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
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ABSTRACT |
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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)-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-
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-B; chemokines; interleukin-6; monocyte
chemotactic protein-1; pancreatitis
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INTRODUCTION |
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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-
(TNF-
) 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- and interleukin-6 (IL-6) and chemokines KC (murine
analog of growth-related protein GRO
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-B
(NF-
B) and activating protein 1 (AP-1) and of p38 mitogen-activated protein kinase (MAPK). NF-
B is a key regulator of cytokine
expression in different cell types (1, 7, 40). Recently,
we found that NF-
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-, IL-1
, 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-, KC, MCP-1, and MIP-2 and that the transcription factors NF-
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-
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-
B. In contrast to isolated pancreatic acini, we detected very little or no cytokine expression and
activation of NF-
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.
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EXPERIMENTAL PROCEDURES |
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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). [-32P]ATP was
from New England Nuclear (Boston, MA), and [
-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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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 -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.
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 -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.
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RESULTS |
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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-, 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-
is expressed in pancreatic acinar
cells.
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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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Inhibition of p38 MAPK and the transcription factor NF-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-
B, we used the proteasomal inhibitor MG-132, which
prevents translocation of activated NF-
B into the nucleus (2,
7). For p38 MAPK inhibition, we applied pyridinyl imidazoles SB-202190 and SB-203580 (9, 24, 35).
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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-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-
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-
B, 30-fold for AP-1, and
37-fold for p38 MAPK.
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DISCUSSION |
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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- (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-B. To inhibit
p38 MAPK, we used pyridinyl imidazoles SB-203580 and SB-202190. These
compounds selectively inhibit the
- and
-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-
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-
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-B DNA binding activity in isolated pancreatic acini. Conversely, MG-132 caused 80% inhibition of NF-
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-
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-
and MIP-2. These results indicate that NF-
B is involved in cytokine
expression. They also suggest that transcription factor(s) other than
NF-
B play a role in the regulation of IL-6, MCP-1, and KC expression
in isolated acini. Alternatively, the residual level of NF-
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-B DNA binding
activity, this does not necessarily mean that p38 MAPK is not involved
in NF-
B activation in isolated acini. For example, in cancer cell
lines stimulated with TNF-
, SB-203580 greatly inhibited NF-
B
transactivation potential without affecting its DNA binding (4,
23). However, our results indicate that NF-
B alone does not
mediate the effects of p38 MAPK on cytokine expression: MG-132, which
almost prevented NF-
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-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-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-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-B inhibition with MG-132. These results suggest a mediatory role
for p38 MAPK and NF-
B in regulation of cytokine expression.
Activation of NF-
B, AP-1, and p38 MAPK and induction of cytokine
expression are caused by the procedure of isolating pancreatic acini
from tissue.
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ACKNOWLEDGEMENTS |
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We thank Yoon Jung and Purvisa Patel for help with PCR experiments and Margaret Chu for preparing the manuscript.
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FOOTNOTES |
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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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