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Originally published In Press as doi:10.1074/jbc.M309152200 on December 1, 2003

J. Biol. Chem., Vol. 279, Issue 8, 7199-7207, February 20, 2004
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p8 Improves Pancreatic Response to Acute Pancreatitis by Enhancing the Expression of the Anti-inflammatory Protein Pancreatitis-associated Protein I*

Sophie Vasseur{ddagger}§, Emma Folch-Puy{ddagger}§, Verena Hlouschek¶, Stephane Garcia{ddagger}||, Fritz Fiedler**, Markus M. Lerch{ddagger}{ddagger}, Jean Charles Dagorn{ddagger}, Daniel Closa§§¶¶, and Juan Lucio Iovanna{ddagger}||||

From the {ddagger}Centre de Recherche INSERM, EMI 0116, 163 Avenue de Luminy, BP172, 13009 Marseille, France, the Medizinische Klinik B, Westfälische Wilhelms-Universität, 48129 Müster, Germany, the ||Hôpital Nord, Chemin des Bourrellys, 13915 Marseille, France, the **Institut für Anästhesie, Klinikum Mannheim, Theodor-Kutzer-Ufer, D-68167, Mannheim, Germany, the {ddagger}{ddagger}Division of Gastroenterology and Endocrinology, Department of Medicine A, Ernst-Moritz-Arndt Universität Greifswald, Friedrich-Löffler-Str. 23A, 17487 Greifswald, Germany, and the §§Department of Experimental Pathology, IIBB-CSIC, c/Rossello 161, 7°, 08036 Barcelona, Spain

Received for publication, August 18, 2003 , and in revised form, November 7, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
p8 is a transcription cofactor whose expression is strongly and rapidly activated in pancreatic acinar cells during the acute phase of pancreatitis. A p8-deficient mouse strain was generated as a tool to investigate its function. Upon induction of acute pancreatitis, myeloperoxidase activity in pancreas and serum concentrations of amylase and lipase were much higher and pancreatic lesions more severe in p8-deficient mice than in wild-type, indicating that p8 expression decreased pancreatic sensitivity to pancreatitis induction. The protective mechanism might involve the pancreatitis-associated protein (PAP I), whose strong induction during pancreatitis is p8-dependent, because administration of anti-PAP I antibodies to rats increased pancreatic inflammation during pancreatitis. In addition, 100 ng/ml PAP I in the culture medium of macrophages prevented their activation by tumor necrosis factor {alpha}, strongly suggesting that PAP I was an anti-inflammatory factor. Finally, PAP I was able to inhibit NF{kappa}B activation by tumor necrosis factor {alpha}, in macrophages and in the AR42J pancreatic acinar cell line. In conclusion, p8 improves pancreatic resistance to inducers of acute pancreatitis by a mechanism implicating the expression of the anti-inflammatory protein PAP I.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathogenesis of acute pancreatitis, one of the most frequent diseases of the pancreas, is not fully understood yet. However, there is good evidence that autodigestion of the gland is involved. A still unknown triggering event converts, within the pancreas, digestive proenzymes into their active forms, leading to cell membrane disruption, edema, interstitial hemorrhage, necrosis, and to activation of other proenzymes. This mechanism is therefore self-propagating and can result in complete destruction of the parenchyma. Yet, most episodes of acute pancreatitis are self-limiting and remain mild. This is due to the rapid build up of pancreatic defense mechanisms involving important reorientation of the gene expression pattern. Genes encoding the potentially harmful digestive enzymes are down-regulated during the acute phase of pancreatitis whereas others, which help curb the extension of the disease, are strongly up-regulated (1, 2). Hence, like other organs including the liver (3-5) the pancreas can initiate in response to acute stress a stringent emergency program intended for protection of the parenchyma.

While studying the stress response of the pancreas, we identified a gene called p8 whose expression is strongly and rapidly activated in acinar cells during the acute phase of pancreatitis (6). In fact, expression of p8 was found induced in almost all tissues in response to stress (7), suggesting an important role in defense mechanisms, inasmuch as orthologues of human p8 (8) were found in rat (6) mouse (9), Xenopus laevis (10), Drosophila melanogaster (GenBankTM accession number NP_609539 [GenBank] ), Bos taurus (GenBankTM accession number BM258976 [GenBank] ), Canis familiaris (GenBankTM accession number AJ536980 [GenBank] ), the zebrafish (GenBankTM accession number BF717555 [GenBank] ), Oryzias latipes (GenBankTM accession number BJ016183 [GenBank] ), Sus scrofa (GenBankTM accession number CB287816 [GenBank] ), and Paralichthys olivaceous (GenBankTM accession number AU091122 [GenBank] ). Secondary structure prediction methods revealed that a basic helix-loop-helix motif characteristic of certain transcription factors was present in the conserved regions of the 11 proteins (6). Although data base searches revealed no significant homology to any protein of known function, the mammalian p8 proteins share biochemical, biophysical, and DNA-binding properties with the HMG-I/Y family (11) even if overall sequence identity is only about 35%. An architectural role of p8 in transcription was proposed by analogy with the HMG-I/Y proteins, and actually received recent support (12).

Several, apparently unrelated functions have already been described for p8 in different cell types, as expected from a ubiquitous transcription co-factor. For instance, transforming growth factor {beta}-1 induces p8 mRNA expression that in turn enhances the Smad-transactivating function responsible for transforming growth factor {beta}-1 activity (13). Also, p8 is involved in cell cycle regulation as a growth promoter (6, 8) or inhibitor (14, 15) or as a mediator of apoptosis (14). In addition, p8 is required for endothelin-induced mesangial cell hypertrophy in the diabetic kidney (16), for initiation of LH{beta} gene expression (17) and, in D. melanogaster to stop cell growth in response to starvation (18). Finally, a particularly attractive role in the control of tumor progression was proposed for p8 (19, 20). None of the functions listed above could easily be attributed to p8 in pancreas during the course of acute pancreatitis. Being strongly activated early in the development of pancreatitis, p8 could be a mediator of disease progression or, conversely, the first occurrence of a protective mechanism. It could also be a simple epiphenomenon. To investigate the role of p8 in vivo, we generated p8-deficient mice by gene targeting. We found that expression of p8 improves the pancreatic response to acute pancreatitis through enhancing the expression of the anti-inflammatory protein pancreatitis-associated protein (PAP)1 I.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Targeted Disruption of the Mouse p8 Gene—The construction of the targeting vector used in targeted disruption of the p8 locus in embryonic stem cells of mouse strain 129/Sv was recently reported (14). Embryonic stem cells from three heterozygous clones for the mutated p8 allele were injected into blastocysts of C57BL/6 mice. We obtained in the littermates 7 highly chimerical male mice. They were intercrossed with wild-type C57BL/6 females and 4 showed germline transmission. Genotype identification was done by PCR and/or Southern blot with genomic DNA prepared from tail biopsies of 10-day-old mice. Heterozygous mice for the mutated p8 allele with mixed genetic background of C57/BL6 and SV129J strains were obtained and used in breeding experiments to generate p8-/- mice. p8-deficient mice were backcrossed to C57/BL6 mice for nine generations so that the p8-deficient mice used in this study have a background closer to that of C57/BL6 mice than to the original C57/BL6 X SV129J-F1 animals.

Induction of Experimental Pancreatitis—Pancreatitis was induced in 4-month-old p8-/- and p8+/+ C57/BL6 mice weighing 20-24 g. After fasting for 18 h with access to water ad libitum, the secretagogue cerulein (Sigma) was administered as seven intraperitoneal injections of 50 µg/kg body weight at hourly intervals, as previously described (21). Saline-injected animals served as controls. All studies were performed in accordance with the European Union regulations for animal experiment.

Preparation of Serum and Tissue Samples—Mice were sacrificed at several time intervals from 3 to 24 h following the first intraperitoneal injection of cerulein. Whole blood samples were centrifuged at 4 °C, and serum was stored at -80 °C for further studies. The pancreas was removed on ice, weighed, immediately frozen in liquid nitrogen, and stored at -80 °C. For measurement of pancreatic enzyme activities, tissue was thawed and homogenized in ice-cold medium containing 5 mM MOPS, 1 mM MgSO4, and 250 mM sucrose (pH 6.5). Samples were sonicated and centrifuged for 5 min at 16,000 x g. Sample preparation for trypsinogen activation peptide (TAP) assay was performed as described previously (22), supplementing the tissue homogenate with 1 mM EDTA and 0.1% Triton X-100 and boiling the samples for 10 min. Supernatants generated at 16,000 x g for 5 min were used in subsequent assays. For the myeloperoxidase assay, samples were macerated with 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0). Homogenates where then disrupted for 30 s using a Labsonic (B Braun) sonicator at 20% power and submitted to three cycles of snap freezing in dry ice and thawing before a final 30-s sonication. Samples were incubated at 60 °C for 2 h and then spun down at 4,000 x g for 12 min. Supernatants were collected for myeloperoxidase assay.

Biochemical Assays—Amylase and lipase activities were determined with commercially available assays (Roche Biochemicals). TAP was assayed by ELISA (Biotrin, Dublin, Ireland). R110-(CBZ-Ile-Pro-Arg)2 and R110-(CBZ-Ala4)2 were used as substrates to monitor trypsin and elastase activities, respectively. Pancreatic homogenates were transferred to 96-well microtiter plates and the kinetics of proteolysis were recorded over 60 min at 37 °C by fluorometry using wavelengths of 485 nm for excitation, 515 nm as cutoff, and 530 nm for emission. To increase specificity, trypsin activity was expressed as the fraction of the increase in fluorescence that can be blocked by phenylmethylsulfonyl fluoride (2 mM) and aprotinin (1 mM). Protein concentrations were determined according to Bradford (23). Myeloperoxidase was measured with 3,3',5,5'-tetramethylbenzidine as substrate as previously described (24). Enzyme activity was recorded at 630 nm. The assay mixture consisted of 20 µl of supernatant, 10 µl of tetramethylbenzidine (final concentration 1.6 mM) dissolved in Me2SO, and 70 µl of H2O2 (final concentration 3.0 mM) diluted in 80 mM phosphate buffer (pH 5.4).

Quantitation of Cerulein-induced Injuries—To evaluate necrosis and inflammation in pancreas of p8-expressing and p8-deficient mice after cerulein-induced pancreatitis, formalin-fixed samples were embedded in paraffin and 5-µm sections were stained with hematoxilin and eosin. Coded sections were evaluated by light microscopy and scored by 3 experienced morphologists.

Transfection and Overexpression of p8 cDNA—The full-length human p8 cDNA was subcloned into the expression vector pcDNA3 (Invitrogen), downstream from the cytomegalovirus promoter, as previously reported (8). The recombinant plasmid was named pcDNA3-hump8. As control, we used the empty pcDNA3 plasmid. The plasmids were transfected into AR42J cells using FuGENE reagent (Roche Diagnostics) as recommended by the manufacturer. To select for stable expression of the pcDNA3-hump8 and of empty pcDNA3, cells were cultivated over 3-4 weeks in media to which G418 (800 µg/ml) was added 48 h after transfection. Surviving colonies were pooled and maintained in standard culture medium supplemented with G418 (400 µg/ml).

Expressions of PAP I, amylase, cathepsin B, and {beta}-actin mRNA were monitored by semiquantitative RT-PCR. Briefly, 1 µg of total RNA was used and the sequence amplified by the Invitrogen One Step RT-PCR system according to the manufacturer's protocol. For PAP I, the forward primer was 5'-CTCCTGCCTGATGCTCTTAT-3' and the reverse primer 5'-TTGTTACTCCACTCCCATCC-3'; for amylase, the forward primer was 5'-AAAATTTGCTCAAGGTCTGG-3' and the reverse primer 5'-TTTATCGAGTGCAAGATCCA-3; for cathepsin B the forward primer was 5'-TGTCGGACGACATGATTAAC-3' and the reverse primer 5'-CATTGGTGTGAATGCAGATT-3'; and for {beta} -actin, a housekeeping gene used as control, the forward primer was 5'-CACGGCATTGTAACCAACTG-3' and the reverse primer 5'-TCTCAGCTGTGGTGGTGAAG-3'. RT-PCR products were resolved by 2% agarose gel electrophoresis and stained with ethidium bromide.

p8 Induces PAP I Expression by Activation of the Promoter—To monitor the induction by p8 of the PAP I promoter, we performed a chloramphenicol acetyltransferase (CAT) assay, using the rat PAP I gene promoter (fragment -1253 to +10 from initiation) positioned upstream from the CAT reporter gene, as previously described (25). One day after seeding AR42J cells stably transfected with pcDNA3-hump8 or the pcDNA3 empty plasmid at a density of 5 x 106 cells per 100-mm Petri dish, the PAP I promoter-CAT reporter construct was transfected using FuGENE reagent as described above. Twenty-four hours later, cells were harvested and extracts were prepared using the reporter lysis buffer (Promega). The pGL3 control plasmid (Promega), which contains the luciferase reporter gene under the control of the SV40 promoter, was used as a transfection control. CAT activity was determined using a phase extraction procedure and luciferase using the Luciferase assay reagent (Promega) according to the manufacturer's instructions.

Rat Model of Pancreatitis—Anesthesia was induced in male Wistar rats (250-300 g) by intraperitoneal injection of 10% urethane (1 ml/100 g). The biliopancreatic duct was cannulated through the duodenum and the hepatic duct was closed by a small bulldog clamp. Acute pancreatitis was induced by retrograde injection of sodium taurocholate (5%) in a volume of 0.1 ml/100 g. Control animals received an infusion of saline solution. In other experiments, animals received immediately after pancreatitis induction an intravenous administration of anti-PAP I IgGs (3 mg/kg) or, as control, Protein A-purified IgGs (3 mg/kg) obtained from non-immunized rabbits. Samples of pancreas where obtained 3 h after induction, immediately frozen and maintained at -80 °C until assayed.

Western Blotting of PAP I—For Western blots, the pancreas of mice and rats were rapidly ground in liquid nitrogen. The resulting powder was reconstituted in an ice-cold solubilization buffer containing 50 mmol/liter Tris/HCl (pH 8), 150 mmol/liter NaCl, 1% Nonidet P-40, 1 mmol/liter phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Sigma) at a concentration of 100 mg/ml. Samples were centrifuged at 4 °C for 10 min at 10,000 x g. Supernatants were recovered, and total amounts of protein were determined using the Bradford method. One hundred µg of protein from pancreas were separated by 12% SDS-PAGE then blotted following standard methods. Nonspecific binding to the membrane was blocked by 5% bovine serum albumin in Tris-buffered saline overnight at 4 °C. Blots were incubated for 1 h at room temperature with an affinity purified rabbit anti-rat PAP I antibody (1:2500) or a rabbit polyclonal antibody specific to human p8 (1:100) diluted in 5% bovine serum albumin. Then, membranes were washed with Tris-buffered saline, 0.1% Triton and incubated with a secondary goat anti-rabbit-HRPO antibody (1:5000) (Amersham Biosciences) diluted in 5% dry nonfat milk in Tris-buffered saline for 1 h at room temperature. Finally, membranes were washed with Tris-buffered saline, 0.1% Triton, developed with the ECL detection system (Santa Cruz Biotechnology, Santa Cruz, CA), quickly dried and exposed to ECL film.

Macrophage Isolation and Treatment—Alveolar macrophages were obtained by bronchoalveolar lavage. Lungs where dissected free of the thoracic cavity and a small length of tubing was inserted into the trachea and ligated. Bronchoalveolar lavage was carried out with 10 ml of cold saline solution instilled and withdrawn from the lungs four times. The cell suspension was centrifuged at 400 x g and the pellet was resuspended in RPMI 1640 medium. Cells were counted, cultured in 12-dish plates (106 cell/ml), incubated for 1 h at 37 °C under 5% CO2 in air, and washed twice with warm medium to remove nonadherent cells. Twenty-four hours after cell isolation, cells were incubated with TNF{alpha} (50 ng/ml) or 0.1 µM fMLP with increasing doses of purified PAP I. After incubating 4 h at 37 °C under 5% CO2 in air, total RNA was obtained to evaluate the changes in TNF{alpha}, interleukin-6 (IL-6), and {beta}-actin mRNA concentrations by the semiquantitative RT-PCR method described above. For TNF{alpha}, the forward primer was 5'-ACTGAACTTCGGGGTGATTG-3' and the reverse primer 5'-GTGGGTGAGGAGCACGTAGT-3'; for IL-6, the forward primer was 5'-CCGGAGAGGAGACTTCACAG-3' and the reverse primer 5'-GAGCATTGGAAGTTGGGGTA-3. {beta}-Actin was also used as a housekeeping control gene.

Immunofluorescence Monitoring of NF{kappa}B—To determine NF{kappa}B translocation, macrophages or AR42J cells were incubated in coverslips overnight at 37 °C under 5% CO2 in air. Cells were treated first with PAP I (100 ng/ml) for 10 min, then TNF{alpha} (50 ng/ml) or fMLP (0.1 µM) were added to the culture medium for 30 min. Cells were fixed with 4% formaldehyde in phosphate-buffered saline for 20 min at 4 °C and permeabilized with 0.1% Triton in phosphate-buffered saline 5 min at room temperature. Saturation was obtained by incubating cells with 5% bovine serum albumin in phosphate-buffered saline for 30 min at room temperature. Cells were stained with anti-p65 NF{kappa}B antibody (Santa Cruz Biotechnology) and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody (Molecular Probes, Eugene, OR). Slides were mounted with Mowiol, and NF{kappa}B localization in the cells was examined by fluorescence microscopy.

ELISA for Activated NF{kappa}B—Activation of NF{kappa}B was measured with a commercial ELISA kit (TransAM, Active Motif, Rixensart, Belgium). In this method, the transcription factor binds to an oligonucleotide containing a consensus NF{kappa}B binding site immobilized on a 96-well plate. It is detected by a primary antibody, which specifically recognizes an epitope accessible only when the factor is activated and bound to its target DNA. A secondary anti-IgG horseradish peroxidase conjugate allows detection of the activated NF{kappa}B-antibody complex by a colorimetric reaction.

Statistical Analysis—Data shown in the figures indicate the mean ± S.E. Differences between groups were compared using Student's t test or one-way analysis of variance test when more than two groups were compared. Asterisks in the figures indicate statistically significant differences (p < 0.05).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Construction of a p8-/- Mouse Model—A p8 null allele was generated in 129/Sv embryonic stem cells by replacing exon 2 of the p8 gene by a neomycin-resistance cassette. Germline transmission was obtained after injection of these embryonic stem cells into C57BL/6 blastocysts. We then intercrossed mice heterozygous (p8+/-) for the targeted disruption to produce homozygous offsprings (p8-/-). These crosses provided litters of normal size, with living, and apparently normal p8-/- offsprings occurring at a frequency consistent with Mendelian inheritance. p8 deficiency was verified by RT-PCR and/or Southern blot analysis and by immunostaining of the pancreas with acute pancreatitis with a p8 polyclonal antibody (data not shown). Given these results and considering the size of the deletion, we concluded that the p8 mutation was null. p8-deficient mice showed normal phenotype at birth, without apparent neurological or behavioral deficits. Postnatal development was indistinguishable from that of heterozygous or wild-type littermates. We concluded that p8 was not involved in crucial developmental processes or that other genes were functionally redundant. Inspection of external and internal organs of p8-/- mice at different ages did not reveal any macroscopic abnormality. Histopathological investigation of pancreas showed an organization of Langerhans islets, secretory ducts, and acini quite similar to that of wild-type animals. p8-/- mice were fertile and reproduced normally. However, compared with wild-type, p8-deficient mice were more sensitive to the noxious effects of lipopolysaccharide with decreased survival, increased levels of serum TNF{alpha} and higher levels of myeloperoxidase and hydroperoxide in liver and pancreas. This suggests the implication of p8 in the defense mechanisms of these organs as recently published (26). Using microarray DNA analysis of liver gene expression we demonstrated that lack of p8 perturbed the expression of a large number of genes (belonging to many cellular pathways), which were improperly up- or down-regulated upon lipopolysaccharide treatment. We concluded that p8, which is probably a transcription factor, is a key regulator of the cellular response to lipopolysaccharide. If its absence is apparently without important consequences in normal conditions, it prevents mice from fully developing their defense program against lipopolysaccharide challenge (26). On the other hand, we have recently demonstrated that although p8-deficient mice develop normally, embryonic fibroblasts obtained from these mice show altered behavior compared with wild-type, p8-/- embryonic fibroblasts growing more rapidly under normal conditions and showing increased resistance to apoptosis after adriamycin-induced DNA damage (14). We also showed that expression of p8 is required for development of tumors because transformed p8-deficient fibroblasts were unable to produce tumors in athymic nude mice when injected subcutaneously, contrary to transformed wild-type fibroblasts (20).

Cerulein-induced Pancreatitis Is More Severe in p8-/- Mice Than in Wild-type—As previously described (21), wild-type mice given intraperitoneal injections of the secretagogue cerulein at a supramaximal dose develop acute necrotizing pancreatitis. We compared the severities of cerulein-induced pancreatitis in wild-type and p8 knock-out mice by measuring amylase and lipase levels in serum and by assessing the extent of acinar cell necrosis. As shown in Figs. 1 and 2, pancreatitis induction was followed by a time-dependent rise in serum amylase and lipase activities, amylasemia and lipasemia being, however, 30 and 50% higher, respectively, in p8 knock-out mice than in wild type (Fig. 1). In addition, histological examination of pancreas sections showed more severe lesions (acinar cell injury/necrosis, edema, inflammation, and cellular vacuolization) in knock-out mice (Fig. 2 and Table I). Acute pancreatitis is therefore more severe in p8-deficient animals, compared with wild-type, suggesting a protective effect of p8. It is known that during acute pancreatitis, neutrophil accumulation increases pancreatic tissue damage. Neutrophil infiltration was assessed by monitoring in pancreas the activity of myeloperoxidase, an enzyme specifically found in polymorphonuclear leukocytes. On that basis, neutrophil sequestration appeared significantly higher in p8-deficient mice than in wild-type (Fig. 2).



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  		FIG. 1.
Serum amylase and lipase profiles in wild-type and p8-/- mice. Serum amylase and lipase levels were measured in control p8+/+ and p8-/- mice and 3, 6, 9, 12, 15, 18, and 24 h following cerulein-induced acute pancreatitis. Results are expressed as mean values ± S.E. (n = 4).

 



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  		FIG. 2.
Acute pancreatitis is more severe in p8-/- mice. A, histologic sections of control pancreas and pancreas after 3 or 18 h following induction by cerulein of acute pancreatitis in p8+/+ and p8-/- mice. Note the significant increase in edema, infiltration, and necrosis in pancreas from p8-/- mice. Bar = 10 µm. B, pancreatic myeloperoxidase activity was measured in the pancreas of control p8+/+ and p8-/- mice and 3, 6, 12, 18, or 24 h following induction by cerulein of acute pancreatitis. Results are expressed as mean values ± S.E. (n = 4).

 


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  		TABLE I
Scoring of histological alterations

The histological alterations were scored by 3 experienced reviewers. The scores range from 0 (absent), 1 (minimal), 2 (moderate), 3 (important) to 4 (maximal). For necrosis and vacuolization, the scores refer to ranges of percentages of cells involved: 0 = 0-5%; 1 = 5-15%; 2 = 15-35%; 3 = 35-50%; 4 >= 50%.

 
The increased severity of pancreatitis in p8-deficient mice can be attributed either to an increased sensitivity to the inducers of pancreatitis or to an altered protective response. Cerulein-induced pancreatitis is the consequence of intra-pancreatic activation of trypsinogen, the resulting trypsin activating other pro-enzymes such as pro-elastase and pro-phospholipase (27-29). The intra-pancreatic activation of digestive proteases is thought to precede the inflammatory response and its intensity correlates to disease severity (30). To monitor the first steps of pancreatitis development in p8-deficient mice, we measured the activities of trypsin and elastase, and the concentration of the TAP in the pancreas during the course of cerulein-induced acute pancreatitis. As expected, trypsin and elastase activities and TAP concentration increased progressively after cerulein treatment (Fig. 3). In the first 12 h, increase was very comparable in the p8-deficient and wild-type pancreas, suggesting that absence of p8 expression does not influence the early steps of the pathogenic mechanism by which supramaximal secretagogue stimulation results in intra-acinar activation of proteolytic enzymes. After 12 h, TAP concentration decreased, showing that the process of pancreatitis induction had stopped. Yet, enzyme activation remained significantly more important at 18 and 24 h in the p8-/- pancreas. Together with the increased enzyme concentrations observed in blood (Fig. 1), this observation indicates that absence of p8 expression is associated with impairment of the acinar cell defense mechanism.



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  		FIG. 3.
Trypsin, TAP, and elastase levels in pancreas following cerulein induction of acute pancreatitis in p8+/+ and p8-/- mice. Trypsin and elastase activities, and TAP concentration were measured in the pancreas from untreated (control) p8+/+ and p8-/- mice and 3, 6, 12, 18, or 24 h following supramaximal cerulein administration. Results are expressed as mean values ± S.E. (n = 4).

 
PAP I Induction Is Strongly Dependent on p8 —During the acute phase of pancreatitis an important reorganization of the gene expression pattern occurs in the gland as part of a well structured response to pancreatitis (2). The PAP I was identified several years ago as the most abundant among proteins overexpressed during the acute phase of pancreatitis. In fact, the PAP I gene encodes a secretory protein that may account for more than 5% of total pancreatic juice protein in animals with acute pancreatitis, while being not detectable in normal juice (31). We compared by Western blot analysis PAP I levels in the pancreas of p8-deficient and wild-type mice during the course of acute pancreatitis. As expected, PAP I was, in both cases, undetectable under normal conditions. Six hours after cerulein administration, a PAP I increase was observed in both groups, although more pronounced in p8-expressing mice. Twelve and 18 h after cerulein injection, a further strong increase in PAP I level was observed, as expected, in the wild-type mouse pancreas, whereas increase was only modest in the pancreas of p8-deficient mice (Fig. 4). These results suggest that p8 is necessary for optimal PAP I gene activation.



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  		FIG. 4.
PAP I expression in the pancreas with acute pancreatitis is p8-dependent. A, the expression levels of p8, PAP I, and {beta}-tubulin in pancreatic homogenates from untreated p8+/+ and p8-/- mice and 6, 12, 18, or 24 h following cerulein-induced pancreatitis were monitored by Western blot analysis as described under "Materials and Methods." B, expressions of PAP I, amylase, cathepsin B, and {beta}-actin mRNA were measured in AR42J cells stably transfected with either an empty vector (AR42J/empty) or a human p8 expression vector (AR42J/p8) as described under "Materials and Methods." RT-PCR products were resolved in 2% agarose gel electrophoresis and stained with ethidium bromide. C, the rat PAP I gene promoter fragment -1253 to +10 positioned upstream from the CAT gene reporter was transfected in AR42J/empty and AR42J/p8 cells using the FuGENE reagent. The pGL3 control plasmid, which contains the luciferase reporter gene, was used as a transfection control. Twenty-four hours later CAT and luciferase activities were determined and expressed as CAT/luciferase ratio. Experiments were performed in triplicate and repeated with similar results.

 
To test this hypothesis in vitro, we induced p8 overexpression in AR42J cells by stable transfection of a human p8 cDNA placed under the control of the cytomegalovirus minimal promoter (8). This cell line, derived from an azaserine-induced tumor of the rat exocrine pancreas, has retained most of the acinar cell phenotype. An empty vector was used as a control. Overexpression of p8 was controlled by Western blotting (data not shown) with specific antibodies to human p8 (8). Expression of PAP I mRNA was measured by semiquantitative RT-PCR. Fig. 4 shows that expression of PAP I remained undetectable after 30 PCR cycles in control cells but was abundant in AR42J cells transfected with p8. As a control, expression of mRNAs encoding amylase, cathepsin B, and {beta}-actin were also monitored and found unaltered by p8 overexpression. In addition, we used CAT as a reporter gene to show that the PAP I promoter was the actual target of p8 expression (Fig. 4). These results confirm that, in pancreatic exocrine cells, PAP I expression is dependent on p8 expression.

PAP I Is an Anti-inflammatory Factor—The strong induction of myeloperoxidase activity observed in the pancreas of p8-/- mice reflects the development of inflammation. Because PAP I is expressed in these mice at low levels (see above), we asked whether polymorphonuclear cell accumulation in the pancreas was in fact made possible by the absence of PAP I induction. To test this hypothesis we injected rats with sufficient anti-PAP I antibodies to completely block circulating PAP I. Then necrotizing acute pancreatitis was induced and pancreatic inflammation was assessed 3 h later, when inflammation is usually established and PAP I strongly expressed. As controls, rats were either injected with saline only or with the same amount of non-relevant IgGs (Fig. 5). In the two control groups pancreatitis was associated with increased myeloperoxidase activity in the pancreas as a consequence of neutrophil infiltration (Fig. 5). However, in animals whose blood PAP I had been captured by the antibodies, pancreatic inflammation following pancreatitis induction was significantly more important than in controls (Fig. 5). In the absence of pancreatitis, administration of anti-PAP I antibodies or of PAP I-unrelated IgGs was without effect. These results suggest that PAP I accumulation in acinar cells in response to acute pancreatitis helps limit pancreatic inflammation.



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  		FIG. 5.
Anti-inflammatory effect of PAP I in rat pancreas with acute pancreatitis. A, the levels of expression of PAP I in pancreatic homogenates from control rats and rats with acute pancreatitis induced by retrograde injection of 5% sodium taurocholate were monitored by Western blotting using specific rat PAP I antibody as described under "Materials and Methods." B, rats received intravenous injection of saline, non-relevant IgGs, or IgGs to PAP I, as described under "Materials and Methods." Then, a retrograde injection of 5% sodium taurocholate (acute pancreatitis) or saline (control)-induced pancreatitis and myeloperoxidase activity was measured in their pancreas. Four animals per group were used. Results are expressed as mean values ± S.E.

 
To further confirm these observations, we looked at the effects of PAP I on the activation of macrophages, which play a key role in the orchestration of the pancreatic inflammatory response during acute pancreatitis (32, 33). Activation in vitro of macrophages by TNF{alpha} (50 ng/ml) or fMLP (0.1 µM) was monitored by measuring the expression of IL-6 and TNF{alpha} mRNAs, {beta}-actin mRNA being used as control. In control experiments, IL-6 and TNF{alpha} mRNAs were both induced (Fig. 6 and data not shown). Interestingly, adding PAP I to the culture medium inhibited that induction in a dose-dependent manner, the minimum concentration having a significant effect being 100 ng/ml (6 nM) (Fig. 6). Macrophage activation is mediated by translocation of the nuclear factor NF{kappa}B to the nucleus. As shown in Fig. 7, the p65 subunit of NF{kappa}B visualized by immunohistochemistry is indeed located to the cytoplasm in untreated macrophages and to the nucleus after exposure to TNF{alpha} or fMLP. The presence of activated NF{kappa}B in the nucleus was confirmed by ELISA. Adding 10 to 500 ng/ml of PAP I to the culture medium of macrophages did not alter the cytoplasmic location of NF{kappa}B. However, concentrations above 100 ng/ml prevented NF{kappa}B translocation in macrophages stimulated by TNF{alpha} or fMLP, as evidenced by immunohistochemistry and ELISA (Fig. 7). It was concluded that inhibition by PAP I of macrophage activation by TNF{alpha} or fMLP occurs upstream from NF{kappa}B.



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  		FIG. 6.
PAP I prevents macrophages activation. Alveolar macrophages obtained by bronchoalveolar lavage were incubated with TNF{alpha} (50 ng/ml) together with increasing doses of purified PAP I (from 0 to 500 ng/ml). TNF{alpha} and IL-6 mRNA expression was measured by semiquantitative RT-PCR using specific primers. {beta}-Actin mRNA was used as a housekeeping control. RT-PCR products were resolved in 1.8% agarose gel electrophoresis and stained with ethidium bromide. Experiments were repeated three times and intensity of bands was measured and expressed as relative intensities of untreated macrophages. Results are expressed as mean values ± S.E.

 



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  		FIG. 7.
PAP I inhibits NF{kappa}B translocation in macrophages. A, macrophages were treated with TNF{alpha} (50 ng/ml) alone or in association with purified PAP I (100 ng/ml) for 30 min. p65 was stained with a specific antibody to visualize its cellular localization. B, macrophages were treated with TNF{alpha} (50 ng/ml) alone or in association with purified PAP I (100 ng/ml) for 30 min and active NF{kappa}B was measured using an oligonucleotide-based specific ELISA (see "Materials and Methods"). Experiments were performed in triplicate and repeated once. Results are expressed as mean values ± S.E.

 
Macrophages are not the only cells that can generate proinflammatory factors in the pancreas during the early stages of pancreatitis. Acinar cells also release cytokines such as TNF{alpha}, through NF{kappa}B activation (34-36). We therefore investigated whether the anti-inflammatory properties of PAP I on macrophages described above would extend to acinar cells. We used the acinar cell line AR42J in the same experimental set-up as for macrophages and obtained comparable results (Fig. 8). In acinar cells, the nuclear translocation of p65 in response to TNF{alpha} was also completely inhibited by PAP I. The prominent anti-inflammatory activity of PAP I observed in our experiments was unexpected. Because PAP I expression is abolished in the absence of p8, increased inflammation observed in p8 knock-out mice is most likely due to the associated impairment in PAP I expression.



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  		FIG. 8.
PAP I inhibits NF{kappa}B translocation in AR42J pancreatic acinar cells. AR42J cells were treated with TNF{alpha} (50 ng/ml) alone or in association with purified PAP I (100 ng/ml) for 30 min. p65 was stained with a specific antibody to visualize its cellular localization. Experiments were repeated two times with similar results.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Induction of experimental acute pancreatitis by repeated intraperitoneal injections of a secretagogue resulted within 6 h in strong activation of p8 expression in the pancreas (Ref. 6 and Fig. 4). Such activation might be of biological significance because we know that the induction of acute pancreatitis triggers important modifications in the pattern of gene expression in the pancreas that aim at protecting parenchyma against injury (2). As p8 was recently defined as a transcription co-factor (12) whose expression is induced at the very beginning of the acute phase of pancreatitis, we speculated that it could play a key role in the establishment of the defense-related gene expression pattern. We therefore constructed a p8-/- mouse strain as a tool to investigate the function of the p8 gene during acute pancreatitis. p8-deficient mice were fertile, developed normally, and produce litters of normal size with a normal sex distribution among pups over 3 generations. The oldest p8-/- homozygotes are aged 18 months and do not exhibit any phenotypic abnormality. Apparently, the complete absence of p8 does not affect their viability, fertility, or health. These findings imply either that p8 is not involved in crucial developmental processes or that other genes are functionally redundant.

We chose for functional studies on p8 a model of secretagogue-induced acute pancreatitis because many of the local and systemic alterations typical of the human disease are observed in this non-invasive animal model, including hyperamylasemia, significant pancreatic tissue necrosis, a systemic inflammatory response, and associated lung injury. Also, like in human pancreatitis, significant intrapancreatic trypsinogen activation precedes acinar cell injury. We observed that the absence of functional p8 altered the course of acute pancreatitis in that knock-out animals developed a more severe disease phenotype with serum levels of amylase and lipase higher than those observed in p8+/+ mice. Scoring histologic lesions showed significantly more edema, vacuolization, necrosis, and inflammation in p8 knock-out animals than in wild-type (Table I). Abundant neutrophil infiltration in the pancreas of p8 knock-out mice was evident by increased pancreatic myeloperoxidase activity in the pancreas (Fig. 2). These data indicate that p8 is directly involved in pancreatic inflammation during pancreatitis. As the absence of p8 worsens the evolution of acute pancreatitis in mice, the next step of the study was to define if p8 was involved in early onset of the disease by preventing premature zymogen activation and/or by inhibiting protease activity, or if it was involved in the pancreatic gene response to acute pancreatitis that provides some protection to the cells. As intensity of intrapancreatic enzyme activation, monitored immediately after cerulein treatment, was similar in p8-deficient and control animals (Fig. 3), p8 is not involved in the initial intra-acinar events associated with premature protease activation and does not affect the susceptibility of acinar cells toward supramaximal secretagogue stimulation. We investigated the alternative possibility by comparing the expression in p8-/- and p8+/+ mice of genes known to be induced during pancreatitis and supposed to be beneficial. One of them, PAP I, which is strongly overexpressed during pancreatitis in p8+/+ animals, showed modest expression changes in p8-/- mice (Fig. 4) suggesting that its expression is strongly dependent on p8 expression. Hence, the PAP I gene could be regarded as a target for p8 and, as such, might mediate the beneficial effects resulting in wild-type animals from p8 induction during acute pancreatitis.

PAP I is a 16-kDa secretory protein synthesized by pancreatic acinar cells during the acute phase of pancreatitis but not detectable in the healthy pancreas. This protein was first described in rat pancreatic juice collected after experimental pancreatitis, where it can account for more than 5% of total protein (31). The PAP I gene was independently identified and cloned by different laboratories and named hepatocarcinoma-intestine-pancreas, p23, or Reg2 (37-39). PAP I, the two other isoforms PAP II and III, and the pancreatic stone protein (PAP/reg) constitute a family of proteins that belongs to the C-type lectin family (40). Besides of overexpression during acute pancreatitis, PAP I is strongly up-regulated in acinar cells (31) by proinflammatory mediators, such as cytokines IL-6 (26, 41), TNF{alpha} (26), interferon {gamma} (25) as well as by free radicals (42), leptin (43), CNTF (44), and the anti-inflammatory cytokine IL-22 (45). PAP I was detected in several human cancer tissues such as liver (37), colon (46), and stomach (47) and in developing and regenerating motor neurons (39, 44). However, PAP I is also expressed in healthy tissues such as the small intestine (48-51), pituitary (52, 53), and uterus (54). Several biological functions have been proposed for PAP I, including induction of bacterial aggregation in vitro (55), stimulation of cell proliferation (39, 56), and inhibition of apoptosis (42, 44, 57, 58). It was also shown to prevent fMLP-induced vasoconstriction and edema formation in isolated rabbit lung (59) suggesting a putative anti-inflammatory function. Yet, its function during the course of pancreatitis remained elusive so far.

As a first step to obtain information on PAP I function during pancreatitis, we used a model of necrohemorrhagic pancreatitis and, because PAP I-/- animals are not available yet, we injected animals with sufficient anti-PAP I antibodies to capture circulating PAP I into an antigen-antibody complex. In animals in which PAP I had been blocked, pancreatitis was associated with increased inflammation as evidenced by more abundant necrosis, increased pancreatic myeloperoxidase levels, and more prominent neutrophil infiltration (Fig. 5). These results suggest that PAP I has anti-inflammatory properties. To confirm these findings, we evaluated the effects of PAP I on the activation of macrophages that, together with neutrophils, are critical in the development of acute inflammation during acute pancreatitis. In rat macrophages in vitro, PAP I concentrations as low as 100 ng/ml could almost completely prevent the induction of TNF{alpha} and IL-6 mRNA expression by TNF{alpha} or fMLP. These inductions are mediated by NF{kappa}B activation. We found that PAP I actually prevents NF{kappa}B translocation/activation in response to TNF{alpha} or fMLP (Fig. 7), indicating that PAP I inhibits macrophage activation upstream from NF{kappa}B in the activation pathway. It is noteworthy that PAP I also prevented NF{kappa}B activation in response to TNF{alpha} in AR42J cells (Fig. 8), which are derived from pancreatic acinar cells, and in HT29 cells, derived from colon cancer cells (data not shown), indicating that the anti-inflammatory effect of PAP I is not restricted to macrophages. Altogether, our results suggest that one of the functions of the p8 protein, which is overexpressed in the pancreas in the early stages of pancreatitis, is to control the development of inflammation during disease development. That control is mediated through the initiation of PAP I gene expression and the PAP I protein inhibits macrophage activating signals upstream from NF{kappa}B activation. Whether PAP I plays that role in the pancreas after it is actively secreted or after passive release consecutive to acinar cell lysis remains to be elucidated.

Acute pancreatitis is characterized by the occurrence of acinar lesions followed by severe inflammation of the parenchyma. The latter is initiated by macrophages and neutrophils, acinar cells being themselves associated with an increased release of proinflammatory factors leading to disease progression and sometimes death. Therefore, the severity of acute pancreatitis should depend in part on the intensity of pancreatic inflammation. Indeed, anti-inflammatory factors such as IL-10 improve acute pancreatitis (60-65), whereas the pro-inflammatory cytokine TNF{alpha} worsens it (66-70). In addition, preventing NF{kappa}B activation strongly improves pancreatitis development (71-76). It is interesting to note that IL-10 (65, 77), TNF{alpha} (35, 36), as well as PAP I (31, 78) are expressed by pancreatic acinar cells during the acute phase of pancreatitis, supporting the idea that acinar cells challenged by pancreatitis activate a defense program that includes PAP I expression in its anti-inflammatory mechanisms. The recent demonstration that PAP I is also a target of the IL-10-related anti-inflammatory cytokine IL-22 in pancreatic acinar cells (45) suggests that PAP I is directly involved in anti-inflammatory processes.

The present results support the concept that p8, as transcription co-factor, is very rapidly overexpressed upon induction of acute pancreatitis (6) to orchestrate the defense of the acinar cell by inducing the expression of anti-inflammatory genes. PAP I is certainly one of those, but other defense genes may also be under the control of p8 and a detailed characterization of the p8 pathway might help to develop more rational therapeutic strategies for the treatment of acute pancreatitis. On the other hand, the evidence presented here for an anti-inflammatory function of PAP I may lead to the development of new treatments of other inflammatory diseases than pancreatitis.


   FOOTNOTES
 
* This work was supported by grants from Association pour le Recherche sur le Cancer and Ligue Contre le Cancer (to J. L. I.) and Fonds de Investigaciones Sanitories PI020286 (to D. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

¶¶ To whom correspondence may be addressed: Dept. of Experimental Pathology, IIBB-CSIC, c/Rossello 161, 7°, 08036 Barcelona, Spain. Tel.: 34-93-3638307; Fax: 34-93-3638301; E-mail: dcabam{at}iibb.csic.es.

|||| To whom correspondence may be addressed: Centre de Recherche INSERM, EMI 0116, 163 Avenue de Luminy, BP172, F-13009 Marseille, France. Tel.: 33-491-827533; Fax: 33-491-826083; E-mail: iovanna{at}marseille.inserm.fr.

1 The abbreviations used are: PAP, pancreatitis-associated protein; MOPS, 4-morpholinepropanesulfonic acid; TAP, trypsinogen activation peptide; ELISA, enzyme-linked immunosorbent assay; RT, reverse transcriptase; TNF{alpha}, tumor necrosis factor {alpha}; fMLP, formylmethionylleucylphenylalanine; IL, interleukin. Back


   ACKNOWLEDGMENTS
 
We thank R. Grimaud and F. Roche for excellent technical assistance.



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