Theresia Reding

Pancreatic stone protein is highly increased during posttraumatic sepsis and activates neutrophil granulocytes.

Objectives:The level of pancreatic stone protein/regenerating protein (PSP/reg), a secretory protein produced in the pancreas, increases dramatically during pancreatic disease. However, after stress (e.g., anesthesia), PSP/reg levels are increased transiently in animals without pancreatic injury. Therefore, we aimed to determine whether PSP/reg is an acute-phase protein after nonpancreatic trauma. Patients:Eighty-three polytraumatic patients without pancreatic damage. Measurements and Main Results:We compared serum PSP/reg levels from polytraumatic patients without pancreatic damage with those in healthy controls (n = 38). C-reactive protein, interleukin-6, procalcitonin, and leukocyte numbers were also compared. The expression of CD62L and CD11b on neutrophils after exposure to PSP/reg was analyzed by flow cytometry. Thirty-three patients (39%) developed sepsis, 32 (38%) had local infections, and 18 (21%) had no infections. At admission, PSP/reg serum levels (10.2 [6.2–14.5] ng/mL; median [interquartile range]) were comparable with those in healthy controls (10.4 [7.5–12.3] ng/mL). During hospital stay, PSP/reg levels were elevated significantly in patients with sepsis (146.4 ng/mL) and in patients with infections (111.4 ng/mL) compared with patients without infections (22.8 ng/mL). Furthermore, binding of fluorescein isothiocyanate–labeled recombinant PSP/reg to human neutrophils was demonstrated. Recombinant PSP/reg elicited a dose-dependent shedding of L-selectin (CD62L) and upregulation of &bgr;2-integrin (CD11b) in neutrophils, which indicates that PSP/reg activates neutrophils. Conclusions:We conclude that PSP/reg is up-regulated in blood after trauma, and the PSP/reg level is related to the severity of inflammation. Furthermore, PSP/reg binds to and activates neutrophils. Therefore, PSP/reg might be an acute-phase protein that could serve as a marker for posttraumatic complications.

A selective COX-2 inhibitor suppresses chronic pancreatitis in an animal model (WBN/Kob rats): significant reduction of macrophage infiltration and fibrosis

Introduction: Therapeutic strategies to treat chronic pancreatitis (CP) are very limited. Other chronic inflammatory diseases can be successfully suppressed by selective cyclooxygenase 2 (COX-2) inhibitors. As COX-2 is elevated in CP, we attempted to inhibit COX-2 activity in an animal model of CP (WBN/Kob rat). We then analysed the effect of COX-2 inhibition on macrophages, important mediators of chronic inflammation. Methods: Male WBN/Kob rats were continuously fed the COX-2 inhibitor rofecoxib, starting at the age of seven weeks. Animals were sacrificed 2, 5, 9, 17, 29, 41, and 47 weeks later. In some animals, treatment was discontinued after 17 weeks, and animals were observed for another 24 weeks. Results: Compared with the spontaneous development of inflammatory injury and fibrosis in WBN/Kob control rats, animals treated with rofecoxib exhibited a significant reduction and delay (p<0.0001) in inflammation. Collagen and transforming growth factor β synthesis were significantly reduced. Similarly, prostaglandin E2 levels were markedly lower, indicating strong inhibition of COX-2 activity (p<0.003). If treatment was discontinued at 24 weeks of age, all parameters of inflammation strongly increased comparable with that in untreated rats. The correlation of initial infiltration with subsequent fibrosis led us to determine the effect of rofecoxib on macrophage migration. In chemotaxis experiments, macrophages became insensitive to the chemoattractant fMLP in the presence of rofecoxib. Conclusion: In the WBN/Kob rat, chronic inflammatory changes and subsequent fibrosis can be inhibited by rofecoxib. Initial events include infiltration of macrophages. Cell culture experiments indicate that migration of macrophages is COX-2 dependent.

Lymphotoxin β Receptor Signaling Promotes Development of Autoimmune Pancreatitis

BACKGROUND & AIMS Little is known about the pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis. Current treatment options are limited and disease relapse is frequent. We investigated factors that contribute to the development of AIP and new therapeutic strategies. METHODS We used quantitative polymerase chain reaction, immunohistochemical, and enzyme-linked immunosorbent analyses to measure the expression of cytokines and chemokines in tissue and serum samples from patients with and without AIP. We created a mouse model of human AIP by overexpressing lymphotoxin (LT)α and β specifically in acinar cells (Ela1-LTab mice). RESULTS Messenger RNA levels of LTα and β were increased in pancreatic tissues from patients with AIP, compared with controls, and expression of chemokines (CXCL13, CCL19, CCL21, CCL1, and B-cell-activating factor) was increased in pancreatic and serum samples from patients. Up-regulation of these factors was not affected by corticosteroid treatment. Acinar-specific overexpression of LTαβ (Ela1-LTαβ) in mice led to an autoimmune disorder with various features of AIP. Chronic inflammation developed only in the pancreas but was sufficient to cause systemic autoimmunity. Acinar-specific overexpression of LTαβ did not cause autoimmunity in mice without lymphocytes (Ela1-LTab/Rag1(-/-)); moreover, lack of proinflammatory monocytes (Ela1-LTab/Ccr2(-/-)) failed to prevent AIP but prevented early pancreatic tissue damage. Administration of corticosteroids reduced pancreatitis but did not affect production of autoantibodies, such as antipancreatic secretory trypsin inhibitor in Ela1-LTab mice. In contrast, inhibition of LTβR signaling reduced chemokine expression, renal immune-complex deposition, and features of AIP in Ela1-LTab mice. CONCLUSIONS Overexpression of LTαβ specifically in acinar cells of mice causes features of AIP. Reagents that neutralize LTβR ligands might be used to treat patients with AIP.

COX-2 is not required for the development of murine chronic pancreatitis.

Chronic pancreatitis is a severe inflammation of the pancreas associated with destruction of the parenchyma, fibrosis, and persistent abdominal pain. Cyclooxygenase-2 (COX-2) and COX-2-derived prostaglandins, key mediators of the inflammatory response, are elevated in patients with chronic pancreatitis. Previous studies investigated COX-2 as a therapeutic target. These reports showed a reduced pathology in COX-2-deficient mice with a better outcome. Here we compared the role of COX-2 in acute and chronic pancreatic inflammation using the same COX-2(-/-) mouse model of cerulein-induced pancreatitis. In a setting of acute pancreatitis, juvenile COX-2(-/-) mice exhibited a reduced histopathological score compared with wild-type littermates; on the contrary, adult mice did not show any difference in the development of the disease. Similarly, in a setting of chronic pancreatitis induced over a period of 4 wk, adult mice of the two strains showed comparable histological score and collagen deposition. However, the abundance of mRNAs coding for profibrotic genes, such as collagen, α-smooth muscle actin, and transforming growth factor-β was consistently lower in COX-2(-/-) mice. In addition, comparable histological scores and collagen deposition were observed in wild-type mice treated with a COX-2 inhibitor. We conclude that, in contrast to what was observed in the rat pancreatitis models, COX-2 has a limited and age-dependent effect on inflammatory processes in the mouse pancreas. These results suggest that COX-2 modulates the inflammatory process during the development of pancreatitis in a species-specific manner. Thus the pathophysiological roles of COX-2 and its therapeutic implications in patients with pancreatitis should be reexamined.

p21WAF1/Cip1 limits senescence and acinar-to-ductal metaplasia formation during pancreatitis

Trans‐differentiation of pancreatic acinar cells into ductal‐like lesions, a process defined as acinar‐to‐ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre‐malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down‐regulation of p21WAF1/Cip1, a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down‐regulation is implicated in controlling the early events of acinar cell trans‐differentiation and ADM formation. p21‐mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein‐induced pancreatitis, using wild‐type (WT) and p21‐deficient (p21−/−) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up‐regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down‐regulation is associated with ADM formation. In support of this hypothesis, p21−/− mice showed a significant increase in number and size of metaplasia. In addition, p21 over‐expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell‐autonomous manner. p21−/− mice displayed increased expression and relocalization of β‐catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate‐keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. Copyright

Inflammation-dependent expression of SPARC during development of chronic pancreatitis in WBN/Kob rats and a microarray gene expression analysis

The pathophysiology of human chronic pancreatitis is not well understood and difficult to follow on a molecular basis. Therefore, we used a rat model [Wistar-Bonn/Kobori (WBN/Kob)] that exhibits spontaneous chronic inflammation and fibrosis in the pancreas. Using microarrays we compared gene expression patterns in the pancreas during development of inflammation and fibrosis of WBN/Kob rats with age-matched healthy Wistar rats. The extracellular matrix protein SPARC (secreted protein, acidic, and rich in cysteines) and other transcripts of inflammatory genes were quantified by real-time PCR, and some were localized by immunohistochemistry. When pancreatic inflammation becomes obvious at the age of 16 wk, several hundred genes are increased between 3- and 50-fold in WBN/Kob rats compared with healthy Wistar rats. Proteins produced by acinar cells and characteristic for inflammation, e.g., pancreatitis-associated protein, are highly upregulated. Other proteins, derived from infiltrating inflammatory cells and from activated stellate cells (fibrosis) such as collagens and fibronectins are also significantly upregulated. SPARC was localized to acinar cells where it increased in the vicinity of inflammatory foci. However, acinar expression of SPARC was lost during destruction of acinar cells. In human pancreatic specimens with chronic pancreatitis, SPARC exhibited a similar expression profile. During chronic inflammation and fibrosis in the WBN/Kob rat, inflammatory genes, growth factors, and structural genes exhibit a high increase of expression. A temporal profile including pre- and postinflammatory phases indicates a concurrent activation of inflammatory and fibrotic changes. Inflammation dependent expression of SPARC appears to be lost during acinar-to-duct metaplasia both in rat and human pancreas.

Class I histone deacetylase inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar‐to‐ductal metaplasia

Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease.

Inactivation of TGFβ receptor II signalling in pancreatic epithelial cells promotes acinar cell proliferation, acinar-to-ductal metaplasia and fibrosis during pancreatitis

Determining signalling pathways that regulate pancreatic regeneration following pancreatitis is critical for implementing therapeutic interventions. In this study we elucidated the molecular mechanisms underlying the effects of transforming growth factor‐β (TGFβ) in pancreatic epithelial cells during tissue regeneration. To this end, we conditionally inactivated TGFβ receptor II (TGFβ‐RII) using a Cre–LoxP system under the control of pancreas transcription factor 1a (PTF1a) promoter, specific for the pancreatic epithelium, and evaluated the molecular and cellular changes in a mouse model of cerulein‐induced pancreatitis. We show that TGFβ‐RII signalling does not mediate the initial acinar cell damage observed at the onset of pancreatitis. However, TGFβ‐RII signalling not only restricts acinar cell replication during the regenerative phase of the disease but also limits ADM formation in vivo and in vitro in a cell‐autonomous manner. Analyses of molecular mechanisms underlying the observed phenotype revealed that TGFβ‐RII signalling stimulates the expression of cyclin‐dependent kinase inhibitors and intersects with the EGFR signalling axis. Finally, TGFβ‐RII ablation in epithelial cells resulted in increased infiltration of inflammatory cells in the early phases of pancreatitis and increased activation of pancreatic stellate cells in the later stages of pancreatitis, thus highlighting a TGFβ‐based crosstalk between epithelial and stromal cells regulating the development of pancreatic inflammation and fibrosis. Collectively, our data not only contribute to clarifying the cellular processes governing pancreatic tissue regeneration, but also emphasize the conserved role of TGFβ as a tumour suppressor, both in the regenerative process following pancreatitis and in the initial phases of pancreatic cancer. Copyright

Serotonin promotes acinar dedifferentiation following pancreatitis-induced regeneration in the adult pancreas

The exocrine pancreas exhibits a distinctive capacity for tissue regeneration and renewal following injury. This regenerative ability has important implications for a variety of disorders, including pancreatitis and pancreatic cancer, diseases associated with high morbidity and mortality. Thus, understanding its underlying mechanisms may help in developing therapeutic interventions. Serotonin has been recognized as a potent mitogen for a variety of cells and tissues. Here we investigated whether serotonin exerts a mitogenic effect in pancreatic acinar cells in three regenerative models, inflammatory tissue injury following pancreatitis, tissue loss following partial pancreatectomy, and thyroid hormone‐stimulated acinar proliferation. Genetic and pharmacological techniques were used to modulate serotonin levels in vivo. Acinar dedifferentiation and cell cycle progression during the regenerative phase were investigated over the course of 2 weeks. By comparing acinar proliferation in the different murine models of regeneration, we found that serotonin did not affect the clonal regeneration of mature acinar cells. Serotonin was, however, required for acinar dedifferentiation following inflammation‐mediated tissue injury. Specifically, lack of serotonin resulted in delayed up‐regulation of progenitor genes and delayed the formation of acinar‐to‐ductal metaplasia and defective acinar cell proliferation. We identified serotonin‐dependent acinar secretion as a key step in progenitor‐based regeneration, as it promoted acinar cell dedifferentiation and the recruitment of type 2 macrophages. Finally, we identified a regulatory Hes1–Ptfa axis in the uninjured adult pancreas, activated by zymogen secretion. Our findings indicated that serotonin plays a critical role in the regeneration of the adult pancreas following pancreatitis by promoting the dedifferentiation of acinar cells. Copyright

Class I HDAC inhibition improves pancreatitis outcome by limiting leukocyte recruitment and acinar-to-ductal metaplasia

Pancreatitis is a common inflammation of the pancreas with rising incidence in many countries. Despite improvements in diagnostic techniques, the disease is associated with high risk of severe morbidity and mortality and there is an urgent need for new therapeutic interventions. In this study, we evaluated whether histone deacetylases (HDACs), key epigenetic regulators of gene transcription, are involved in the development of the disease.