Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappa B activation

Bing Han and Craig D. Logsdon

Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Inflammatory mediators are involved in the early phase of acute pancreatitis, but the cellular mechanisms responsible for their generation within pancreatic cells are unknown. We examined the role of nuclear factor-kappa B (NF-kappa B) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokine expression in pancreatic acinar cells in vitro. Supraphysiological, but not physiological, concentrations of CCK-8 increased inhibitory kappa B (Ikappa B-alpha ) degradation, NF-kappa B activation, and mob-1 gene expression in isolated pancreatic acinar cells. CCK-8-induced Ikappa B-alpha degradation was maximal within 1 h. Expression of mob-1 was maximal within 2 h. Neither bombesin nor carbachol significantly increased mob-1 mRNA or induced Ikappa B-alpha degradation. Thus the concentration, time, and secretagogue dependence of mob-1 gene expression and Ikappa B-alpha degradation were similar. Inhibition of NF-kappa B with pharmacological agents or by adenovirus-mediated expression of the inhibitory protein Ikappa B-alpha also inhibited mob-1 gene expression. These data indicate that the NF-kappa B signaling pathway is required for CCK-8-mediated induction of mob-1 chemokine expression in pancreatic acinar cells. This supports the hypothesis that NF-kappa B signaling is of central importance in the initiation of acute pancreatitis.

pancreas; pancreatitis; cytokines; adenovirus


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CELLULAR MECHANISMS that initiate the development of acute pancreatitis are uncertain. It has been very difficult to investigate the cellular mechanisms involved in acute pancreatitis in humans because of its multifaceted clinical manifestations and the difficulty of early diagnosis. A number of animal models of experimental pancreatitis have been developed to investigate the biochemical, morphological, and pathophysiological changes that accompany this disease (15, 17, 41). Although progress in understanding the pathophysiology of acute pancreatitis has been achieved using these models, the cellular mechanisms and factors that initiate the disease remain unclear, partly because cellular mechanisms are obscured by the complications of events outside the pancreas in vivo. In vitro models offer advantages for mechanistic studies.

The development of acute pancreatitis is often considered as consisting of two phases: an early phase involving pancreatic acinar cell damage and a secondary phase involving an inflammatory response. Mechanisms that lead to acinar cell damage observed during acute pancreatitis have been well studied and are thought to include the intracellular activation of digestive enzymes (41). In contrast, the cellular mechanisms responsible for the initiation of the inflammatory response are less well understood. Once initiated, the inflammatory response may spread to other organs, particularly the lungs, and is the primary cause of mortality associated with the disease (42). Evidence is accumulating that an important event in the evolution of the inflammatory phase of acute pancreatitis is release of endogenous inflammatory mediators, such as cytokines and chemokines (23). The serum levels of interleukin-6, interleukin-8, tumor necrosis factor-alpha , and platelet-activating factor are known to increase during acute pancreatitis and are well correlated with the severity of the disease (18, 22, 32, 45). The source of these cytokines has not been clear, and it is likely that many of these inflammatory regulators are released from infiltrating immune cells as a late response (12). However, it has recently been suggested that cytokines may originate within the pancreas (19, 33). We found that acinar cells themselves express chemokines, a class of cytokines known for their chemoattractive functions, early in the course of experimental acute pancreatitis (16).

Chemokines attract and activate inflammatory cells (29, 38). Therefore, chemokines are ideally suited to play a critical role in the earliest initiating events in pancreatitis. We found a dramatic increase in the expression of mob-1, a member of the alpha -chemokine (C-X-C) family, and mcp-1, a member of the beta -chemokine (C-C) family, in pancreatic acinar cells early in the course of experimental acute pancreatitis (16). These observations suggested that general cellular mechanisms leading to the expression of a variety of chemokine genes were likely activated in an early phase of acute pancreatitis. However, little is known concerning the cellular mechanisms involved in chemokine gene expression in pancreatic acinar cells.

In the present study we investigated the cellular mechanisms involved in cholecystokinin octapeptide (CCK-8)-induced chemokine gene expression in pancreatic acinar cells in the absence of the confounding factors existing in vivo by utilizing an in vitro preparation of isolated pancreatic acini. Expression of chemokines and other cytokines is widely regulated by the transcription factor nuclear factor-kappa B (NF-kappa B) (4). Recently, it has been reported that cerulein hyperstimulation activates NF-kappa B in vivo in a manner that correlates with the development of pancreatitis (20, 43). In the present study we analyzed the relationship between the activation of NF-kappa B and the expression of mob-1 in vitro. We also utilized several independent methods to inhibit NF-kappa B activation, including the overexpression of the inhibitory protein Ikappa B-alpha , using adenovirus-based gene transfer. Our results indicate that mob-1 chemokine gene expression is an early and specific response to CCK-8 hyperstimulation and that NF-kappa B activation is required for the regulation of this chemokine gene in pancreatic acinar cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Chromatographically purified collagenase was purchased from Worthington Biochemical (Freehold, NJ). Soybean trypsin inhibitor (SBTI), carbachol, bombesin, beta -mercaptoethanol, phenylmethylsulfonyl fluoride, sodium orthovanadate, HEPES, glutamine, and pyrrolidine dithiocarbamate (PDTC) were obtained from Sigma Chemical (St. Louis, MO). CCK-8 was purchased from Research Plus (Bayonne, NJ). Enhanced chemiluminescence detection reagents, [gamma -32P]ATP, d-[alpha -32P]CTP, and goat anti-rabbit IgG horseradish peroxidase conjugate were from Amersham (Arlington Heights, IL). The electrophoretic mobility shift assay (EMSA) systems kit was obtained from Promega (Madison, WI). The rabbit polyclonal antibodies to Ikappa B-alpha and NF-kappa B p65, p50, and c-Rel subunits were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Eagle's minimum essential amino acids, guanidine thiocyanate, and agarose were purchased from GIBCO BRL Life Technologies (Gaithersburg, MD).

Pancreatic acini isolation and treatments. Pancreatic acini were prepared by a modification of previous methods (27). Briefly, pancreata from male Wistar rats were quickly (<10 min) injected with collagenase (100 U/ml) in a Krebs-Henseleit bicarbonate medium to which SBTI (0.1 mg/ml) and minimal essential amino acids had been added. Injected pancreata were then minced into three to five pieces and incubated at 37°C for 45-50 min with shaking (120 cycles/min). Acini were then dispersed using an electric pipette aid gently triturating the pancreas through polypropylene pipettes with decreasing orifice (3.0, 2.4, and 1.2 mm). After filtration through a 150-µm nylon mesh, acini were purified by centrifugation at 10 g for 5 min in a solution containing 4% BSA and resuspended in HEPES-buffered Ringer solution (pH 7.5) supplemented with 0.2% glucose, Eagle's minimum essential amino acids, 2 mM glutamine, SBTI (0.1 mg/ml), and 0.5% BSA. The dispersed acini were aliquoted and treated with various hormones and pharmacological agents at indicated concentrations in HEPES-buffered Ringer solution for specified times in 100-mm tissue culture dishes. All treatments and incubations were conducted in a cell culture incubator at 37°C in a humidified atmosphere.

Adenoviral infection. An adenoviral vector with a cytomegalovirus promoter driving expression of a full-length Ikappa B-alpha cDNA modified by the addition of a nuclear translocation sequence (AdIkappa B-alpha ) was a kind gift of Dr. J. Anrather (Beth Israel Deaconess Medical Center, Boston, MA). A previously described adenovirus bearing the bacterial beta -galactosidase gene (AdLacZ) (3) obtained from Dr. B.J. Roessler (University of Michigan, Ann Arbor, MI) was utilized as a control. Acini prepared as described above were infected with this adenovirus, as described previously (31). Adenovirus titers ranged from 107 to 109 plaque-forming units/mg acinar protein (multiplicity of infection = 10-1,000) as indicated. Acini were then incubated for 8 or 20 h before the addition of CCK-8 for indicated times.

Isolation of RNA and analysis of mob-1 mRNA expression. Total RNA was isolated by a modified acid guanidinium-thiocyanate-phenol-chloroform extraction (8). Briefly, after incubation with or without indicated agonists, pancreatic acini pellets were homogenized in 3 ml of 4 M guanidine thiocyanate buffer containing 8% beta -mercaptoethanol with use of a Polytron (Brinkman Instruments, Westbury, NY). Phenol-chloroform extraction was performed immediately, and the aqueous phase was precipitated with isopropanol. The pellets were dissolved into 4 M guanidine thiocyanate buffer, and the RNA was reextracted with phenol-chloroform and precipitated with isopropanol at -20°C overnight. RNA was quantitated spectrophotometrically, and 25 µg from each sample were electrophoresed in 1% agarose and 2.2 M formaldehyde gels in 1× MOPS buffer and transferred to a Hybond N+ membrane (Amersham). Mob-1 mRNA was detected using a full-length 1.2-kb cDNA of rat mob-1 (gift from Dr. P. Liang, Vanderbilt University, Nashville, TN). Membranes were hybridized at high stringency using QuickHyb solution (Stratagene, La Jolla, CA) with the d-[alpha -32P]CTP-labeled mob-1 probe at 68°C for 2 h. After hybridization the membranes were exposed to a B-1 phosphor-imaging screen and visualized using the GS-250 Molecular Imaging System (Bio-Rad Laboratories, Richmond, CA). Ethidium bromide staining of total RNA was used as a loading control.

Ikappa B-alpha immunoblot analysis. Dispersed acini were treated as described in Figs. 1-8. The treatments were terminated by washing the acini with ice-cold PBS containing 1 mM Na3VO4. The pellets were lysed by sonication for 5 s in a solution containing 50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail containing 10 µg/ml each of aprotinin, leupeptin, and pepstatin. The samples were then set on ice for 15 min and centrifuged at 14,000 g for 15 min at 4°C. The supernatant was removed as whole cell lysate and assayed for protein by protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (20 µg) were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Ikappa B immunoblot analysis was performed as described previously (21) and visualized with enhanced chemiluminescence reagent on film or screen.

Preparations of nuclear extracts. Nuclear extracts were prepared using a modified version of the method of Maire et al. (28). After the different treatments described in the experimental design, pancreatic acini were collected by brief centrifugation and washed with ice-cold PBS containing 1 mM EDTA. The pellets were then resuspended in 0.5 ml of homogenization buffer containing 10 mM HEPES (pH 7.9), 2 M sucrose, 10% glycerol (vol/vol), 25 mM KCl, 150 mM spermine, 500 mM spermidine, and 2 mM EDTA, to which 1 mM dithiothreitol (DTT) and the protease inhibitor cocktail described above were added before use. Cells were homogenized with a motor-driven pestle for 5-10 strokes on ice. Nuclei were collected by centrifugation at 62,000 g for 30 min at 4°C and washed with 1 ml of PBS containing 1 mM EDTA, then centrifuged at 14,000 g for 5 min at 4°C. The nuclei were resuspended in an appropriate volume (~100 µl) of ice-cold high-salt buffer containing 10 mM HEPES (pH 7.9), 10% glycerol (vol/vol), 0.42 M NaCl, 100 mM KCl, 3 mM MgCl2, and 0.1 mM EDTA, to which DTT and the protease inhibitor cocktail described above were added. The nuclear suspension was incubated on ice for 15-30 min with intermittent mixing and centrifuged at 14,000 g for 5 min at 4°C. Protein concentration in the nuclear extract was determined using the Bio-Rad protein assay reagent. The same procedure was also utilized for preparation of nuclear extract from untreated pancreatic tissue.

EMSA. Aliquots of nuclear extract with equal amounts of protein (6-12 µg) were utilized in 10-µl reactions in a buffer containing 10 mM HEPES (pH 7.9), 10% glycerol (vol/vol), 1 mM DTT, 1 µg of poly(dI-dC), and 5 µg of nuclease-free BSA. The binding reaction was started by addition of 10,000 cpm of the 22-bp oligonucleotide 5'-AGT TGA <UNL>GGG GAC TTT CC</UNL>C AGG C-3' containing the NF-kappa B consensus sequence (underlined) or the 22-bp oligonucleotide 5'-TGT CGA ATG CAA ATC ACT AGA A-3' containing the OCT1 sequence (Promega) that had been labeled with [gamma -32P]ATP (10 mCi/mmol) by T4 polynucleotide kinase. The reaction was allowed to proceed for 30 min at room temperature. For cold competition experiments, unlabeled NF-kappa B oligonucleotide or OCT1 oligonucleotide as a nonspecific competitor (300×) was added to the binding reaction 5 min before the addition of the radiolabeled probe. For antibody supershift assays, 2 µl of specific antibodies to NF-kappa B protein subunits p65, p50, and c-Rel were incubated with nuclear extracts for 1 h at room temperature before the addition of labeled probe. All reaction mixtures were subjected to PAGE on 4.5% gel in 0.5× Tris base-EDTA-boric acid buffer at 200 V. Gels were dried and directly exposed to B-1 phosphor-imaging screen, as indicated previously for Northern blots.

NF-kappa B immunoblotting analysis. Nuclear extracts were prepared as described above. Aliquots with equal amounts of nuclear protein from treated acinar cells or intact pancreata were subjected to SDS-PAGE on a 10% gel and transferred onto a nitrocellulose membrane. The NF-kappa B protein was detected by a specific antibody against the p65 subunit of NF-kappa B.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CCK-8 activates NF-kappa B in pancreatic acini in vitro. On the basis of the known role of NF-kappa B in inducing the expression of a wide variety of inflammatory mediators, we hypothesized that NF-kappa B might mediate the expression of the chemokine mob-1 stimulated by supraphysiological concentrations of CCK-8 in the pancreas. Activation of NF-kappa B involves the phosphorylation and degradation of the inhibitory protein Ikappa B-alpha and the nuclear translocation and DNA binding of NF-kappa B. To determine the ability of CCK-8 to activate NF-kappa B in pancreatic acini in vitro, we examined the effects of CCK-8 on nuclear translocation of the NF-kappa B p65 subunit and on NF-kappa B DNA binding. Only low levels of NF-kappa B consensus site binding were observed in EMSAs with use of nuclear extracts from untreated pancreatic tissue (Fig. 1A, lane 2) and control acini (Fig. 1A, lane 3), indicating that the procedure for isolation of acini did not activate this signaling pathway. Likewise, only low levels of p65 NF-kappa B were detected in Western blots of nuclear protein extracts from control acini (Fig. 2). Treatment with CCK-8 (100 nM) for 1 h increased the amount of p65 subunit detected in the nuclear fraction by 5.3 ± 0.6-fold (n = 6; Fig. 2) and led to a marked increase (4.9 ± 0.9-fold, n = 6) in NF-kappa B consensus site binding (Fig. 1A, lane 4). These data indicate that treatment of isolated pancreatic acini with supraphysiological concentrations of CCK-8 activates NF-kappa B, as has recently been reported by others (20, 43).


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Fig. 1.   Cholecystokinin octapeptide (CCK-8) induces nuclear factor-kappa B (NF-kappa B) consensus site binding in electrophoretic mobility shift assays (EMSAs). A: effects of CCK-8 on NF-kappa B consensus site binding. HeLa cell nuclear extract was run as a positive control (lane 1). Nuclear extracts from untreated pancreas (lane 2) and isolated pancreatic acini that were treated without (lane 3) or with 100 nM CCK-8 (lanes 4-13) for 1 h were subjected to EMSA with equal amount of nuclear protein per sample. NF-kappa B-DNA complexes are indicated by open arrowhead at left. For supershift analysis (filled arrowhead), nuclear extracts were incubated for 1 h at room temperature in presence of anti-p65, p50, or c-Rel NF-kappa B subunit antibodies before addition of labeled kappa B oligonucleotide (lanes 5-10). In competition experiments, 300-fold excess of unlabeled OCT1 (lanes 11 and 12) or kappa B (lane 13) oligonucleotide was added to reaction before addition of labeled kappa B oligonucleotide. B: effects of CCK-8 treatment on OCT1 consensus site binding. Aliquots of nuclear extracts utilized for lanes 3 and 4 in A were subjected to EMSA for OCT1 (lanes 2 and 3 in B). HeLa cell nuclear extract is included as a positive control (lane 1). OCT1-DNA complexes are indicated by open arrowhead at right. Data are representative of 3-5 independent experiments.



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Fig. 2.   CCK-8 stimulates nuclear translocation of NF-kappa B p65 subunit in pancreatic acini in vitro. Dispersed rat pancreatic acini were incubated for 1 h in absence or presence of CCK-8. Top: nuclear extracts were prepared, and samples (20 µg protein) were subjected to SDS-PAGE and probed with a specific anti-p65 subunit antibody. Bottom: translocated p65 levels were quantified by Bio-Rad Multi-Analyst and presented as fold of control (mean ± SD) for 2-6 experiments.

To characterize the proteins associated with binding to the NF-kappa B consensus site, supershift analysis was carried out using antibodies specific for NF-kappa B subunits. Preincubation of nuclear extracts with antibodies specific for p65 or p50, but not c-Rel, NF-kappa B subunits resulted in the formation of a supershift band (Fig. 1A, lanes 5-10). Thus p65/p50 and p50/p50, but not NF-kappa B complexes involving c-Rel are activated in pancreatic acinar cells by CCK-8. Specificity of the NF-kappa B band was verified by incubation with excesses of unlabeled kappa B oligonucleotide or an unrelated oligonucleotide containing an OCT1 consensus sequence. Excess of unlabeled kappa B oligonucleotide (Fig. 1A, lane 13) but not of OCT1 oligonucleotide (Fig. 1A, lanes 11 and 12) inhibited the appearance of the NF-kappa B consensus site binding band. As a separate control, the effects of CCK-8 on the nuclear transcription factor OCT1 were examined in nuclear extracts from the acinar cells. Treatment of acini with a supraphysiological concentration of CCK-8 did not result in regulation of OCT1 (Fig. 1B). Thus supraphysiological concentrations of CCK-8 specifically activate NF-kappa B in pancreatic acinar cells.

CCK-8 induces chemokine gene expression and Ikappa B-alpha degradation in a dose- and time-dependent manner in dispersed rat pancreatic acini. Similar to what is observed with other NF-kappa B activators, CCK-8 activation of NF-kappa B was associated with a decrease in cytoplasmic levels of the inhibitory component Ikappa B-alpha (Fig. 3). To define the concentration and time dependence of CCK-8-mediated NF-kappa B activation and mob-1 gene expression, we analyzed samples of treated acini for Ikappa B-alpha protein levels and mob-1 mRNA. There was minimal Ikappa B-alpha degradation (Fig. 3A) and mob-1 gene expression (Fig. 3B) in freshly isolated pancreatic acini (lane 2). This was similar to observations in undisturbed pancreatic tissue (Fig. 3, A and B, lane 1). After treatment with CCK-8 (100 nM), Ikappa B-alpha was degraded, as evidenced by loss of the Ikappa B-alpha band in Western blots (16 ± 6% of control acini, n = 9), and mob-1 gene expression was induced, as evidenced by an increased amount of mob-1 mRNA in Northern blot analysis (4.5 ± 0.6-fold, n = 9).


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Fig. 3.   CCK-8 causes a concentration-dependent degradation of Ikappa B-alpha and increase of mob-1 gene expression. Pancreas from a male Wistar rat was divided into two parts: one part was processed immediately (lane 1); other part was used for isolation of pancreatic acini (lanes 2-8). Isolated acini were processed immediately (lane 2) or incubated with different concentrations of CCK-8 (lanes 3-8) for 1 h, then samples were analyzed for Ikappa B-alpha levels by Western blotting (A) or for 2 h, then total RNA was utilized for detection of mob-1 mRNA levels by Northern blotting (B). C: ethidium bromide staining of total RNA as a loading control. Data are representative of 3 experiments.

The degradation of Ikappa B-alpha and the induction of mob-1 mRNA by CCK-8 were concentration and time dependent. Ikappa B-alpha degradation (Fig. 3A) and mob-1 mRNA induction (Fig. 3B) were markedly induced by supraphysiological concentrations (>1 nM) but not by physiological concentrations of CCK-8. CCK-8 (100 nM) induced noticeable Ikappa B-alpha degradation within 10 min, maximal effects were observed within 1 h, and no recovery was observed for >4 h (Fig. 4A). These effects on Ikappa B-alpha slightly preceded the effects of CCK-8 on mob-1 gene expression. Mob-1 mRNA levels were significantly increased after treatment of the acini with 100 nM CCK-8 for 30 min, were maximal after 2 h, and remained high for >= 4 h (Fig. 4B). Thus the concentration and time dependence of CCK-8-induced mob-1 gene expression and Ikappa B-alpha degradation were well correlated.


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Fig. 4.   CCK-8 induces Ikappa B-alpha degradation and mob-1 gene expression in a time-dependent manner in pancreatic acini in vitro. Protein (A) and RNA (B) were prepared from isolated pancreatic acini incubated in presence of 100 nM CCK-8 for indicated times. Whole cell lysates (20 µg protein) were run on an SDS gel and probed with anti-Ikappa B-alpha antibody. RNA extracts were analyzed by Northern blotting with cDNA specific for mob-1. C: ethidium bromide staining of total RNA as a loading control. Blots are representative of 4 experiments.

Bombesin and carbachol do not cause Ikappa B-alpha degradation or mob-1 expression. To determine the specificity of the ability of CCK-8 to degrade Ikappa B-alpha and induce mob-1 gene expression, we tested the effects of other secretagogues (Fig. 5). After treatment of dispersed pancreatic acini with supraphysiological concentrations of bombesin (100 nM) or carbachol (1 mM) for 1 h in vitro, most of Ikappa B-alpha protein [92.5 ± 14.4% (n = 4) and 87.5 ± 16.5% (n = 4) of control, respectively] remained undegraded. There was also little or no increase in mob-1 mRNA levels induced by treatment with bombesin or carbachol for 2 h. These results support the hypothesis that mob-1 gene expression is associated with pancreatitis and not simply secretagogue hyperstimulation.


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Fig. 5.   Neither bombesin nor carbachol stimulates Ikappa B-alpha degradation or mob-1 chemokine expression in pancreatic acini in vitro. Pancreatic acini were incubated in absence of agonist (control, lane 1) or in presence of a supramaximal concentration of CCK-8 (100 nM, lane 2), bombesin (100 nM, lanes 3 and 4), or carbachol (1 mM, lanes 5 and 6). To detect Ikappa B-alpha degradation, samples (20 µg protein) were prepared after 1 h of incubation and analyzed in Western blots with an anti-Ikappa B-alpha antibody (A). For detection of mob-1 gene expression, RNA was extracted after 2 h of treatment and Northern blotting (25 µg RNA) was performed with a cDNA for mob-1 (B). C: ethidium bromide staining of total RNA as a loading control. Images are representative of 3-4 experiments.

PDTC pretreatment inhibits NF-kappa B activation and blocks induction of mob-1 chemokine expression in isolated pancreatic acini. An inhibitor for NF-kappa B activation, PDTC, was utilized to explore the relationship between CCK-8-induced NF-kappa B activation and mob-1 gene expression. PDTC pretreatment prevented the degradation of Ikappa B-alpha induced by supraphysiological concentrations of CCK-8, as evidenced by Western blotting (Fig. 6A), and the increase of mob-1 mRNA, as evidenced by Northern blot analysis of total RNA isolated from acini of the same experiment (Fig. 6C). PDTC pretreatment also blocked NF-kappa B p65 subunit translocation to the nucleus (data not shown) and the appearance of a NF-kappa B consensus site binding band (Fig. 6B). Thus PDTC blocked NF-kappa B activation and mob-1 gene expression stimulated by supraphysiological concentrations of CCK-8, suggesting a role for NF-kappa B in CCK-8-mediated mob-1 gene expression.


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Fig. 6.   Pyrollidine dithiocarbamate (PDTC) pretreatment blocks effects of a supraphysiological concentration of CCK-8 on Ikappa B-alpha degradation (A), NF-kappa B activation (B), and mob-1 chemokine expression (C) in pancreatic acini in vitro. Pancreatic acini from a male Wistar rat were preincubated without (lanes 1 and 2) or with (lane 3) 10 mM PDTC for 1 h, then incubated without (lane 1) or with 10 nM CCK-8 (lanes 2 and 3). A: to detect Ikappa B-alpha degradation, samples (20 µg protein) were analyzed in Western blots with an anti-Ikappa B-alpha antibody. PDTC blocked CCK-8-induced degradation of Ikappa B-alpha . B: PDTC blocked ability of CCK-8 to induce NF-kappa B consensus site binding. Nuclear extracts (10 µg protein/sample) were subjected to EMSA. C: for detection of mob-1 gene expression, RNA was extracted and Northern blotting (25 µg RNA) was performed with a cDNA for mob-1. Blots are representative of 3-4 experiments.

Adenovirus-mediated expression of Ikappa B-alpha inhibits CCK-8-induced mob-1 gene expression. To verify the role of NF-kappa B in mob-1 gene expression, a more specific approach using adenovirus-mediated gene transfer of the inhibitory Ikappa B-alpha protein was utilized. In this approach, acini were infected with an adenovirus bearing an Ikappa B-alpha gene modified to localize to the cell nucleus (44). In previous studies, adenovirus-mediated gene transfer to isolated acinar cells was found to be highly efficient and titer dependent (31, 34). To analyze the effects of adenoviral infection on Ikappa B-alpha levels, we determined the effects of infection with various titers of a control adenovirus bearing the beta -galactosidase gene and those of infection with adenovirus bearing the Ikappa B-alpha gene (Fig. 7). Infection with the control virus led to a decrease in basal levels of acinar Ikappa B-alpha , and these levels were further decreased by treatment of acini with CCK-8 (Fig. 7A). In contrast, infection with adenovirus bearing Ikappa B-alpha led to a titer-dependent increase in the levels of Ikappa B-alpha . At lower titers, treatment with supraphysiological concentrations of CCK-8 continued to cause a significant decrease in Ikappa B-alpha levels. However, at higher titers of adenovirus bearing Ikappa B-alpha , no effect of CCK-8 on Ikappa B-alpha levels was noted (Fig. 7B). Thus, after adenoviral delivery of the modified Ikappa B-alpha gene, CCK-8 was no longer able to cause a major decrease in whole cell Ikappa B-alpha levels.


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Fig. 7.   Adenoviral infection leads to titer-dependent effects on Ikappa B-alpha levels. Dispersed pancreatic acini were infected with recombinant adenovirus bearing a modified Ikappa B-alpha gene (AdIkappa B-alpha ) or a control virus bearing beta -galactosidase (AdLacZ) at indicated titers (plaque-forming units/mg acinar protein) for 20 h. Acini were subsequently treated with or without CCK-8 (100 nM) for 1 h, and Ikappa B-alpha protein levels were determined in Western blots. Blots are representative of 3 experiments.

Throughout this study, mob-1 mRNA levels were inversely related to the levels of Ikappa B-alpha . Thus it was not surprising that acini infected with control adenovirus that showed a slight reduction in the basal levels of Ikappa B-alpha protein (Fig. 8A) also showed a corresponding increase in the basal levels of mob-1 mRNA (Fig. 8B). CCK-8 treatment of uninfected acini or acini infected with control adenovirus led to a much larger decrease in Ikappa B-alpha and a correspondingly large increase in mob-1 mRNA. In contrast, in acini infected with adenovirus bearing Ikappa B-alpha , basal levels of Ikappa B-alpha were high and levels of mob-1 mRNA were low. Furthermore, in acini overexpressing Ikappa B-alpha , CCK-8 was unable to cause a significant increase in the levels of mob-1 mRNA (Fig. 8B). This inhibitory effect was observed by 8 h after infection and continued for >= 20 h after infection. In control experiments, infection with adenovirus expressing Ikappa B-alpha had no observable effect on the ability of CCK-8 to stimulate intracellular Ca2+ release or amylase release (data not shown). Therefore, these data provide direct evidence for a role of NF-kappa B activation in CCK-8-mediated mob-1 gene expression in pancreatic acinar cells.


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Fig. 8.   Infection with adenovirus bearing Ikappa B-alpha leads to high levels of Ikappa B-alpha expression and completely blocks CCK-8 induction of mob-1 gene expression. Dispersed pancreatic acini were infected with recombinant adenovirus bearing beta galactosidase or a modified Ikappa B-alpha gene at 108 plaque-forming units/mg protein (multiplicity of infection ~ 100) for 8 or 20 h. Acini were subsequently treated with or without CCK-8 (100 nM) for 1 h (for analysis of Ikappa B-alpha protein levels) or 2 h (for analysis of mob-1 mRNA levels). Samples were probed with an antibody specific for Ikappa B-alpha in Western blots (A) or with a cDNA for mob-1 in Northern blots (B). C: ethidium bromide staining of total RNA as a loading control. Data are representative of 2 or 3 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We recently demonstrated that chemokine expression occurs early in the course of acute pancreatitis and originates from acinar cells themselves (16). We showed a correlation between the expression of two chemokine genes, mob-1 and mcp-1, and the manifestations of experimental acute pancreatitis in two separate models, secretagogue hyperstimulation and retrograde bile infusion. In the secretagogue hyperstimulation model, mob-1 gene expression and the severity of pancreatitis shared secretagogue and concentration dependence. We have also observed an association of mob-1 gene expression and the severity of pancreatitis in an arginine model of acute pancreatitis (Han, Tashiro, Williams, and Logsdon; unpublished observation). Recently, it has been shown that systemic manifestations of acute pancreatitis, especially the associated lung inflammation, were significantly reduced in animals lacking one of the chemokine receptors (39). Taken together, these observations and the known role of chemokines in other inflammatory diseases suggest that chemokines are likely important in the initiation of the inflammatory component of acute pancreatitis. However, the cellular mechanisms involved in pancreatic acinar cell chemokine expression have not been elucidated.

The transcription factor NF-kappa B is considered of central importance in inflammation for its ability to induce expression of a variety of genes that amplify and perpetuate inflammatory responses. That NF-kappa B was activated during cerulein-induced pancreatitis was suggested by the ability of inhibitors of NF-kappa B to block mob-1 gene expression and early parameters of pancreatitis (16). More recently, activation of NF-kappa B during experimental pancreatitis has been reported by several groups (11, 20, 43). NF-kappa B regulates several families of rapid-response and inflammatory genes, including those for chemokines, cytokines, inflammatory enzymes, adhesive molecules, and growth factors (1, 4, 14). Therefore, we hypothesized that NF-kappa B would be involved in the regulation of acinar cell mob-1 gene expression. Consensus NF-kappa B binding sites have been identified within the promoter region of the mob-1 gene, but direct regulation by NF-kappa B has not been shown for mob-1.

The activation of NF-kappa B generally involves the phosphorylation, dissociation, and proteolytic degradation of inhibitory proteins of the Ikappa B family. Ikappa B proteins function to mask the nuclear localization signals found in the NF-kappa B subunits that prevent the NF-kappa B dimers from translocating to the nucleus in unstimulated cells (40). Therefore, in some experiments we utilized Ikappa B-alpha protein levels as an indirect marker for NF-kappa B activation. In the present study conducted in vitro, Ikappa B-alpha levels were rapidly reduced by CCK-8 within 10 min, and no recovery was observed for >= 4 h. This is similar to in vivo observations reported by Steinle et al. (43) but differs from the in vivo observations of Gukovsky et al. (20), who observed a rapid recovery of Ikappa B-alpha levels within 3 h. Differences in Ikappa B-alpha recovery between in vitro and in vivo models might be explained by the influence of infiltrating inflammatory cells at later times in the in vivo situation. However, the explanation for the observed differences in Ikappa B-alpha recovery between the in vivo studies is unknown.

We found that CCK-8 treatment of dispersed acinar cells led to a dose- and time-dependent decrease in Ikappa B-alpha protein. Significant decreases of Ikappa B-alpha levels were only observed in acinar cells stimulated with supraphysiological concentrations of CCK-8, correlating with the known concentration dependence of CCK-8 on pancreatitis in vivo. Furthermore, the concentration dependence of Ikappa B-alpha degradation and mob-1 mRNA expression were identical. We also observed a direct correlation between Ikappa B-alpha degradation and NF-kappa B nuclear translocation and DNA binding. The correlation between CCK-8-induced Ikappa B-alpha protein degradation and mob-1 gene expression in the same preparation supports the hypothesis that the activation of NF-kappa B is involved in the regulation of mob-1 expression.

In contrast to the effects of CCK-8, neither Ikappa B-alpha degradation nor mob-1 expression was induced by the secretagogues bombesin and carbachol. Bombesin and carbachol are equivalent to CCK-8 in terms of their abilities to stimulate acinar cell secretion. However, bombesin does not cause pancreatitis in this model (35). Whether carbachol administration can induce pancreatitis in the rat is controversial. Bilchik et al. (5) found that administration of carbachol, a cholinergic agonist, produced a mild form of pancreatitis that was partially blocked by a CCK-8 receptor antagonist. Robert et al. (37) found that carbachol treatment did not induce pancreatitis, even when rats were treated with toxic concentrations. We were unable to induce pancreatitis in the rat by intraperitoneal administration of carbachol (unpublished observation). Others have shown that the CCK analog JMV-180, which is fully efficacious as a secretagogue but does not cause pancreatitis, also does not activate NF-kappa B when administered at high concentrations to rats (20). Thus there appears to be an excellent correlation between the ability of secretagogues to induce pancreatitis and their abilities to activate NF-kappa B and trigger expression of proinflammatory mediators.

To directly demonstrate a role for NF-kappa B in the induction of mob-1 expression in pancreatic acinar cells, we utilized a variety of methods to inhibit NF-kappa B activation. Previously, we reported that PDTC, a known inhibitor of NF-kappa B activation in a variety of cell models (40), inhibited mob-1 gene expression in vivo (16). In the present study we found that PDTC blocked NF-kappa B activation in acinar cells in vitro. Recently, it has been reported that PDTC and N-acetylcysteine, another known inhibitor of NF-kappa B activation, inhibited the ability of cerulein hyperstimulation to activate NF-kappa B in vivo (20, 43). However, PDTC is also known to be an antioxidant and an iron chelator (6) and may have nonspecific actions on cell function. Likewise, N-acetylcysteine is a powerful antioxidant with multiple effects on cell function. These antioxidants have recently been shown to have posttranscriptional effects that do not affect NF-kappa B activity (7). Therefore, in the present study, to more directly demonstrate a role for NF-kappa B in mob-1 gene expression, we utilized a molecular approach.

Adenoviral vectors have recently been shown to be a highly efficient method for gene transfer to isolated acini (31, 34). These studies have shown that infection of acini with adenovirus bearing beta -galactosidase has no obvious deleterious effects on acini, as assessed by effects on lactate dehydrogenase release or the ability of CCK-8 to stimulate increases in intracellular Ca2+ or amylase release or to activate MAP kinases. However, the data in the present study suggest that adenoviral infection may acutely activate NF-kappa B and stimulate mob-1 expression in pancreatic acinar cells. It has previously been reported that adenoviral infection at high titer stimulates NF-kappa B expression in other cells, including human vascular smooth muscle cells (9) and hepatocytes (25). This effect may account for some of the inflammation previously noted when adenovirus was utilized to deliver genes to the pancreas in vivo (36).

In contrast to the effects of infection with a control adenovirus bearing beta -galactosidase, infection with adenovirus bearing Ikappa B-alpha did not raise basal levels of mob-1 expression. Furthermore, adenovirus-mediated expression of Ikappa B-alpha blocked the effects of supraphysiological concentrations of CCK-8 on mob-1 expression. This inhibitory effect was noted within 8 h of adenoviral infection, a time at which no effects were noted on other CCK-8-mediated effects, including the stimulation of Ca2+ release or amylase secretion (data not shown). Expression of Ikappa B-alpha acts as a very specific inhibitor of NF-kappa B activation and has been widely utilized as a means of determining the effects of NF-kappa B activation on cellular activation and gene expression (14, 26, 30, 44). The adenovirally delivered Ikappa B-alpha utilized in this study was modified by the addition of a nuclear translocation signal, such that this molecule localizes at least in part to the cell nucleus (44). Ikappa B-alpha has a high affinity for the NF-kappa B family of proteins and can prevent their binding to DNA (10). This construct was previously shown to block NF-kappa B activation of cytokines and cell adhesion molecules in endothelial cells without affecting gene expression mediated by other transcription factors or interfering with other cellular functions not linked to NF-kappa B activation (44). We observed no effect of this virus on CCK-8-mediated Ca2+ signaling or secretion from isolated acini after 8 or 20 h (data not shown). Thus the present data strongly support the hypothesis that NF-kappa B activation is directly involved in the induction of mob-1 chemokine expression by CCK-8 in pancreatic acinar cells.

Much is yet to be discovered about the identities and roles of inflammatory mediators involved in the local and systemic responses that occur during acute pancreatitis. Significant differences exist between rodents and humans in terms of the cytokines/chemokines produced and the specific roles of proinflammatory molecules. Mob-1 is a chemokine found in the rat that has significant structural homology with the human chemokine IP-10. However, whether these two molecules function in a homologous manner is unknown. Chemokines are small secreted molecules that are chemotactic for inflammatory cells and are typically induced and released at the site of injury (2, 38). According to the evidence from clinical and experimental studies, it has recently come to be well accepted that inflammatory mediators and cells are involved in the development of acute pancreatitis. The levels of several cytokines have been reported to be increased in serum of the patients with acute pancreatitis (18, 22, 32, 45). However, cytokines are a general feature of the inflammatory response that leads to a continuing infiltration of activated leukocytes and secondary cascade of cytokines. The mechanisms and proximal mediators underlying the inflammatory response in pancreatitis remain obscure. Thus it is necessary to distinguish between the secondary effects of cytokines released from infiltrated leukocytes and those initially released from damaged acinar cells.

Our results from isolated rat pancreatic acinar cells demonstrate that injured acinar cells themselves are the source of chemokines. Because they originate from acinar cells and are known to initiate leukocyte infiltration and activation in other tissues, it is highly likely that chemokines may be an important initiating signal for the development of pancreatitis. Chemokines can also be released into the general circulation and can influence cells at distant sites. It was recently reported that the inflammatory response often observed in the lungs of animals during acute pancreatitis was absent in animals in which the CCR1 chemokine receptor had been knocked out by homologous recombination (13). The CCR1 receptor is one of several receptors that recognize beta -chemokines. The source and identity of the chemokines that activate this receptor in the lungs during acute pancreatitis are unknown, but it is likely that chemokines released from the pancreas itself may be involved.

The present data indicate that NF-kappa B activation is critical for the expression of a proinflammatory mediator in rat pancreatic acinar cells. It seems highly likely that these same cellular mechanisms are important for the inflammatory response in human pancreatitis. Thus these observations may be relevant to new approaches to intervention and therapy for acute pancreatitis.


    ACKNOWLEDGEMENTS

We thank Drs. J. A. Williams, B. Nicke, and D. Simeone for critical review of the manuscript.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-52067 and University of Michigan Gastrointestinal Peptide Center Grant DK-34933.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: C. Logsdon, Dept. of Physiology, Box 0622, University of Michigan, 7710 Medical Sciences Bldg. II, Ann Arbor, MI 48109-0622 (E-mail: clogsdon{at}umich.edu).

Received 29 January 1999; accepted in final form 5 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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