7 and -9.^{9, 19} It was suggested that cancer cells, for example, of pancreatic carcinoma,^{20, 21} can counteract apoptotic signals by the expression of IAPs. We found a pattern of fibrosis-associated enhancement of cIAP1 and survivin protein expression similar to RelA emerge in islets. Our finding of cIAP1 protein being undetectable in NP is at variance with published data suggesting that mRNA is present in all adult tissues.²² The low levels or lack of survivin expression in NP is in line with data on the absence of mRNA in normal adult tissues²² and the absence of protein in NP reported by Satoh et al.²¹ However, survivin expression has been reported in human fetal islets.²³ We show for the first time a fibrosis-associated dramatic induction of both cIAP1 and survivin in CP, again, especially in islet cells.

Our findings suggest that sustained enhanced expression of NFB subunits and, consequently, IAPs are lifeguards for endocrine cells in the adverse microenvironment of CP, while the pattern of NFB activation is likely to be deleterious to exocrine parenchyma. One of the key inductors of this reprogramming might be TRAIL. Interestingly enough, TRAIL was shown in two independent experimental studies to protect islet cells in experimental type I diabetes in mice.²⁴ Mi et al reported TRAIL gene upregulation in islet cells during the development of diabetes of nonobese diabetic (NOD) mice. Further, treatment of freshly isolated islet cells with TRAIL did not result in apoptosis. Finally, TRAIL blockade exacerbated the onset of type I diabetes in NOD Scid recipients of transferred diabetogenic T cells. Lamhamedi-Cherradi et al²⁵ treated normal and TRAIL-deficient mice with multiple low-dose streptozotocin to induce diabetes. They went on to treat NOD mice with soluble TRAIL blocker. Both the incidence and the degree of insulitis in both disease models were enhanced in the absence, or blockade, of TRAIL. Although both groups of authors attribute the protective TRAIL effect to an inhibition of proliferation of diabetogenic T cells, it seems, against the background of our findings, worthwhile considering TRAIL-mediated protection of the islet cells as an alternative possibility.

The two major epithelial components of pancreas meet different fates in different chronic inflammatory diseases. In CP, where the exocrine parenchyma is progressively lost, the endocrine islets remain intact and functional, even in advanced stages of fibrous replacement. Vice versa, in type I diabetes, the islet cells vanish in the course of insulitis, while the exocrine parenchyma is unaffected. In both processes, apoptotic cell death is the key event.²⁶ Current understanding suggests that immune attacks are cell type specific, and there is little left to doubt this. Nevertheless, chronic inflammation functions via local release of chemokines, cytokines and soluble death receptor ligands, which lack the specificity of cognate T-cell–target cell interaction. These short-range acting molecules not only fuel the directed attack but are also likely to inflict collateral damage,²⁷ all the more if targets and innocent bystanders are so intimately assembled. Therefore, survival programs run by exocrine and endocrine cells regulating their fate in terms of apoptosis resistance or susceptibility must fundamentally differ in CP and type I diabetes, although the death pathways and their interfering inhibitors are very likely just the same in both cell types.

Summarizing our previous findings^{3, 4} and integrating our new data, we propose the following model of CP leading to enforced death of exocrine cells and at the same time to armored survival of endocrine cells (Figure 5). Initiated by IFN locally released by CD4⁺Th1 cells, exocrine pancreatic epithelia lose CD95L and neoexpress the functional death receptors CD95, TRAIL-R1 and TRAIL-R2. This renders them vulnerable to CD95L-mediated T-cell attack and/or to action of soluble TRAIL released by IFN-triggered PSCs. The effects of IFN and TRAIL on endocrine cells are completely opposite. In the setting of CP, islet cells retain their protective CD95L⁺, death receptor^- state but respond to IFN/TRAIL via TRAIL-R4 signalling with NFB activation and subsequent neoexpression of IAPs. Thus, islet cells at risk of succumbing to collateral damage caused by local release of death-inducing molecules by the inflammatory infiltrate augment their functional repertoire ensuring survival in a locally adverse environment.

Figure 5.

Hypothetical model of dichotomy of fates of pancreatic epithelia in CP.

Full figure and legend (18K)

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Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) SFB 518/A13 to P Möller and a grant from the Deutsche Krebshilfe (Str 10-1644-St1) to J Sträter.

Supplementary Information accompanies the paper on Laboratory Investigation website (http://www.nature.com/labinvest)