Calpain mediates caspase-dependent apoptosis initiated by hydrogen peroxide in pancreatic acinar AR42J cells.

Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H2O2-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H2O2 induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H2O2-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.

Differentiation potential of pancreatic fibroblastoid cells/stellate cells: effects of peroxisome proliferator-activated receptor gamma ligands.

Pancreatic stellate cells have been investigated mostly for their activation process, supposed to support the development of pancreatic disease. Few studies have been presented on reversal of the activation process in vitro. Thiazolidinediones (TZDs) have been used as antidiabetics and have now been reported to exert antifibrotic activity. We tested effects of natural and synthetic ligands of peroxisome proliferator-activated receptor gamma (PPARγ) on human pancreatic fibroblastoid cells (hPFCs) in search for specificity of action. Ciglitazone, as a prototype of TZDs, was shown to have reversible growth inhibitory effects on human pancreatic fibroblastoid cells/stellate cells. Cells treated with ciglitazone for three days showed enhanced lipid content and induction of proteins involved in lipid metabolism. Collagen synthesis was reduced in hPFC. Interaction of PPARγ with DNA binding sites upon ligand binding was shown by gel shift analysis. These findings point toward a potential for adipocyte differentiation in human pancreatic fibroblastoid cells.

Granzyme K: a novel mediator in acute airway inflammation.

BACKGROUND: Granzymes are a subfamily of serine proteases involved in the pathogenesis of many inflammatory disorders. In contrast with granzyme A and B, the role of granzyme K (GrK) in human lung diseases is unknown. Therefore, the release and expression of GrK in allergic asthma, chronic obstructive pulmonary disease (COPD) and bronchopneumonia were investigated. METHODS: Soluble GrK was quantified using an enzyme linked immunosorbent assay in the bronchoalveolar lavage fluid of patients with allergic asthma (before and after segmental allergen challenge), and in patients with mild COPD, pneumonia and in healthy controls. The molecular form of GrK was analysed by western blot. Flow cytometry was performed to determine the cellular expression of GrK. RESULTS: Compared with healthy controls, there were normal levels of soluble GrK in the bronchoalveolar lavage fluid of patients with COPD, and patients with allergic asthma before allergen challenge. In contrast, soluble GrK was strongly increased in the bronchoalveolar lavage fluid of patients with acute bronchopneumonia. In patients with allergic asthma, there was a significant increase in soluble GrK as well as in GrK expressing CD8(+) T cells in the bronchoalveolar lavage fluid 24 h and 72 h after allergen challenge. After allergen challenge, soluble GrK correlated with the percentage of GrK expressing CD8(+) T cells. Finally, it was shown that the endobronchial release of the CCR5 ligand CCL3 might be a mechanism for the recruitment of GrK(+)CD8(+) T cells after allergen challenge. CONCLUSION: These data provide the first evidence that expression of GrK is upregulated in acute airway inflammation, both in infectious and non-infectious diseases.

Extracellular signal regulated kinases are key mediators of mitogenic signals in rat pancreatic stellate cells.

BACKGROUND: Pancreatic stellate cells (PSCs) have been implicated in pancreatic fibrosis as they synthesise increased amounts of extracellular matrix proteins in response to activation by profibrogenic mediators such as cytokines. AIMS: The purpose of this study was to analyse cytokine receptor stimulated signalling pathways involved in PSC activation. Using a rat culture model of PSCs, we have also tested the potential of the platelet derived growth factor (PDGF) antagonist trapidil and PD98059, a specific inhibitor of extracellular signal regulated kinase (ERK) activation, to suppress PSC growth. METHODS: Cultured PSCs were stimulated with PDGF, and the signal transduction pathways activated in response to the mitogen were analysed by immunoblotting, kinase assays, and electrophoretic mobility shift assays. Furthermore, comparison of signalling cascades activated in PSCs before and after completing transdifferentiation to alpha-smooth muscle actin expressing myofibroblasts was performed. Biological effects of PDGF, trapidil, and PD98059 were analysed by proliferation assays and correlated with molecular effects of the substances. RESULTS: PDGF induced rapid activation of Raf-1, ERKs 1 and 2, as well as AP-1 proteins. The transforming growth factor beta activated transcription factor Smad2 was found to be constitutively phosphorylated in PSCs of different transdifferentiation grades. Furthermore, the results indicate a correlation between ERK activities and induction of PSC activation. Trapidil efficiently inhibited both PDGF induced ERK activation and, in common with PD98059, PSC proliferation. CONCLUSIONS: Our data suggest that ERKs play a key role in the regulation of PSC growth and that inhibition of the ERK signalling pathway may become a strategy to prevent activation of these cells.

Expression of hepatocyte growth factor, keratinocyte growth factor and their receptors in experimental chronic pancreatitis.

BACKGROUND: Hepatocyte (HGF) and Keratinocyte growth factors (KGF) are key factors of tissue organization and regeneration. These peptide growth factors and their receptors c-met and keratinocyte growth factor receptor (KGFR) are overexpressed in pancreatic cancer. AIM: Expression and localization of ligands and receptors were investigated during the development of experimental chronic pancreatitis. METHODS: Chronic pancreatitis was induced in rats by intravenous injection of dibutyltin dichloride. One to 60 days after treatment, the expression of growth factors and receptors was analysed by competitive polymerase chain reaction, Western blot analysis and immunohistochemistry. RESULTS: HGF mRNA expression increased (10-fold) until days 7-14 followed by a decrease to control level. Expression of c-met mRNA constantly increased (15-fold). KGF and KGFR mRNA expression were increased after 14-28 days (5-fold) and then returned to control levels. mRNA expression patterns correlated with changes in the protein expression, whereas protein levels of KGF remained unchanged. Ligands were localized in mesenchymal cells and their receptors on epithelial cells. CONCLUSIONS: The significant increase of HGF and c-met expression suggests an essential role of this growth factor in the morphological changes during the development of chronic pancreatitis. Changes in the expression of KGF and KGFR are less pronounced.

Cytokine mRNA levels and lymphocyte infiltration in pancreatic tissue during experimental chronic pancreatitis induced by dibutyltin dichloride.

There is little information available regarding the role of inflammatory cells in the pathogenesis of chronic pancreatitis. Therefore, we analyzed the local cytokine profile and infiltrating lymphocytes in a rat model of chronic pancreatitis. Experimental pancreatitis was induced by a single intravenous application of dibultyltin dichloride (DBTC). During a time course of two months we observed the mRNA expression of cytokines using competitive RT-PCR. Lymphocytes were characterized by immunohistochemistry, FACS analysis, and the lymphocyte proliferation test. IL-1beta, IL-6, IL-5, and IL-10 were immediately up-regulated in the acute phase of disease, while lymphocyte-restricted expression of IL-2, IL-2R, and IFN-y was only found in the chronic course. Among the infiltrating lymphocytes, CD4+ cells dominated, but during the chronic process there was an increase of CD8+ cells, resulting in a reduced CD4/CD8 ratio. Mitogen-induced activation of isolated mesenteric lymph node cells increased during the chronic inflammation. Our results suggest that in experimental pancreatitis acute inflammatory reactions are followed by a T-lymphocyte-mediated process.

Gut-derived effector T cells circulating in the blood of the rat: preferential re-distribution by TGFbeta-1 and IL-4 maintained proliferation.

Effector T cells generated in mesenteric lymph nodes (mLN) preferentially accumulate in mLN and sites drained by them, such as Peyer's patches and the lamina propria of the gut, after circulation in the blood. The molecular mechanisms mediating this re-distribution are poorly understood. To study this, rat T cells from mLN were activated via the T cell receptor and CD28, and injected either intravenously into congenic recipients, or maintained in culture in the presence of various cytokines. Three days later effector T cells were identified in vivo and in vitro, and surface molecule expression and proliferation rate was determined. The data show that in vivo effector mLN T cells express significantly higher levels of activation markers and maintain a higher proliferation rate after entering the mLN environment (tissue of origin) than after entering the peripheral LN environment (unrelated site). The proliferation is mediated by TGFbeta-1 and IL-4 present in mLN. The requirement for these cytokines is imprinted on effector mLN T cells during the initial activation. Thus, the preferential proliferation of effector mLN T cells in milieus providing the cytokine mixture experienced during activation ensures a privileged accumulation at sites where they are most needed. This can be used to manipulate the effector phase of an immune response.

The course of pancreatic fibrosis induced by dibutyltin dichloride (DBTC).

In summary, in addition to an acute interstitial pancreatitis the organotin compound DBTC induced a pancreatic fibrosis in rats. The course of the pancreatic fibrosis was studied 2-36 weeks after single i.v. treatment of rats with 6 or 8 mg/kg DBTC. The pancreatic fibrosis induced by DBTC differs from other experimental models of acute pancreatitis. Extensive infiltration by mononuclear cells is present in fibrotic areas without pancreatic atrophy or lipomatosis. The presence of chronic inflammatory lesions characterized by the destruction of exocrine parenchyma and fibrosis and in the later stages the endocrine parenchyma, indicate a chronic pancreatitis. In completion of the experimental model of the DBTC-induced acute interstitial pancreatitis in rats, the described late fibrotic effects on rat pancreas may be used as an experimental model of chronic pancreatitis.

Immunohistochemical characterization of the pancreatic cellular infiltrate in normal pancreas, chronic pancreatitis and pancreatic carcinoma.

BACKGROUND/AIMS: Chronic pancreatitis is histologically characterized by an extended fibrosis and infiltration of leukocytes. We intended to differentiate the infiltration to evaluate the inflammatory process. METHODS: Samples of tissues of normal pancreas (NP, n = 12), of chronic pancreatitis (CP, n = 7), and pancreatic tissues surrounding pancreatic carcinoma (CA, n = 7) were investigated by immunohistochemical staining using the APAAP technique. RESULTS: In normal pancreas, mononuclear cells (47.1 +/- 26.0 cells/mm2) were observed with a predominance of macrophages (56.3%) and T lymphocytes (31.3%) which were differentiated in CD8+ lymphocytes (9.3 +/- 7.2 cells/ mm2) and CD4+ lymphocytes (6.7 +/- 3.2 cells/mm2). Rarely, plasma cells (5.3%) and B lymphocytes (7.1%) could be detected. In pancreatic tissue of patients with CP and in CA there was a significant increase of mononuclear cells to 264.4 +/- 120.3 cells/mm2 and 284.3 +/- 67.8 cells/mm2, respectively. In both diseases percentages of T lymphocytes (CP: 50.5%; CA: 48.1%) were higher than in normal controls. CD4+/CD8+ ratio of 0.77 in CP and 0.82 in CA demonstrated a predominance of CD8+ cells compared to the peripheral blood. In NP and CA, nearly all T lymphocytes expressed CD45R0 identifying memory cells, while only 58% of T lymphocytes were CD45R0 positive in CP. CONCLUSION: Our data suggest that the investigated cases of CP were of a common inflammatory type rather than due to an autoimmunological reaction. CD8+ T lymphocytes were the predominant T cell subset in the inflammatory infiltrates in both CP and CA.

Secretory immunoglobulin A in pancreatic juice and pancreatic tissue of patients with chronic pancreatitis.

BACKGROUND: The predominance of secretory IgA (S-IgA) in intestinal secretions compared with blood is well established, but concentrations of this protein in pancreatic juice and its origin, especially in chronic pancreatitis, are unknown. AIMS: To investigate the role of S-IgA in chronic pancreatitis. PATIENTS: Twenty one patients with chronic pancreatitis (group I), three patients with proven malignancies (group II), and 12 patients without pancreatic disease (group III). METHODS: Pure human pancreatic juice was collected endoscopically in four fractions after consecutive stimulation with secretin and cholecystokinin (CCK). Samples were analysed for S-IgA, protein, trypsinogen, and proteolytic activity. RESULTS: The S-IgA level was significant increased in fraction 1 of pancreatic juice of group I (1210 (1411) ng/ml) compared with controls (33 (70) ng/ml). Protein concentrations and trypsinogen content were lower in group I than in the other groups. Proteolytic activity could be observed in 53% of all 133 pancreatic juice samples, but in 87% of fraction 1. In pancreatic tissue of three patients with chronic pancreatitis both IgA and secretory component were detected by immunohistology. Expression of the secretory component by human pancreatic epithelial cells was increased in patients with chronic pancreatitis compared with normal controls. The concentration of S-IgA in pancreatic juice did not correlate with the serum S-IgA level. In contrast, serum levels of S-IgA were decreased in patients with chronic pancreatitis. CONCLUSION: There are high levels of S-IgA in human pancreatic juice following chronic inflammation and a protective role is suggested for this immunoglobulin.

Caerulein pancreatitis increases mRNA but reduces protein levels of rat pancreatic heat shock proteins.

We have recently reported that preconditioning through hyperthermia induces expression of pancreatic heat shock proteins (HSPs) and protects against caerulein pancreatitis. Here, we investigate caerulein-mediated effects on pancreatic HSPs without prior hyperthermia. Caerulein time and dose dependently increased pancreatic mRNA levels of the constitutive isoform of HSP70 (HSC70). However, pancreatic HSC70 protein levels were decreased, as were HSP60 protein levels. Also, in contrast to hyperthermia preconditioning, caerulein did not induce measurable levels of mRNA or protein of the inducible isoform of HSP70. Thus the pancreas reacts to different kinds of stress (hyperthermia vs. hyperstimulation) with differential induction of HSP mRNAs. Clearly, hyperthermia leads to induction of HSP protein expression, whereas caerulein treatment does not. Therefore, our current study further supports the idea that hyperthermia-induced protection against caerulein pancreatitis may be mediated through increased protein levels of pancreatic HSPs. It is further tempting to hypothesize that failure to appropriately increase HSP protein levels in response to high doses of caerulein might be a factor in the development of pancreatitis.

Pancreatic fibrosis in experimental pancreatitis induced by dibutyltin dichloride.

BACKGROUND & AIMS: Regulatory mechanisms in chronic pancreatitis finally resulting in pancreatic fibrosis cannot be studied sufficiently in human pancreas. Results of a new pancreatitis model in rats suitable for investigation of the processes leading to pancreatic fibrosis are presented. METHODS: Experimental pancreatitis was induced by intravenous application of 8 mg/kg body wt dibutyltin dichloride. Pancreatitis was characterized by histology, serum parameters, and immunohistochemistry, detecting inflammatory cells. Gene expression of collagen type I and transforming growth factor beta1 was shown by Northern blot analysis. RESULTS: Dibutyltin dichloride induced an acute edematous pancreatitis within 24 hours. Extensive infiltration with mononuclear cells could be observed after day 7 followed by the development of fibrosis. Parallel to the cell infiltration, an upregulation of messenger RNA-encoding collagen type I and transforming growth factor beta1 could be shown. An active inflammatory process could be shown until the end of the observation period, i.e., 2 months. CONCLUSIONS: The findings suggest that dibutyltin dichloride-induced pancreatitis in rats is suitable to study cellular interactions and mediators involved in the development of pancreatic fibrosis.

Conditional expression of human TNF-alpha: a system for inducible cytotoxicity.

Tumour necrosis factor-alpha (TNF-alpha) is currently being used in clinical trials for cancer treatment, but toxic side effects, due to systemic administration and high doses, are observed. Inducible expression of TNF may permit selective killing of tumour cells in gene therapy protocols without need for prolonged and/or high-level TNF expression. A conditional TNF expression vector has been constructed in which the coding sequences of human TNF have been placed under the transcriptional control of the glucocorticoid-regulated murine mammary tumour virus long terminal repeat (MMTV-LTR). Negligible levels of TNF expression, associated with no phenotypic alterations, are observed in cells transfected with MMTV-TNF vectors in the absence of glucocorticoid. Expression levels could be stimulated by the addition of the synthetic glucocorticoid dexamethasone. Increasing expression levels of TNF were associated with enhanced cytotoxicity. Our results suggest the potential use of inducible TNF systems for the treatment of tumours in gene therapy protocols.

Interaction of adrenal and pancreatic hormones in the control of hepatic enzymes during development.

In the liver of suckling rats, the synthesis of hepatic tyrosine aminotransferase, serine dehydratase, and phosphofructokinase 2 as well as of renal beta-glucosidase is controlled by the circulating concentrations of adrenal and pancreatic hormones. Glucagon is capable of stimulating enzyme synthesis only in the presence of a steroid hormone. Dexamethasone and estradiol have been found to exert a permissive function on the inducibility of the studied enzymes by glucagon. Between the hormones of the adrenal medulla and glucagon antagonistic effects in enzyme induction were observed. Obviously, this antagonism is mediated by the alpha 1-adrenergic signal transferring system. A characteristic age dependence of enzyme induction by dexamethasone has been established. This might be correlated to alterations in the degree of methylation of the respective promoters. The methylation inhibitor 5-azacytidine influences significantly the enzyme induction by glucocorticoid hormones.

Biochemistry of liver development in the perinatal period.

Just before birth, changes occur in the metabolic capacities of rat liver so that the animal can adapt to changes in the substrate supply. In utero, glucose is the main energy-generating fuel and the liver metabolism is directed towards glucose degradation. The activities of the rate-limiting enzymes of glycolysis, hexokinase and phosphofructokinase, are high. In preparation for post-natal life, when the continuous glucose supply from the mother is interrupted, very large amounts of glycogen are stored in the late fetal liver. With the intake of the fat-rich and carbohydrate-poor milk diet, the animal develops the ability to synthesize glucose de novo from non-carbohydrate precursors. During suckling, metabolic energy is derived mainly from the beta-oxidation of fatty acids, which in turn is an essential prerequisite for the high rate of gluconeogenesis, by yielding acetyl-CoA for the activation of pyruvate carboxylase and by generating a high NADH/NAD ratio for the shift of the glyceraldehyde 3-phosphate dehydrogenase reaction in the direction of glucose formation.--The developmental adaptation of metabolism and the process of enzymatic differentiation are closely connected with the maturation of the endocrine system and the changes in the concentration of circulating hormones. The neonatal regulation of phosphoenolpyruvate carboxykinase and of tyrosine aminotransferase by variations in the hormonal milieu around birth, and also the interaction of hormonal and nutritional factors in the induction of serine dehydratase and glucokinase at the end of the suckling period, will be discussed in detail.