Activation of pancreatic acinar cells on isolation from tissue: cytokine upregulation via p38 MAP kinase

Thane A. Blinman1, Ilya Gukovsky2, Michelle Mouria2, Vjekoslav Zaninovic2, Edward Livingston1, Stephen J. Pandol2, and Anna S. Gukovskaya2

Departments of 2 Medicine and 1 Surgery, Veterans Affairs Greater Los Angeles Healthcare System, and the University of California Los Angeles, Los Angeles, California 90073


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytokines produced by pancreatic acinar cells may mediate cell death and recruitment of inflammatory cells into pancreas in pancreatitis and other disorders. Here, we demonstrate mRNA expression for a number of cytokines in acini isolated from rat pancreas. Using RNA from microscopically selected individual cells, we confirmed the acinar cell as a source for cytokine expression. Competitive RT-PCR, Western blot analysis, and immunocytochemistry showed large amounts of monocyte chemotactic protein-1 and interleukin-6 compared with other cytokines. Cytokine expression was inhibited by either inhibitors of p38 mitogen-activated protein kinase (MAPK), SB-202190 and SB-203580, or (less strongly) by the transcription factor nuclear factor (NF)-kappa B inhibitor MG-132. A combination of SB-203580 and MG-132 inhibited mRNA expression of all cytokines by >90%. The results suggest a major role for p38 MAPK and involvement of NF-kappa B in cytokine expression in pancreatic acinar cells. In contrast to isolated acini, we detected no or very low cytokine expression in normal rat pancreas. Our results indicate that activation of p38 MAPK, transcription factors, and cytokines occurs during removal of the pancreas from the animal and isolation of acini.

nuclear factor-kappa B; chemokines; interleukin-6; monocyte chemotactic protein-1; pancreatitis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

EXPRESSION OF CYTOKINES AND chemokines by pancreatic acinar cells is of interest from several points of view. First, the ability of epithelial cells (as opposed to immune and inflammatory cells) to produce cytokines has been recognized only recently (8). It remains to be determined whether the pancreatic acinar cell, a typical exocrine secretory epithelial cell, can express a whole spectrum of cytokines and chemokines as do inflammatory cells and what signaling mechanisms mediate cytokine expression in acinar cells. We and others showed recently that this cell can express tumor necrosis factor-alpha (TNF-alpha ) and a chemokine, Mob-1 (14, 16, 19).

Second, the ability of acinar cells to produce cytokines may play critical roles in pancreatic diseases, especially pancreatitis (29). Cytokines are upregulated in both human and experimental pancreatitis (14-17, 20, 29, 31, 34), and blockade of proinflammatory cytokines by using various strategies has been shown to ameliorate pancreatitis in experimental models (16, 17, 29, 34). However, it is generally believed that cytokines in pancreatitis are derived from inflammatory cells infiltrating the pancreas in later stages of the disease. Cytokines and chemokines produced by acinar cells may provide the first signals required for recruiting inflammatory cells into the pancreas during the initiation of pancreatitis.

Third, the pancreatic acinar cell has been a model cell type with which to study the mechanisms of protein secretion, hormone action, and stimulus-secretion coupling. These and other studies mostly use primary cultures of acinar cells isolated from pancreatic tissue because there are no cell lines that authentically reproduce the function of the acinar cell. However, the basal activation status of acinar cells in primary culture is not well characterized. The standard procedure for preparation of primary cell cultures from various organs, including pancreas, involves dissection of the tissue from the animal followed by collagenase digestion of tissue extracellular matrix (22, 30, 33, 37). The effect of these environmental stresses on signaling pathways and, in particular, cytokine expression has not been addressed.

Fourth, the process of obtaining tissue for transplantation is often similar to that applied for cell isolation. For example, for isolation and transplantation of islets of Langerhans, the pancreas is removed from the organism and digested with collagenase to provide a purified population of islets (25, 36). The level of expression of cytokines and other inflammatory mediators in the transplantation material may be critical for success of transplantation.

Thus one aim of the present study was to determine whether the pancreatic acinar cell expresses various cytokines and chemokines and whether the process of isolating pancreatic acini from the whole pancreas upregulates cytokine expression. For this study, we chose cytokines TNF-alpha and interleukin-6 (IL-6) and chemokines KC (murine analog of growth-related protein GROalpha and IL-8), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2). These cytokines and chemokines are of importance for pancreatitis (14, 16, 17, 20, 29).

The second aim of our study was to determine signaling mechanisms underlying cytokine expression in pancreatic acinar cells. We considered the involvement of transcription factors nuclear factor-kappa B (NF-kappa B) and activating protein 1 (AP-1) and of p38 mitogen-activated protein kinase (MAPK). NF-kappa B is a key regulator of cytokine expression in different cell types (1, 7, 40). Recently, we found that NF-kappa B is activated in experimental pancreatitis and mediates the induction of IL-6 and KC in pancreas (17). AP-1 may also regulate cytokine (e.g., IL-8) expression (3, 39).

Recent data provide evidence that p38 MAPK mediates the expression and/or production of TNF-alpha , IL-1beta , IL-6, and IL-8 in some cells (4, 27, 28). The mechanisms of this regulation are only beginning to be elucidated. p38 MAPK is activated by various cellular stresses (9, 13, 32). Recently, p38 MAPK was shown to be activated in pancreas from rats with experimental pancreatitis (38) and in vitro in pancreatic acinar cells under the action of CCK (35).

We found that rat pancreatic acinar cells express the cytokines/chemokines IL-6, TNF-alpha , KC, MCP-1, and MIP-2 and that the transcription factors NF-kappa B and AP-1, as well as p38 MAPK, are activated in isolated pancreatic acini. Cytokine expression in pancreatic acini was inhibited >90% by application of the inhibitors for NF-kappa B and p38 MAPK together. The results suggest that cytokine expression in acinar cells is mediated by two separate pathways, one involving p38 MAPK and the other, NF-kappa B. In contrast to isolated pancreatic acini, we detected very little or no cytokine expression and activation of NF-kappa B, AP-1, and p38 MAPK in normal pancreas. Thus a standard procedure of isolating epithelial cells from tissue can activate various signaling mechanisms, including protein kinases, transcription factors, and expression of cytokine genes.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents. Antibodies against MCP-1, IL-6, p38 MAPK, and active (phosphorylated) p38 MAPK were from Santa Cruz Biotechnology (Santa Cruz, CA). Pyridinyl imidazoles SB-202190 and SB-203580 were from Calbiochem (San Diego, CA). The proteasomal inhibitor Z-Leu-Leu-Leu-H (MG-132) was from Peptide International (Louisville, KY). [gamma -32P]ATP was from New England Nuclear (Boston, MA), and [alpha -32P]dCTP was from ICN Pharmaceuticals (Costa Mesa, CA). Enhanced chemiluminesence (ECL) kit and protein A-Sepharose were from Pierce (Rockford, IL). Poly [d (I-C)] was from Boehringer Mannheim (Indianapolis, IN). Chromatographically purified collagenase (CLSPA) was from Worthington (Freehold, NY), and type IV collagenase (C-5138) was from Sigma (St. Louis, MO). Precast Tris-glycine gels were from Novex (San Diego, CA). Protein assay dye reagent was from Bio-Rad (Hercules, CA). TRIzol reagent, SuperScript II Preamplification System, and Taq DNA polymerase were from GIBCO-BRL (Gaithersburg, MD). SYBR Green I was from Molecular Probes (Eugene, OR). Exo(-) Klenow DNA polymerase I was from Stratagene (La Jolla, CA). All other chemicals were from Sigma.

Isolation of dispersed pancreatic acini. Dispersed rat pancreatic acini were regularly prepared by a collagenase digestion technique as previously described (30). Briefly, pancreas was dissected and injected with 5 ml of solution A containing CLSPA from Worthington (~135 U/5 ml). Solution A was composed of (in mM) 110 NaCl, 5 KCl, 25 HEPES (pH 7.4), 2 NaH2PO4, 1 MgCl2, 1 CaCl2, 12 glucose, 4 Na-pyruvate, 4 Na-fumarate, and 4 Na-glutamate as well as 0.01% (wt/vol) soybean trypsin inhibitor and 0.2% (wt/vol) BSA. For measuring the effect of inhibitors, solution A containing collagenase was supplemented with the inhibitors or vehicle (for control). Pancreas was subjected to three successive 15-min incubations in 5 ml of solution A containing collagenase with vigorous shaking at 37°C. For each 15-min incubation, solution A containing collagenase was replaced with a fresh oxygenated aliquot. To obtain dispersed acini, the treated tissue was passed through pipettes with narrow bores. Acini were then washed twice with solution A containing 4% (wt/vol) BSA and once in solution A with 0.2% BSA, followed by resuspension in 199 medium. This isolation procedure takes 45-50 min.

In some experiments, a modified cell-isolation procedure (5) was applied. Pancreas was dissected, minced in 4 ml of solution A, and transferred to a conical tube. One milligram (~125 U) of crude type IV collagenase (no. C-5138, Sigma) was added, and the suspension was vigorously shaken by hand for 8-10 min until the tissue was digested. Acini were then washed twice with solution A containing 4% BSA and once in solution A with 0.2% BSA, filtered through a 190-µm nylon mesh, and resuspended in 199 medium. The whole procedure was performed at room temperature and took 15-20 min.

To measure cytokine expression in tissue, pancreas was dissected from normal rat and either immediately processed or incubated in solution A with or without CLSPA (~400 U/pancreas) under conditions strictly imitating the procedure of isolating pancreatic acini.

Detection and quantitation of cytokine mRNA by RT-PCR. The procedures were as we described previously (16, 17, 31). Briefly, total RNA was obtained from isolated pancreatic acini or pancreatic tissue with TRIzol reagent (GIBCO-BRL), and its quality was verified by ethidium bromide staining of rRNA bands on a denaturing agarose gel. RNA was reverse-transcribed with the SuperScript II preamplification kit (GIBCO-BRL) and subjected to PCR with rat gene-specific, intron-spanning primers described in Table 1. Target sequences were amplified at 56°C by using the same amount of cDNA for all primer sets. The RT-PCR products were all of expected size. Their identity was confirmed by direct sequencing. Negative controls were performed by omitting the RT step or cDNA template from PCR amplification.

                              
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Table 1.   Rat gene-specific, intron-spanning PCR primers

For semiquantitative RT-PCR, the cycle number was adjusted between 22 and 31 cycles to yield visible products within the linear amplification range. Resulting RT-PCR products were run on agarose gel and visualized by staining with ethidium bromide or another fluorescent dye, SYBR Green I (Molecular Probes). Their densities were quantified by using the image-analysis system AMBIS (Scanalytics, San Diego, CA) and normalized to that of RT-PCR product of the housekeeping acidic ribosomal phosphoprotein P0 gene (ARP) in the same sample.

We used quantitative competitive PCR (17) to quantify mRNA levels for IL-6, MCP-1, and ARP. Briefly, a homologous external standard DNA (mimic) was created by using a "composite" primer that was designed consisting of the forward PCR primer spliced to a sequence 50-100 bp downstream in the target cDNA. Coamplification of a constant amount of target cDNA (derived from 0.5 µg of total RNA) with serial dilutions of its mimic allowed accurate quantitation of starting amount of target cDNA. Because the target cDNA and its mimic are coamplified with the same primer pair, share the same sequence, and have similar sizes, the difference in the efficiencies of their amplification is expected to be minimal.

The amount of the mRNA of interest was determined from a competition curve derived by plotting the ratio of the densities of the target to mimic RT-PCR products coamplified in the same tube vs. the concentration of the mimic (cf. Fig. 2, A and B). The equivalence point, where this ratio equals 1.0, was estimated, and a correction was made for the difference in the target and mimic sizes. In most cases, the competition curve was well described by a straight line with the slope close to the theoretical value of -1.0, indicating similar amplification efficiencies for the target and mimic (cf. Fig. 2B). The reproducibility of our quantitative competitive PCR was tested by performing parallel PCR reactions on one and the same cDNA sample.

Immunoprecipitation. To extract proteins, freshly prepared pancreatic acini were washed twice with PBS and lysed by incubating for 20 min at 4°C in a lysis buffer containing 0.15 M NaCl, 50 mM Tris (pH 7.2), 1% deoxycholic acid (wt/vol), 1% Triton X-100 (wt/vol), 0.1% SDS (wt/vol), 1 mM phenylmethylsulfonyl fluoride (PMSF), as well as 5 µg/ml each of protease inhibitors pepstatin, leupeptin, chymostatin, antipain, and aprotinin. Then the cell lysates were centrifuged for 20 min at 15,000 g at 4°C, and the supernatants were used for immunoprecipitation.

For immunoprecipitation, the supernatants from cell lysates were incubated at 4°C overnight with primary antibodies, then for 1 h with protein A-Sepharose. The protein A-Sepharose-antigen precipitates were separated by centrifugation, washed three times with the lysis buffer, and resuspended in a sample buffer containing 10% (vol/vol) glycerol, 2% (wt/vol) SDS, and 0.0025% (wt/vol) bromophenol blue in 63 mM Tris (pH 6.8). The antigen was eluted from protein A-Sepharose by heating for 5 min at 100°C. Samples were centrifuged, and supernatants containing the antigen were collected.

Western blot analysis. Protein from cell lysates or immunoprecipitated proteins were analyzed by immunoblotting. Proteins were separated by SDS-PAGE at 120 V by using precast gels and a minigel apparatus (Novex). Separated proteins were electrophoretically transferred to polyvinylidene difluoride membranes for 2 h at 30 V by using a blot module (Novex). Nonspecific binding was blocked by 1-h incubation with 5% (wt/vol) nonfat dry milk in Tris-buffered saline (TBS; pH 7.5). Blots were then incubated overnight with primary antibodies in the antibody buffer containing 1% (wt/vol) nonfat dry milk in TTBS (0.05% vol/vol Tween-20 in TBS), washed three times in TTBS, and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody in the antibody buffer. Blots were developed with Supersubstrate Ultra ECL kit (Pierce).

Immunocytochemistry. Dispersed pancreatic acini were allowed to adhere to polylysine-coated slides and fixed in 5% paraformaldehyde in PBS. The slides were washed three times in PBS, and the nonspecific binding was blocked by incubation in a blocking buffer containing 1.5% (vol/vol) normal goat serum, 1% BSA, and 0.1% (wt/vol) saponin. Intrinsic peroxidase activity was quenched by 10-min incubation in horseradish peroxidase blocking buffer (DAKO, Carpinteria, CA). After three washes with PBS, endogenous biotin was blocked by incubating the slides first in avidin and then in biotin solution from the Avidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA), each time for 15 min. Then the primary antibody against rat MCP-1 was applied (1:100 dilution) and allowed to incubate overnight at 4°C. Biotin-conjugated secondary antibody was applied for 30 min and developed by using the Immuno Pure Ultra-Sensitive ABC Staining Kit (Pierce) according to the manufacturer's instructions. Samples were observed by using a Nikon Diaphot microscope.

Preparation of nuclear extracts and electrophoretic mobility shift assay. Nuclear protein extracts were prepared as described (16, 17). Briefly, isolated acini or pancreatic tissue was lysed on ice in a hypotonic buffer (17) supplemented with 1 mM PMSF and 1 mM dithiothreitol (DTT) and with the protease inhibitor cocktail containing 5 µg/ml each of pepstatin, leupeptin, chymostatin, antipain, and aprotinin. The resulting nuclear pellet was collected by microcentrifugation for 30 s. The supernatant was removed, and the nuclear pellet was resuspended in the high-salt buffer containing 20 mM HEPES (pH 7.6), 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 MgCl2, 0.2 mM EDTA, 20 mM beta -glycerophosphate, 10 mM Na2MoO4, 50 µM Na3VO4, 1 mM DTT, 1 mM PMSF, and the protease inhibitor cocktail described above. Nuclear membranes were pelleted by microcentrifugation for 10 min, and the clear supernatant (nuclear extract) was aliquoted and stored at -80°C. Protein concentration in the nuclear extract was determined by the Bio-Rad protein assay.

For the electrophoretic mobility shift assay, aliquots of nuclear extracts with equal amounts of protein (2-10 µg) were mixed in 20-µl reactions in a buffer containing (in mM) 10 HEPES (pH 7.6), 50 KCl, 0.1 EDTA, and 1 DTT as well as 10% (vol/vol) glycerol and 3 µg poly (dI-dC). Binding reactions were started by addition of 32P-labeled DNA probe and incubated at room temperature for 30 min. The sequences of the oligonucleotides used to determine the DNA binding activity of NF-kappa B and AP-1 were as follows (binding sites are underlined): NF-kappa B, 5'-GCAGAGGGGACTTTCCGAGA-3'; AP-1, 5'-GGCTTGATGAGTCAGCCGGAA-3'. The oligonucleotides were annealed with their complementary strands bearing a 5' G overhang, and end-labeled by using Klenow DNA polymerase I. Samples were electrophoresed at room temperature in 0.5× TBE buffer (1× TBE: 89 mM Tris base, 89 mM boric acid, 2 mM EDTA) with loading dye on nondenaturing 4.5% polyacrylamide gel at 200 V. Gels were dried and directly analyzed in the PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Kinase assays. To measure p38 MAPK activity, we used a p38 MAPK assay kit (New England BioLabs, Beverly, MA). Immunoprecipitated p38 MAPK was used to phosphorylate 2 µg glutathione-S-transferase-ATF-2 fusion protein in 50 µl of kinase buffer containing (in mM) 25 Tris (pH 7.5), 5 beta -glycerophosphate, 2 DTT, 0.1 Na3V04, and 10 MgCl2 as well as 200 µM ATP. The reaction mixture was incubated at 30°C for 30 min with shaking. Reactions were terminated by addition of 3× SDS sample buffer, and the samples were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membrane and probed with phospho-ATF-2 antibody. Protein detection was performed by using ECL.

MAPK-activated protein kinase-2 (MAPKAPK-2) activity was measured with a MAPKAPK-2 immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY). Immunoprecipitated MAPKAPK-2 was used to phosphorylate a specific MAPKAPK-2 substrate peptide (KKLNRTLSVA) in 30 µl of kinase buffer containing (in mM) 20 MOPS (pH 7.2), 25 beta -glycerophosphate, 5 EGTA, 1 Na3V04, 1 DTT, and 10 MgCl2 as well as 50 µM ATP and 10 µCi/assay of [gamma -32P]ATP. The reaction mixture was incubated at 30°C for 30 min with shaking, and 25 µl of the supernatant were transferred onto Whatman P81 paper. The P81 paper was then washed three times with 0.75% phosphoric acid and once with acetone. Radioactivity was determined by liquid scintillation counting.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pancreatic acinar cells express mRNA for cytokines and chemokines. Pancreatic acini were prepared from rat pancreas by a standard collagenase digestion procedure (30). To determine whether pancreatic acinar cells express messages for cytokines, total RNA was extracted from the isolated acini, and RT-PCR was performed by using rat gene-specific, intron-spanning primers (Table 1). Prominent RT-PCR products were observed for cytokines IL-6, TNF-alpha , and chemokines KC, MCP-1, and MIP-2 (Fig. 1A). Identity of the bands was confirmed by direct sequencing. Earlier we showed (16) that TNF-alpha is expressed in pancreatic acinar cells.


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Fig. 1.   Rat pancreatic acinar cells express a variety of cytokines and chemokines. Representative RT-PCR for the expression of cytokines and chemokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha ), KC, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and the housekeeping gene ARP in pancreatic acinar cells. Total RNA was extracted from pancreatic acini isolated from rat pancreas by a standard collagenase digestion technique (A) and from ~100 individual acinar cells selected under the microscope (B) from a preparation of pancreatic acini. RNA was reverse-transcribed and PCR amplified as described under EXPERIMENTAL PROCEDURES. The RT-PCR products were resolved on agarose gel stained with ethidium bromide (A) or SYBR Green (B), and their identities were verified by sequencing. The last lane in (A) shows DNA size markers. Note that in A, to illustrate band intensities used in semiquantitative RT-PCR analysis, different cycle numbers (from 22 for ARP to 31 for TNF-alpha ) were employed to amplify different mRNAs, whereas data in B were obtained at a constant number of cycles (n = 36).

Our preparation of dispersed pancreatic acini consists of >95% acinar cells (16). To ensure that the observed cytokines and chemokines were indeed expressed in acinar cells, 100-200 individual acinar cells were collected under the microscope from the preparation of dispersed acini by using a micromanipulator and a fine Hamilton syringe. RNA was extracted and screened by RT-PCR as above. Clear bands for IL-6, KC, MCP-1, and fainter bands for TNF-alpha and MIP-2 were amplified from RNA obtained from microscopically selected acinar cells (Fig. 1B). In another control experiment, we showed (12) that our preparations of microscopically selected acinar cells do not express markers specific for endothelial cells: type 1 receptor for vascular endothelial growth factor and platelet-endothelial cell adhesion molecule-1 (PECAM-1), indicating that these preparations are not contaminated by endothelial cells, a potential source for cytokines. These results confirm pancreatic acinar cells as a source of cytokine/chemokine expression.

Using quantitative competitive PCR (17), we estimated the amount of mRNA for the two more abundant cytokines/chemokines, IL-6 and MCP-1, as well as for the housekeeping gene ARP (Fig. 2). Exogenous homologous standards (mimics) were created for IL-6, MCP-1, and ARP on the basis of the published rat cDNA sequences (see EXPERIMENTAL PROCEDURES). Serial dilutions of a known concentration of the mimic were coamplified with a constant amount of reverse-transcribed RNA, and a competition curve was derived from densitometric analysis of the PCR products (Fig. 2, A and B). The amount of ARP or cytokine mRNA in a sample was estimated from the equivalence point where the ratio of the densities of the target to mimic RT-PCR products was equal to 1.0 (Fig. 2B), after correcting for the differences in the target and mimic sizes. This analysis was done for each RNA sample. Our results indicated that in isolated pancreatic acini ARP was expressed at a level of 20 amol/µg of total RNA, MCP-1 was expressed at ~2 amol/µg, and IL-6 at ~0.2 amol/µg total RNA (Fig. 2C).


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Fig. 2.   Quantitation of cytokine mRNA levels in rat pancreatic acini by competitive PCR. A: representative gel showing detection of target ARP cDNA and its mimic, which was made as described in EXPERIMENTAL PROCEDURES. RNA was extracted from rat pancreatic acini, and a constant amount of cDNA derived from 0.5 µg total RNA was coamplified with sequential 1:5 dilutions of ARP mimic. RT-PCR products were resolved on agarose gel stained with ethidium bromide, and band intensity was quantified by densitometry. B: ratio of the target to mimic band intensities was plotted against amount of mimic in each PCR reaction for ARP (), MCP-1 (black-triangle), and IL-6 (), yielding a straight line with a slope of approximately -1.0. Absolute amount of cytokine cDNA in each sample was calculated from the equivalence point where intensities of target and mimic bands were equal, after correction for differences in target and mimic size. C: the levels of cytokine mRNA expression were quantified as shown in A and B and then normalized to the amount of total RNA in the sample. Values are means ± SE from 3 independent experiments.

Cytokine proteins are present in pancreatic acinar cells. To detect IL-6 protein in pancreatic acinar cells, we first enriched the IL-6 amount by immunoprecipitation. Specific antibody against IL-6 recognized a 23-kDa band in the immunoprecipitate, which corresponded to that for recombinant IL-6 (Fig. 3A). MCP-1 protein was detected by direct immunoblotting without immunoprecipitation. Antibody against MCP-1 recognized one prominent band at 7 kDa, corresponding to that for the recombinant MCP-1 (Fig. 3B). Higher levels of MCP-1 were detected in the cytosolic fractions compared with total cell lysates, suggesting cytosolic localization of MCP-1 (Fig. 3B).


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Fig. 3.   A: IL-6 and MCP-1 proteins are present in isolated rat pancreatic acini. IL-6 was immunoprecipitated from a whole cell lysate obtained from isolated rat pancreatic acini; immunoprecipitate was fractionated on 4-20% SDS-polyacrylamide gel and immunoblotted with the same anti-rat IL-6 monoclonal antibody. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL). RP, recombinant IL-6. B: samples of whole cell and cytosolic protein extracts were obtained from rat pancreatic acini, adjusted to equal protein concentration, fractionated on 4-20% SDS-polyacrylamide gel, and immunoblotted with anti-MCP-1 antibody. Immunoreactive bands were visualized by ECL. RP, recombinant MCP-1. C: isolated rat pancreatic acini were plated on glass coverslips, permeabilized, and stained for MCP-1 by using the avidin-biotin system. Slides were visualized with light microscopy at ×40. Left: primary and secondary antibodies; right: secondary antibody only.

To confirm that MCP-1 is localized to acinar cells, we applied immunocytochemistry with the same antibody we used in Western blot. Figure 3C shows cytosolic and perimembrane staining for MCP-1 throughout the cell. Staining was observed in the majority of acinar cells in our preparations of dispersed pancreatic acini.

Inhibition of p38 MAPK and the transcription factor NF-kappa B attenuates cytokine expression in pancreatic acini. We used inhibitory analysis to determine the role of transcription factors and p38 MAPK in cytokine expression in pancreatic acini. To inhibit NF-kappa B, we used the proteasomal inhibitor MG-132, which prevents translocation of activated NF-kappa B into the nucleus (2, 7). For p38 MAPK inhibition, we applied pyridinyl imidazoles SB-202190 and SB-203580 (9, 24, 35).

In our previous studies (16, 17), we showed NF-kappa B DNA binding activity in isolated pancreatic acini. Figure 4 in the present study demonstrates that the NF-kappa B activity was inhibited by ~80% in acinar cells isolated in the presence of 10 µM MG-132. Higher (50 µM) doses of the inhibitor did not cause further inhibition of NF-kappa B binding activity (not shown). NF-kappa B activation was not significantly affected by specific inhibitors of p38 MAPK, SB-202190 and SB-203580. A combination of MG-132 and SB-203580 had the same inhibitory effect as MG-132 alone (Fig. 4B).


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Fig. 4.   Effects of proteasomal inhibitor MG-132 and p38 mitogen-activated protein kinase (MAPK) inhibitors SB-202190 and SB-203580 on activation of transcription factors nuclear factor (NF)-kappa B and activating protein 1 (AP-1) in isolated rat pancreatic acini. A: electromobility shift assays (EMSA) were performed on nuclear extracts containing 7 µg protein from rat pancreatic acini isolated in the absence or presence of 10 µM proteasomal inhibitor MG-132 or 20 µM p38 MAPK inhibitors SB-202190 and SB-203580. The oligonucleotide probes used for NF-kappa B and AP-1 EMSA contained a consensus kappa B site and a phorbol ester-responsive (TRE) site, respectively. The results are representative of 3 similar experiments performed on different preparations of acini. B: intensities of the NF-kappa B and AP-1 bands presented in lanes 2-5 were normalized to the intensity of the corresponding band in control, untreated acini (lane 1). Band intensities were quantified in a PhosporImager. Values are means ± SE from 3 independent experiments. *P < 0.05 compared with control.

Figure 4 demonstrates that the transcription factor AP-1 was also activated in isolated pancreatic acini. Neither MG-132 nor the SB compounds significantly inhibited AP-1 binding to its phorbol ester-responsive binding site. However, when added together, MG-132 and SB-203580 diminished AP-1 binding activity by >75% (Fig. 4B), showing synergism in their action on AP-1 in pancreatic acini.

Using Western blot analysis with antibodies against p38 MAPK and against phosphorylated p38 MAPK, we found that p38 MAPK is partially phosphorylated (activated) in isolated pancreatic acini (data not shown). These results are in agreement with the recent publication of Schäfer et al. (35).

The pyridinyl imidazoles inhibit p38 MAPK by competitive binding to the ATP-binding domain of the kinase (42). Therefore, to assess the inhibitory effect of SB-202190 and SB-203580 on the activity of p38 MAPK in pancreatic acinar cells, we measured the activity of a downstream phosphorylation target, MAPKAPK-2, a specific substrate for p38 MAPK (9, 13, 32). The results (Fig. 5) showed that the SB compounds at a concentration of 20 µM inhibited MAPKAPK-2 activity by 70-80%. MG-132 did not significantly affect MAPKAPK-2, and the combination of SB-203580 plus MG-132 produced about the same inhibitory effect on MAPKAPK-2 activity as did SB-203580 alone (Fig. 5).


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Fig. 5.   Effects of proteasomal inhibitor MG-132 (MG) and p38 MAPK inhibitors SB-202190 and SB-203580 on MAPK-activated protein kinase-2 (MAPKAPK-2) activity in pancreatic acini. Acini were isolated with or without 10 µM MG-132 or 20 µM SB-202190 (SB202) and SB-203580 (SB203). MAPKAPK-2 was immunoprecipitated from cell lysates, and its activity was assessed by measuring phosphorylation of MAPKAPK-2-specific substrate in the presence of [gamma -32P]ATP, as described in EXPERIMENTAL PROCEDURES. Data are expressed as a percentage of the control value (without inhibitors). Each point represents mean ± SE (n = 3). *P < 0.05 compared with control.

The results in Figure 6 show the effects of the inhibition of p38 MAPK and NF-kappa B on cytokine/chemokine expression in pancreatic acini. p38 MAPK inhibitors produced a dramatic inhibition of mRNA expression for all cytokines and chemokines under study. This inhibition was to the same (or even higher) extent that these inhibitors blocked the activity of p38 MAPK (Fig. 5). In particular, 20 µM SB-203580 inhibited steady-state mRNA levels for the different cytokines/chemokines by 70-95% (Fig. 6). The same inhibitory effect on cytokine expression was observed with 10 µM SB-203580, indicating that the results presented in Fig. 6 show maximal inhibition of the cytokines by the p38 MAPK inhibitors. These results demonstrate that signaling pathway(s) involving p38 MAPK mediate cytokine expression in pancreatic acinar cells.


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Fig. 6.   Effects of proteasomal inhibitor MG-132 and p38 MAPK inhibitors SB202190 and SB203580 on the expression of cytokines and chemokines in isolated rat pancreatic acini. A: representative RT-PCR for expression of cytokines IL-6, TNF-alpha , chemokines KC, MCP-1, and MIP-2 and the housekeeping gene ARP in pancreatic acini isolated in the absence (control) or presence of 10 µM MG-132 or 20 µM SB-202190 and SB-203580. RT-PCR products were resolved on agarose gel stained with ethidium bromide, and band intensity was quantified by densitometry in the image-analysis system AMBIS. The results are representative of 3 similar experiments performed on different preparations of acini. B: densities of cytokine and chemokine RT-PCR products were normalized to that of housekeeping gene ARP in the same sample. Cytokine mRNA expression levels are given as means ± SE (n = 3). *P < 0.05 compared with control (without inhibitors).

The effect of NF-kappa B inhibition with MG-132 on cytokine/chemokine mRNA expression was less pronounced. The highest level of inhibition with 10 µM MG-132 was for MIP-2 (~65%), the lowest (<30%), for IL-6 and MCP-1. Greater doses of MG-132 did not further increase its inhibitory action on cytokine expression (not shown).

For all cytokines and chemokines, the maximal inhibition (>90%) was achieved by the combination of MG-132 and SB-203580. The results suggest that two pathways are required for cytokine expression in acinar cells. One of these pathways is inhibited by SB-203580, the other by MG-132.

Cytokine expression is upregulated during preparation of pancreatic acini. We asked whether the transcription factors, p38 MAPK, and the cytokines/chemokines under study were activated in the acinar cells by the procedure of their isolation from pancreatic tissue. The results in Fig. 7A demonstrate drastic differences in activation status of NF-kappa B, AP-1, and p38 MAPK between normal pancreatic tissue and isolated pancreatic acini. For these experiments, pancreas was removed from rat and immediately homogenized or processed to obtain nuclear protein; part of the same pancreas was used for preparation of dispersed acini. Normal pancreatic tissue did not display any activated complexes of NF-kappa B or AP-1, as measured by their binding to consensus sites (Fig. 7A). We determined kinase activity of p38 MAPK by measuring phosphorylation of its specific substrate, ATF-2. ATF-2 phosphorylation was almost undetectable in pancreatic tissue lysate but was greatly upregulated in lysates from pancreatic acini (Fig. 7A). The extent of activation of transcription factors and p38 MAPK in isolated pancreatic acini was very dramatic: ~15-fold for NF-kappa B, 30-fold for AP-1, and 37-fold for p38 MAPK.


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Fig. 7.   Activities of transcription factors NF-kappa B and AP-1 as well as p38 MAPK in normal rat pancreatic tissue and isolated acini. Effect of different steps in the process of cell isolation on cytokine expression in pancreatic acini. A: NF-kappa B, AP-1, and p38 MAPK activities were measured in normal pancreatic tissue immediately after dissection and in isolated acinar cells. EMSA were performed on nuclear extracts containing 7 µg protein from tissue and cells. To measure p38 MAPK activity, p38 was immunoprecipitated from tissue and cell lysates, and the immunoprecipitates were used in a kinase reaction with glutathione-S-transferase-ATF-2 as substrate. Samples were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted with antibody against phosphorylated ATF-2. The results are representative of 3 similar experiments performed on different cell preparations. B: representative RT-PCR for expression of cytokines IL-6, TNF-alpha , KC, MCP-1, and MIP-2 and the housekeeping gene ARP in pancreatic tissue immediately after dissection (tissue); in pancreatic tissue incubated for 45 min at 37°C without (tissue+inc) or with (tissue+coll) collagenase; and in freshly isolated acinar cells. The results are representative of 2 similar experiments performed on different cell preparations.

In normal pancreatic tissue, we detected no significant messages for IL-6, KC, and MIP-2. Similarly, mRNA expression of TNF-alpha and MCP-1 was much less pronounced in pancreatic tissue than in isolated acini (Fig. 7B).

As described in EXPERIMENTAL PROCEDURES, the standard technique for preparation of pancreatic acini includes removal of pancreas followed by collagenase digestion of pancreatic extracellular matrix (ECM). In an attempt to imitate various factors operating during the process of cell isolation, we performed the following experiments. We measured cytokine expression in freshly dissected pancreas; pancreas incubated for 45 min at 37°C in the isolation solution A without collagenase; pancreas incubated for 45 min at 37°C in solution A containing collagenase; and in isolated acini. These conditions simulate different steps in the procedure of cell isolation. For example, during the incubation of undigested tissue, pancreatic cells experience lack of trophic factors, nutrients, and/or oxygen. Tissue digestion with collagenase results in the loss of cell-ECM contacts, and at the same time facilitates the access of oxygen and nutrients. The difference between the last two conditions is that an additional mechanical stress is applied at the final step of isolation of the pancreatic acini.

Figure 7B demonstrates that even incubation of normal tissue at 37°C in the absence of collagenase resulted in the expression of cytokines and chemokines. mRNA expression levels for the cytokines/chemokines under study were almost the same in tissue incubated without collagenase, with collagenase, and in isolated pancreatic acini. This indicates that the various above-specified stresses (which are common in the process of any cell isolation from tissue) activate the expression of cytokine and chemokine genes.

With semiquantitative RT-PCR, we measured that the expression of activated MCP-1 mRNA did not change, and that of IL-6 decreased only slightly, over 6-h incubation of isolated acini at 37°C (Fig. 8A). It has been reported (14, 19) that mRNA expression of chemokine Mob-1 is upregulated in isolated pancreatic acini but greatly decreases after >2-h incubation of acini at 37°C. The data in Fig. 8A show that different cytokines vary in their sensitivity to such "pacification by preincubation": activated MCP-1 and IL-6 could not be "pacified" in this way.


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Fig. 8.   Cytokine expression in pancreatic acini isolated with different procedures. A: time course of MCP-1 and IL-6 mRNA expression was measured by semiquantitative RT-PCR in acini isolated with the regular procedure (using purified collagenase) and incubated at 37°C. Cytokine mRNA expression levels were normalized to that of housekeeping gene ARP in the same sample. There was no significant change in MCP-1 and IL-6 expression over 6-h period of incubation (n = 4 for each time point). B: cytokine expression as detected by RT-PCR in acini isolated at room temperature using crude collagenase (lanes 1 and 2) and after their subsequent 2-h incubation at 37°C (lanes 3 and 4). Shown are the results from 2 different acini preparations.

We also measured whether modification of the isolation procedure affected cytokine expression. We applied a procedure (5) using a crude type IV collagenase, which digests the pancreas quickly (in 20 min) and works at room temperature. Acini obtained with the "short" procedure had the same percentage of acinar cells (>97%) as those isolated with the regular procedure. When this procedure was applied at 37°C, we detected the same high level of cytokine activation in isolated acini (not shown). The only factor we found to affect cytokine expression was temperature. Acini obtained at room temperature by using the crude collagenase did not display cytokine expression on isolation; however, subsequent incubation at 37°C resulted in the expression of cytokines and chemokines (Fig. 8B). Accordingly, NF-kappa B and p38 MAPK displayed little activity in acini isolated at room temperature but quickly (within 15 min) became activated on being switched to 37°C (data not shown).

The isolation procedure could affect acinar cells directly or indirectly, through soluble mediator(s) produced by various cell populations present in the pancreas. The fact that cytokine expression was not detected in acini isolated at room temperature but was activated on subsequent incubation at 37°C suggests that cytokine upregulation is caused by direct action of the isolation procedure on acinar cells rather than by soluble mediator(s) produced in the pancreas.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results obtained in the present study show that isolated pancreatic acini express mRNA for a variety of cytokines and chemokines. Recently, pancreatic acinar cells were shown to express TNF-alpha (16) and the chemokine Mob-1 (14, 19). The present study expands the list of cytokines known to originate within pancreatic acinar cells.

Using RNA extracted from individual acinar cells that were selected under the microscope, we confirmed that the acinar cell itself expresses the cytokines/chemokines. Further proof that the cytokines and chemokines originate within acinar cells comes from our negative control experiments with PCR amplification of markers specific for endothelial cells, a potential contaminating source for cytokines. In these control experiments (12), we did not detect messages for vascular endothelial growth factor receptor 1 and platelet-endothelial cell adhesion molecule-1 in the microscopically selected acinar cells, even after two successive rounds of PCR.

We measured the amounts of mRNA for IL-6 and MCP-1 in isolated pancreatic acini by using quantitative competitive PCR. The steady-state level of IL-6 mRNA was approximately two orders of magnitude less, and that of MCP-1 message only one order of magnitude less than the expression of the housekeeping gene, ARP. This shows a relative abundance of messages for these cytokines in isolated pancreatic acini. The presence of the proteins for the more abundant cytokines, IL-6 and MCP-1, was demonstrated by Western blot analysis and immunocytochemistry.

Our results link cytokine expression in pancreatic acinar cells to activation of p38 MAPK and the transcription factor NF-kappa B. To inhibit p38 MAPK, we used pyridinyl imidazoles SB-203580 and SB-202190. These compounds selectively inhibit the alpha - and beta -isoforms of p38 MAPK but not other protein kinases (9, 13), and they are widely applied to determine a role of p38 MAPK in cell functioning (9, 27, 28, 35). For NF-kappa B inhibition, we used peptide proteasomal inhibitor MG-132 (2, 7, 11, 21, 43). MG-132 has recently been used to demonstrate regulation by NF-kappa B of the expression of intercellular adhesion molecule-1 in intestinal epithelial (21) and pancreatic acinar cells (43).

The pyridinyl imidazoles significantly inhibited p38 MAPK activity (in agreement with the data of Ref. 35) but had no effect on NF-kappa B DNA binding activity in isolated pancreatic acini. Conversely, MG-132 caused 80% inhibition of NF-kappa B binding activity but did not affect p38 MAPK. Cytokine expression in isolated pancreatic acini was strongly inhibited by the pyridinyl imidazoles, suggesting that p38 MAPK is a major mediator of cytokine expression in these cells. NF-kappa B inhibition with MG-132 also decreased cytokine mRNA expression in pancreatic acini, but to a lesser extent than the inhibition of p38 MAPK. More than 50% inhibition by MG-132 was only observed for TNF-alpha and MIP-2. These results indicate that NF-kappa B is involved in cytokine expression. They also suggest that transcription factor(s) other than NF-kappa B play a role in the regulation of IL-6, MCP-1, and KC expression in isolated acini. Alternatively, the residual level of NF-kappa B activity remaining in the presence of MG-132 may be enough to support the expression of these cytokines.

When given in combination, MG-132 and SB-203580 produced maximal inhibitory effect (>90%) on the expression of all cytokines and chemokines under study. The inhibitory effects of MG-132 and SB-203580 on cytokine expression were additive. These results suggest that activation of two separate signaling pathways is required for cytokine expression: one of them is inhibited by MG-132, the other by the pyridinyl imidazoles.

Although p38 MAPK inhibitors had no effect on NF-kappa B DNA binding activity, this does not necessarily mean that p38 MAPK is not involved in NF-kappa B activation in isolated acini. For example, in cancer cell lines stimulated with TNF-alpha , SB-203580 greatly inhibited NF-kappa B transactivation potential without affecting its DNA binding (4, 23). However, our results indicate that NF-kappa B alone does not mediate the effects of p38 MAPK on cytokine expression: MG-132, which almost prevented NF-kappa B activation, was less potent in inhibiting cytokine expression in pancreatic acini than the pyridinyl imidazoles.

Neither MG-132 nor SB-203580 had a significant effect on AP-1 binding activity toward the phorbol ester-responsive site. The AP-1 activation in pancreatic acini was, however, synergistically inhibited by a combination of SB-203580 and MG-132. Although the mechanism of this inhibition remains to be determined, it suggests that AP-1 may be involved in cytokine expression.

We compared the expression of cytokines/chemokines, and the activities of transcription factors and p38 MAPK, in normal pancreas and in isolated acini. We detected no or very little activity of NF-kappa B, AP-1, p38 MAPK, and cytokine mRNA expression in normal pancreatic tissue. Thus these key signaling pathways are all activated during the preparation of pancreatic acini. Because similar procedures are used for cell isolation from different organs (22, 33, 37) and for obtaining transplantation material (25, 36), the results we obtained are of importance and should be taken into account when data using primary cultures of various cell types are analyzed.

Pancreatic acini preparations are used in numerous studies of physiological and pathological effects of hormones, neurotransmitters, growth factors, etc. Sustained activation of cytokine expression and underlying signaling mechanisms may modulate acinar cell responses to physiological and pathological factors. If these mechanisms have already been activated in the process of cell isolation, the response to the stimulus can be blunted. It has been reported, for example, that the induction of chemokine Mob-1 expression by CCK is blunted in freshly isolated acini (14, 19), probably due to activation of NF-kappa B (19) as well as other transcription factors during cell isolation. Mob-1 expression greatly decreased after >2-h preincubation of the isolated acini at 37°C (14, 19); however, we observed no such "pacification" for IL-6 and MCP-1.

The procedure of cell isolation involves removal of tissue from the animal, followed by digestion of ECM to release cells from the tissue. We found that just removal of pancreatic tissue from the animal resulted in cytokine expression. Activation of cytokines in donor tissue should be taken into account during transplantation procedures (25, 36). For example, inhibition of cytokine production in donor tissue with p38 MAPK inhibitors may improve results of transplantation.

The ability of acinar cells to express and produce cytokines may play a role in various pathological conditions. In particular, cytokines have been shown to play a critical role in the development of acute pancreatitis (14-17, 20, 29, 31, 34). They attract inflammatory cells to the damaged area and mediate apoptotic and necrotic death of acinar cells (16, 29, 34). It has recently been shown that activation of NF-kappa B (17) and p38 MAPK (38) are early events in the development of pancreatitis. Our study provides further evidence that acinar cells themselves are able to activate signaling mechanisms mediating cytokine expression, which may initiate the inflammatory response in pancreatitis.

In conclusion, this study demonstrates expression of cytokines and chemokines by isolated pancreatic acini at both mRNA and protein levels. Cytokine expression in pancreatic acini is greatly inhibited by p38 MAPK inhibitors S-B202190 and SB-203580, and to a lesser extent, by NF-kappa B inhibition with MG-132. These results suggest a mediatory role for p38 MAPK and NF-kappa B in regulation of cytokine expression. Activation of NF-kappa B, AP-1, and p38 MAPK and induction of cytokine expression are caused by the procedure of isolating pancreatic acini from tissue.


    ACKNOWLEDGEMENTS

We thank Yoon Jung and Purvisa Patel for help with PCR experiments and Margaret Chu for preparing the manuscript.


    FOOTNOTES

This work was supported by the Department of Veterans Affairs, the National Institutes of Health, and the Andy Barnes Family Foundation.

Address for reprint requests and other correspondence: A. S. Gukovskaya, VA Greater Los Angeles Healthcare System, Bldg. 258, Rm. 340, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: agukovsk{at}ucla.edu).

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. Section 1734 solely to indicate this fact.

Received 9 February 2000; accepted in final form 13 July 2000.


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