Fibroblasts stimulate acinar cell proliferation through IGF-I during regeneration from acute pancreatitis

C. U. Ludwig, A. Menke, G. Adler, and M. P. Lutz

Department of Internal Medicine I, University of Ulm, D-89070 Ulm, Germany


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Pancreatic regeneration after caerulein-induced pancreatitis is characterized by transient fibroblast proliferation followed by replication of acinar cells. The mechanisms that coordinate regeneration are incompletely understood. In this study, we examine the role of insulin-like growth factor I (IGF-I). Acute edematous pancreatitis was induced in rats by 12 h caerulein infusion. Pancreatic IGF-I mRNA levels increased over 50-fold during regeneration, reaching a maximum at day 2. Immunohistochemically, IGF-I was localized to fibroblasts within the areas of interstitial tissue. IGF-I mRNA was demonstrated in primary cultures of pancreatic fibroblasts but not in cultured pancreatic acinar cells. However, with the use of Western blotting acinar cells did express IGF-I receptors. IGF-I stimulated 5-bromo-2'-deoxyuridine uptake and increased numbers of acinar cells in a dose-dependent manner. Stimulation was half maximal at 1.1 nM and completely inhibited by an IGF-I antagonist and by IGF binding protein-3 (IGFBP-3). Possible paracrine regulation was confirmed by stimulation of acinar cell proliferation with fibroblast-conditioned medium, which was partially inhibited by IGF-I antagonist or by IGFBP-3. We conclude that acinar cell proliferation during late regeneration from pancreatitis is mediated at least in part by paracrine release of IGF-I from fibroblasts.

pancreas; insulin-like growth factor I; insulin-like growth factor binding protein-3; injury


    INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References

COMPLETE PANCREATIC regeneration is observed within 2 wk after caerulein-induced acute edematous pancreatitis (5). The sequence of events during regeneration includes inflammation, transient activation and proliferation of fibroblasts, deposition of extracellular matrix, and proliferation of acinar cells (6). Accordingly, thymidine incorporation into DNA exhibits a biphasic distribution, with a first peak of activity localized to fibroblasts at day 1 and a second peak after 4-7 days that is localized to acinar cells (5, 6). The mechanisms that control this sequence are only partially understood. During early regeneration, transforming growth factor-beta seems to be one of the most important regulatory factors and is thought to stimulate the transient increase in fibroblast proliferation and the deposition of extracellular matrix in a paracrine manner (20). The factors that may stimulate acinar cell proliferation during late regeneration are less well characterized. Acinar cell proliferation in vitro is stimulated by epidermal growth factor (17), basic fibroblast growth factor (12), hepatocyte growth factor (23), or insulin-like growth factor I (IGF-I) (23), the latter being one of the most efficient inducers of DNA synthesis (17). IGF-I is a polypeptide hormone that stimulates cell growth and differentiation largely through high-affinity binding to the IGF-I receptor (1). During fetal development, IGF-I expression is localized to connective tissue or mesenchymal cells and probably regulates proliferation of neighboring cells through paracrine mechanisms (10). After injury, IGF-I mRNA is upregulated in a variety of tissues, including skin (8), kidney (19), muscle (13, 16), or bone (2) and is thought to regulate tissue remodeling. There is increasing evidence that IGF-I may have a similar role in pancreatic regeneration, which is mainly based on the observation that IGF-I mRNA levels are elevated after partial pancreatectomy (3, 11, 22).

Therefore, we more closely examined the role of IGF-I during regeneration from acute edematous pancreatitis. We suggest that the acinar cell proliferation during late regeneration is mediated by paracrine release of IGF-I from fibroblasts.


    MATERIALS AND METHODS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

Materials

Male Wistar rats weighing between 150 and 200 g were bred at the animal care and treatment facility of the University of Ulm. Caerulein was purchased from Farmitalia (Freiburg, Germany). An IGF-I cDNA probe was obtained from American Type Culture Collection (no. 63070; Manassas, VA). Mouse epidermal growth factor was from Molecular Probes (Göttingen, Germany), and insulin was from Sigma (Deisenhofen, Germany). IGF-I was purchased from Saxon Biochemicals (Hannover, Germany). The IGF-I antagonist (21) and human IGF binding protein-3 (IGFBP-3) were purchased from Biomol (Hamburg, Germany). Polyclonal anti-IGF-I antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany).

Methods

Experimental pancreatitis. Rats were infused via tail vein catheter with 10 µg · kg-1 · h-1 caerulein for 12 h. Control rats were infused with saline. Rats were killed under ether anesthesia by exsanguination at different time points after the start of caerulein infusion. Explanted pancreatic tissue was snap frozen in liquid nitrogen or fixed in 4% paraformaldehyde for further studies. The experimental protocol was approved by the governmental animal care review committee.

Northern blots. RNA was extracted from frozen tissue as described (20). RNA from cultured cells was extracted using the Quiagen RNeasy Midi kit (Hilden, Germany) according to the manufacturer's guidelines. For Northern blot analysis, 30 µg of total RNA were separated by gel electrophoresis in 1% agarose-2.2 M formaldehyde gels and transferred by capillary elution to Hybond N membranes (Amersham-Buchler, Braunschweig, Germany). Hybridization with the purified [alpha -32P]dCTP-labeled IGF-I cDNA probe was performed as described (20). To verify equal loading and blotting of total RNA, ethidium bromide staining of the agarose gels and hybridization with an 18S rRNA probe were used. After autoradiography, mRNA expression was quantified densitometrically using the Sigma Plot software.

Immunohistochemistry. Five-micrometer sections of paraffin-embedded pancreatic tissue were blocked with PBS without calcium or magnesium containing 3% BSA. Anti-IGF-I antibody in PBS-0.3% BSA was added for 1 h at 37°C. Antibody staining was visualized using Cy3-conjungated secondary antibody (Biomol).

Western blot analysis. Total cellular protein extracts were examined for IGF-I receptor beta -chain expression by Western blotting as described (18) using a polyclonal rabbit anti-IGF-I receptor beta -antibody (clone C20, Santa Cruz Biotechnology) in Tris-buffered saline-2% BSA-2% nonfat dry milk.

Isolation and culture of pancreatic acinar cells. Dispersed rat pancreatic acini were prepared under sterile conditions by sequential enzymatic and mechanical dissociation as described (18). The viability of acini after this preparation procedure remained in excess of 95%, as assessed by trypan blue exclusion. After two washes with Waymouth medium containing 10% FCS, acini were plated on eight-well LabTec chamber slides (Nunc, Wiesbaden, Germany) or on Primaria dishes (Becton Dickinson, Heidelberg, Germany) in Waymouth medium supplemented with 10% FCS, 1.6 nM epidermal growth factor, 0.8 µM insulin, 10 nM cholera toxin, 1 µg/ml hydrocortisone, and antibiotics modified according to the methods of Hoshi and Logsdon (12) and Hall and Lemoine (9). Cell density was adjusted to obtain 30-50% confluency after 4 days of culture. Cells were grown at 37°C in 5% CO2. Medium was exchanged every second day. The epithelial origin of cultured cells was confirmed using immunocytological staining for E-cadherin (Transduction Laboratories), and, to demonstrate acinar cell characteristics, the presence of zymogen granules was shown by transmission electron microscopy. In addition, fibroblasts were differentiated by their relative abundance of stress fibers using rhodamine-phalloidin. As judged by these criteria, contamination with fibroblasts was <2% under all culture conditions. Conditioned medium was obtained from rat pancreatic acinar cells in primary culture after six days of culture. After two washing steps in Waymouth medium for 30 min, Waymouth medium was added for another 24 h and frozen at -20°C before further use.

Isolation and culture of rat pancreatic fibroblasts. Pancreatic tissue samples of ~1 mm3 were obtained from untreated male Wistar rats after exsanguination under ether anesthesia; samples were placed on cell culture dishes in DMEM containing 10% FCS under standard culture conditions. Fibroblasts were allowed to grow out of the tissue specimens and were subcloned twice in DMEM-10% FCS. Purity was confirmed by negative staining for E-cadherin and by the abundance of actin stress fibers. To generate conditioned medium, adherent cells were washed twice for 30 min with Waymouth medium. Four milliliters of Waymouth medium were added to each 250-ml flask for 24 h. The conditioned media were stored at -20°C until further use.

Proliferation assays. After 4 days of culture, cells were washed two times for 30 min with Waymouth medium. Waymouth medium-1% FCS or fibroblast acinar cell-conditioned medium was added for 24 h with different concentrations of IGF-I, IGFBP-3, or IGF-I antagonist. FCS (1%) had to be added to the Waymouth medium because cell numbers decreased with lower concentrations of FCS. Next, 5-bromo-2'-deoxyuridine (BrdU; Serva, Heidelberg, Germany) was added together with 2'-deoxycytidine (Serva) to obtain a final concentration of 20 µM each. After 18 h, cells were fixed by adding -20°C methanol for 6 min; cells were then treated with 1 M HCl for 20 min. Monoclonal anti-BrdU antibody (DAKO, Hamburg, Germany) was added in PBS-0.5% Tween 80 for 40 min at 37°C. Staining was visualized using biotinylated rabbit anti-mouse immunoglobulin (DAKO), peroxidase-conjugated streptavidin (DAKO), and 3,3'-diaminobenzidine tetrahydrochloride as color substrate. Cells were counterstained with eosin.

Statistics

The total cell number in each well was counted under ×40 magnification using a Zeiss Axiophot microscope. The percentage of proliferating cells was calculated as the number of BrdU-positive nuclei × 100 divided by the total cell number. Numbers are given as means ± SE. For comparisons, the two-tailed Student's t-test for unpaired samples was used. P values below 0.05 were judged as significant.


    RESULTS
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Abstract
Introduction
Materials and methods
Results
Discussion
References

IGF-I mRNA Expression During Regeneration From Acute Pancreatitis

Expression of IGF-I mRNA was examined by Northern blotting after induction of caerulein-induced acute edematous pancreatitis. As shown in Fig. 1 (left), IGF-I mRNA levels were barely detectable in control organs. Within 1 day after the induction of acute pancreatitis, levels of four IGF-I mRNA transcripts increased significantly. IGF-I mRNA transcripts migrated at ~1.2, 1.9, 4.7 and 7.5 kb. Expression of all transcripts increased to over 50-fold of basal levels, and a maximum increase was observed 2 days after the start of caerulein infusion during regeneration from acute pancreatitis. During further regeneration, IGF-I mRNA levels decreased continuously.


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Fig. 1.   Northern blot analysis of insulin-like growth factor I (IGF-I) mRNA in pancreatic tissue during regeneration from acute caerulein-induced pancreatitis (left) or in cultured pancreatic fibroblasts and acinar cells (right). Time points after the start of caerulein infusion are indicated (left). Arrows indicate IGF-I transcripts. Bottom panels: control hybridizations were performed using an 18S ribosomal RNA probe. Results are representative of 3 independent experiments.

IGF-I mRNA Expression in Primary Cultures

To determine the possible source of IGF-I production, we compared IGF-I mRNA expression in primary cultures of pancreatic acinar cells or pancreatic fibroblasts. As shown in Fig. 1 (right), IGF-I mRNA was not detectable in cultured acinar cells, whereas fibroblasts demonstrated a strong signal for IGF-I mRNA at 4.7 kb and demonstrated less prominent but clearly detectable transcripts migrating at 1.2 and 1.9 kb. The difference in transcript expression in cultured cells compared with tissue extracts likely is due to altered processing (3). Control hybridizations were unable to detect an IGF-II mRNA signal in cultured acinar cells or fibroblasts.

Localization of IGF-I in the Pancreas After Acute Edematous Pancreatitis

To determine the pancreatic source of IGF-I, tissues from rats were collected at various time points after the start of caerulein infusion. After immunohistochemical staining for IGF-I, the fluorescent signal was restricted to the areas of connective tissue adjacent to exocrine and endocrine parts of the gland. Because these areas mainly contain fibroblasts and some macrophages, counterstaining with FITC-coupled anti-CD68 antibody was performed to demonstrate that most of the IGF-I-labeled cells did not costain with the macrophage marker. Again, maximum labeling was detected on the second day after induction of pancreatitis, which is demonstrated in Fig. 2.


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Fig. 2.   IGF-I expression in rat pancreatic tissue 2 days after induction of pancreatitis. Left: immunohistochemical staining for IGF-I. Right: phase contrast micrograph of the same section (magnification = ×400). Arrows indicate IGF-I-positive cells.

Expression of the IGF-I Receptor in Cultured Pancreatic Acinar Cells

The presence of the IGF-I receptor in cultured acinar cells was demonstrated by Western blot analysis. As shown in Fig. 3, staining with antibodies against the IGF-I receptor beta -chain revealed two bands representing the IGF-I receptor beta -subunit and its precursor protein migrating at 205 kDa.


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Fig. 3.   IGF-I receptor (R) expression in primary cultures of rat pancreatic acinar cells. Total cellular extracts of acinar cell cultures were analyzed by Western blotting. Arrows indicate the beta -subunit of the IGF-I receptor [relative molecular weight (Mr) = 95,000] and its precursor protein (Mr = 205,000).

Growth Stimulation of Cultured Pancreatic Acinar Cells by IGF-I

To demonstrate that IGF-I was indeed able to stimulate proliferation of pancreatic acinar cells in culture, we measured IGF-I-induced changes of the BrdU labeling index. IGF-I significantly increased the BrdU labeling index in a concentration-dependent manner (Fig. 4). Maximum labeling was 265 ± 37% of control and was reached at a concentration of 10 nM IGF-I. Labeling did not increase further with higher concentrations of IGF-I. Under similar conditions, relative cell number increased to 194 ± 45%. Half-maximal stimulation of BrdU labeling was calculated at 1.1 nM using Sigma Plot software. Of note, this concentration is consistent with the activation of a specific IGF-I receptor and is clearly different from binding characteristics obtained with the insulin or IGF-II receptors (15). In addition, specificity of IGF-I stimulation was confirmed using a competitive IGF-I peptide antagonist (21). Addition of the IGF-I antagonist significantly (P = 0.02) inhibited IGF-I-stimulated proliferation in a dose-dependent manner (Fig. 5). Inhibition was complete and was half maximal at 76 nM, i.e., an eightfold excess of inhibitor over the IGF-I concentration. When IGFBP-3 was used as IGF-I scavenger, the mitogenic activity of IGF-I was significantly decreased by up to 20 nM IGFBP-3 (P = 0.006) (Fig. 5). IGFBP-3 alone had no effect on basal acinar cell growth.


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Fig. 4.   Growth stimulation of primary acinar cell cultures in response to IGF-I. Four-day cultures of pancreatic acini were stimulated with increasing concentrations of IGF-I for 42 h. Fraction of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells was determined after an 18-h incubation with BrdU. Results are given as means ± SE of at least 3 independent experiments.


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Fig. 5.   Inhibition of IGF-I-stimulated acinar cell proliferation by a peptide antagonist of IGF-I or by IGF binding protein-3 (IGFBP-3). Four-day cultures of pancreatic acini were stimulated with 10 nM IGF-I in the presence of the indicated concentrations of the antagonist or of IGFBP-3 for 42 h. Fraction of BrdU-labeled cells was determined after an 18-h incubation with BrdU. Results are given as means ± SE of at least 3 independent experiments.

Growth Stimulation of Acinar Cells by Fibroblast-Conditioned Medium

We next examined whether fibroblasts were able to stimulate growth of pancreatic acinar cells. Fibroblast-conditioned medium (without FCS) increased the labeling index of rat pancreatic acinar cells in primary culture to 355 ± 90% (P = 0.003) of control and total cell number to 333 ± 108% of control (P = 0.07; Fig. 6). This increase was similar to that seen in the presence of 1% FCS and 10 nM IGF-I (265 ± 37%). To demonstrate that IGF-I in the conditioned medium was responsible for the effects observed, 10 µM IGF-I antagonist was added. In the presence of the antagonist, there was partial inhibition of the growth stimulatory effects of the conditioned medium by 60 ± 6% (P = 0.06). In similar experiments, the addition of 20 nM IGFBP-3 decreased the stimulated proliferation rate significantly by 75 ± 17% (P = 0.01). To exclude autocrine stimulation, 20 nM IGFBP-3 or 10 µM IGF-I antagonist was added to acinar cell-conditioned medium. Both inhibitors had no effects on either the proliferation rate (labeling index under basal conditions = 8.6 ± 0.8%; 8.9 ± 0.1% with IGF-I antagonist, 10.4 ± 2.1% with IGFBP-3, and 25.4 ± 1.7% with 10 nM IGF-I) or the total cell count of cultured acinar cells.


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Fig. 6.   Growth stimulation of rat pancreatic acinar cells by pancreatic fibroblast-conditioned medium and inhibition by a peptide antagonist of IGF-I or by IGFBP-3. Four-day cultures of pancreatic acini were incubated with rat pancreatic fibroblast-conditioned medium alone or with conditioned medium in the presence of 10 µM IGF-I antagonist or 20 nM IGFBP-3 for 42 h. Fraction of BrdU-labeled cells was determined after an 18-h incubation with BrdU. Results are given as means ± SE of at least 3 independent experiments.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

The mechanisms that coordinate pancreatic regeneration after pancreatitis are incompletely understood. In this study, we examined the potential function of IGF-I. IGF-I is an important physiological mitogen for epithelial cells (15) that acts not only through the systemic circulation but is widely synthesized locally and has important paracrine and autocrine actions (4, 10). Locally produced IGFs are thought to regulate regeneration after acute kidney or muscle injury, where they are produced by the regenerating cells (13, 19) or by renal cortical fibroblasts (14). Similar mechanisms may occur in the pancreas, where IGF-I mRNA is overexpressed during regeneration after partial pancreatectomy (3, 11) in the focal areas of regeneration (22).

The role of IGF-I during regeneration from pancreatitis is less clear. In a model of acute edematous pancreatitis in which organ damage was induced by repeated injection of supramaximal secretory concentrations of caerulein over 2 days, mRNA levels of IGF-I started to increase 24 h after the start of the first injection (3). However, interpretation is difficult, since maximum increases of IGF-I mRNA levels occurred during ongoing caerulein stimulation and thus might be a direct effect of caerulein stimulation or, as proposed by the authors (3), might be related to regeneration. In our model, pancreatitis was induced by continuous infusion of supramaximal secretory concentrations of caerulein for 12 h. Maximum expression of IGF-I mRNA was observed at day 2 after the start of infusion and corresponded to the start of replication of acinar cells (5), suggesting a role for IGF-I in proliferation of these cells and in regeneration of the organ.

The possible pancreatic sources of IGF-I production have been examined by Smith et al. (22) after partial pancreatectomy. These authors detected maximum labeling of IGF-I mRNA in connective tissue cells and in proliferating ductule epithelial cells within the focal areas of regeneration. Likely sources of IGF-I within the connective tissue included fibroblasts and macrophages, which are known to secrete IGF-I (10). In our study, IGF-I protein expression was maximal in the areas of connective tissue between lobules. These areas contain mainly proliferating fibroblasts (5) and some macrophages, which did not express significant amounts of IGF-I. Because acinar cells do express functional IGF-I receptors, we hypothesized that fibroblasts stimulate proliferation of acinar cells during regeneration from acute caerulein-induced pancreatitis through paracrine secretion of IGF-I. This hypothesis is supported by the sequence of proliferation in the regenerating pancreas, where maximum fibroblast proliferation clearly precedes acinar cell replication (7) and maximum IGF-I expression. Of note, whereas IGF-I and IGF-I receptor expression in regenerating muscle tissue is maximal in the proliferating cell compartment and thus is thought to demonstrate autocrine stimulation (13), regeneration of injured kidney is regulated by autocrine and paracrine mechanisms (14, 19), and regeneration of exocrine pancreatic cells seems to be regulated in a paracrine fashion mainly because acinar cells do not express detectable amounts of IGF-I.

To further support this hypothesis, the basic mechanisms of paracrine stimulation were confirmed in cell culture as follows: 1) expression of IGF-I mRNA was demonstrated in primary cultures of pancreatic fibroblasts, 2) primary cultures of pancreatic exocrine cells were shown to express the IGF-I receptor, 3) their proliferation could be stimulated by IGF-I in a specific manner, and 4) proliferation could be stimulated by fibroblast-conditioned medium, an effect that was partially inhibited by using either a competitive IGF-I antagonist or IGFBP-3. In contrast, growth stimulation by autocrine mechanisms was likely not relevant, since growth of acinar cell cultures was not influenced by the IGF-I antagonist or by IGFBP-3 alone. In addition, IGF-I mRNA expression was not detectable by Northern blotting in our acinar cell cultures or by in situ hybridization in acinar cells of the intact pancreas (22).

In summary, we provide evidence for paracrine stimulation of acinar cell proliferation by release of IGF-I from fibroblasts. We propose that this is one of the mechanisms that regulates pancreatic regeneration from acute edematous pancreatitis. Within this model, the transient increase of activated fibroblasts at days 1 and 2 would lead to enhanced secretion of IGF-I. Paracrine stimulation then initiates acinar cell replication and ultimately results in complete organ reconstitution. We postulate that deregulation of this sequence could be responsible for incomplete regeneration and might be involved in the pathogenesis of chronic pancreatitis.


    ACKNOWLEDGEMENTS

We thank B. B. M. Flossmann-Kast, R. Voisard, H. Lührs, E. Wolff-Hieber, and C. Längle for suggestions and expert technical assistance.


    FOOTNOTES

This work was supported in part by the Deutsche Forschungsgemeinschaft (Lu 441/2-2 and Kn 200/4-5).

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: M. P. Lutz, Dept. of Internal Medicine I, Univ. of Ulm, Robert-Koch-Str. 8, D-89070 Ulm, Germany.

Received 1 May 1998; accepted in final form 1 October 1998.


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Abstract
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Materials and methods
Results
Discussion
References

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Am J Physiol Gastroint Liver Physiol 276(1):G193-G198
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