Cathy M. Mueller

Pancreatitis-Associated Protein 2 Modulates Inflammatory Responses in Macrophages

Pancreatitis-associated proteins (PAP) are stress-induced secretory proteins that are implicated in immunoregulation. Previous studies have demonstrated that PAP is up-regulated in acute pancreatitis and that gene knockdown of PAP correlated with worsening severity of pancreatitis, suggesting a protective effect for PAP. In the present study, we investigated the effect of PAP2 in the regulation of macrophage physiology. rPAP2 administration to clonal (NR8383) and primary macrophages were followed by an assessment of cell morphology, inflammatory cytokine expression, and studies of cell-signaling pathways. NR8383 macrophages which were cultured in the presence of PAP2 aggregated and exhibited increased expression of IL-1, IL-6, TNF-α, and IL-10; no significant change was observed in IL-12, IL-15, and IL-18 when compared with controls. Chemical inhibition of the NFκB pathway abolished cytokine production and PAP facilitated nuclear translocation of NF-κB and phosphorylation of IκBα inhibitory protein suggesting that PAP2 signaling involves this pathway. Cytokine responses were dose dependent. Interestingly, similar findings were observed with primary macrophages derived from lung, peritoneum, and blood but not spleen. Furthermore, PAP2 activity was inhibited by the presence of serum, inhibition which was overcome with increased PAP2. Our results demonstrate a new function for PAP2: it stimulates macrophage activity and likely modulates the inflammatory environment of pancreatitis.

Pancreatic Reg I Binds MKP-1 and Regulates Cyclin D in Pancreatic-Derived Cells

BACKGROUND The pancreatic regenerating (reg I) gene and its protein product are derived from acinar cells and are mitogenic to beta- and ductal cells. We studied the mechanism of this mitogenic response. MATERIALS AND METHODS ARIP (rat ductal) and RIN 1046-38 (rat beta-) cell lines were exposed to exogenous reg I in culture or transfected with a reg I expression vector. Mitogenesis was assessed by MTS assay (CellTiter 96; Promega, Inc., Madison, WI), and cellular mRNA was subjected to gene microarray analysis to determine signal transduction pathways. Yeast two-hybrid technology was then used to determine intracellular binding of reg I protein. RESULTS Cells exposed to exogenous reg I showed a mitogenic response; cells transfected with reg I expression vector showed inhibited growth. Microarray analysis of the former showed induction of cyclin pathways and mitogen-activated protein kinase phosphatase (MKP-1); cyclins were inhibited in the latter. Northern analysis confirmed gene induction of cyclin D1 and MKP-1; JNK was phosphorylated prior to expression of both. Yeast two-hybrid analysis confirmed a protein-protein interaction with MKP-1; this was confirmed by immunoprecipitation. CONCLUSIONS Pancreatic-derived cells exposed to reg I grow by activation of signal transduction pathways involving the mitogen-activated protein kinase phosphatases and cyclins, with concomitant induction of MKP-1. However, high intracellular levels of reg I lead to decreased growth, likely via a binding to and inactivation of MKP-1. Inhibition of cell growth, and possible induction of apoptosis, may lead to differentiation of these cells to other cell types.

PANCREATIC REGENERATING PROTEIN I IN CHRONIC PANCREATITIS AND AGING IMPLICATIONS FOR NEW THERAPEUTIC APPROACHES TO DIABETES

Objectives: We investigated the relationship of pancreatic regenerating protein (reg) in models of acinar cell atrophy and aging, and the effect of reg I protein replacement on glucose tolerance. Methods: Rats underwent pancreatic duct ligation (PDL) and were followed through 12 months. Aging rats were studied at 12 and 20 months. Intraperitoneal glucose tolerance tests (IPGTTs) were performed, pancreatic reg I, reg I receptor, insulin gene expression, and reg I protein levels were measured. Pancreatic duct ligation and aged animals were treated with exogenous reg I protein and assessed for glucose metabolism. Results: After PDL, chronic atrophic pancreatitis developed, with a progressive loss of acinar cells and pancreatic reg I. During aging, a similar depression of reg I gene expression was also noted. The reg I levels correlated with pancreatic insulin levels. Twelve months after PDL, IPGTT results were abnormal, which were significantly improved by administration of reg I protein. Aged animals demonstrated depressed IPGTT, which marginally improved after reg I administration. Anti-reg antibody administration to young rats depressed IPGTT to elderly levels. Conclusions: Depletion of the acinar product reg I is associated with the pathogenesis of impaired glucose tolerance of pancreatitis-associated diabetes and aging, and replacement therapy could be useful in these patients. Reg I is an acinar cell product, which affects islet function.

Small-interference RNA gene knockdown of pancreatitis-associated proteins in rat acute pancreatitis.

Objectives: Pancreatitis-associated proteins (PAPs) are induced in acute pancreatitis and antisense-mediated gene knockdown of PAP decreased PAP gene expression and worsened pancreatitis. Here, we investigated the effect of a more stable inhibition of PAP using small-interference RNA gene knockdown in vitro and in an in vivo model of experimental pancreatitis. Methods: Pancreatitis-associated protein-specific siRNA was administered to AR42J cell cultures or rats induced with pancreatitis. Controls included administration of scrambled siRNA or vehicle alone. After 24 hours, cells and pancreata were harvested and assessed for PAP (PAP 1, PAP 2, PAP 3) gene expression and pancreatitis severity. Results: In vitro, PAP protein, and mRNA levels were reduced (PAP 1, 76%; PAP 2, 8%; PAP 3, 24%) in cells treated with PAP siRNA. In vivo, PAP 1, and PAP 3 expressions were reduced (PAP 1, 36%; PAP 3, 66%) in siRNA-treated rats; there was no difference in PAP 2 isoform mRNA expression and serum protein levels. Serum amylase and lipase levels decreased (≥50%) after administration of siRNA; interleukin (IL) 1&bgr;, IL-4, and IL-6 increased, whereas C-reactive protein and tumor necrosis factor-&agr; decreased when compared with vehicle control. Administration of PAP siRNA correlated with worsening histopathology. Conclusions: siRNA-mediated gene knockdown of PAP worsens pancreatitis. Differences in gene knockdown technology may provide different approaches to study gene function.

Mutational Characterization of Pancreatitis-Associated Protein 2 Domains Involved in Mediating Cytokine Secretion in Macrophages and the NF-κB Pathway

Pancreatitis-associated protein 2 (PAP2) is a member of the Reg3 gene family and is classified as a group 7 C-type lectin-like protein. In rats, each of the three PAP isoforms has independent immunologic functional effects on macrophages. We have previously shown that PAP2 up-regulates inflammatory cytokines in macrophages in a dose-dependent manner and acts through NF-κB mechanisms. In this study, we aimed to determine protein domains that are essential for the immunologic function of PAP2 by mutational or chemical analysis. The protein activity for each mutant was determined by measuring TNF-α, IL-6, or IL-1 production in macrophages. Truncation of the first 25 residues on the N terminus of PAP2 did not affect protein activity whereas truncation of the last 30 residues of the C terminus of PAP2 completely inactivated the function of PAP2. Additionally, reduction of three disulfide bonds proved to be important for the activity of this protein. Further investigation revealed two invariant disulfide bonds were important for activity of PAP2 while the disulfide bond that is observed in long-form C-type lectin proteins was not essential for activity. Coupling the ability of PAP2 to up-regulate inflammatory cytokines via NF-κB with its associated expression in acute pancreatitis, a condition with aberrant concentrations of inflammatory cytokines, we investigated whether PAP2 mutants mechanistically activate the NF-κB-signaling pathway and demonstrate that preincubation with select rPAP2 mutant proteins affect translocation of this transcription factor into the nucleus.

Acute pancreatitis in aging animals: Loss of pancreatitis-associated protein protection?

AIM To investigate the effect of age on severity of acute pancreatitis (AP) using biochemical markers, histology and expression of the protective pancreatitis-associated proteins (PAPs). METHODS AP was induced via intraductal injection of 4% sodium taurocholate in young and old rats. Sera and pancreata were assayed at 24 h for the parameters listed above; we also employed a novel molecular technique to assess bacterial infiltration using polymerase chain reaction to measure bacterial genomic ribosomal RNA. RESULTS At 24 h after induction of AP, the pancreata of older animals had less edema (mean ± SE histologic score of young vs old: 3.11 ± 0.16 vs 2.50 ± -0.11, P < 0.05), decreased local inflammatory response (histologic score of stromal infiltrate: 3.11 ± 0.27 vs 2.00 ± 0.17, P < 0.05) and increased bacterial infiltration (174% ± 52% increase from sham vs 377% ± 4%, P < 0.05). A decreased expression of PAP1 and PAP2 was demonstrated by Western blotting analysis and immunohistochemical staining. There were no differences in serum amylase and lipase activity, or tissue myeloperoxidase or monocyte chemotactic protein-1 levels. However, in the most-aged group, serum C-reactive protein levels were higher (young vs old: 0.249 ± 0.04 mg/dL vs 2.45 ± 0.68 mg/dL, P < 0.05). CONCLUSION In older animals, there is depressed PAP expression related to a blunted inflammatory response in AP which is associated with worsened bacterial infiltration and higher C-reactive protein level; this may explain the more aggressive clinical course.

Acinar cell injury induced by inadequate unfolded protein response in acute pancreatitis

Acute pancreatitis (AP) is an inflammatory disorder of pancreatic tissue initiated in injured acinar cells. Severe AP remains a significant challenge due to the lack of effective treatment. The widely-accepted autodigestion theory of AP is now facing challenges, since inhibiting protease activation has negligible effectiveness for AP treatment despite numerous efforts. Furthermore, accumulating evidence supports a new concept that malfunction of a self-protective mechanism, the unfolded protein response (UPR), is the driving force behind the pathogenesis of AP. The UPR is induced by endoplasmic reticulum (ER) stress, a disturbance frequently found in acinar cells, to prevent the aggravation of ER stress that can otherwise lead to cell injury. In addition, the UPR’s signaling pathways control NFκB activation and autophagy flux, and these dysregulations cause acinar cell inflammatory injury in AP, but with poorly understood mechanisms. We therefore summarize the protective role of the UPR in AP, propose mechanistic models of how inadequate UPR could promote NFκB’s pro-inflammatory activity and impair autophagy’s protective function in acinar cells, and discuss its relevance to current AP treatment. We hope that insight provided in this review will help facilitate the research and management of AP.

635 Acute Pancreatitis (AP) in Aging Animals-Loss of PAP Protection?

Background: Although acute inflammation is a critical protective response against injury and infection, persistent inflammation and the resulting oxidative stress drive the pathogenesis of numerous chronic degenerative diseases, including carcinogenesis. In human pancreatic ductal adenocarcinoma (PDA) and in animal models that recapitulate the disease progression, an intense fibroinflammatory reaction composed of stromal and immune cells accompanies the progression from normal histology to PDA. Angiotensin II (AngII), the principal hormone of the renin angiotensin system, is actively generated in the pancreas and has been proposed as a key mediator of inflammation. Monocyte chemoattractant protein(MCP)-1 is a chemokine that plays an important role in the recruitment of mononuclear cells into sites of inflammation. Objective: To investigate the potential proinflammatory role of AngII in PDA through studying its effect on MCP-1. Methods: PDA cells (AsPC-1, HS766T, MiaPaca) were cultured and treated with or without Ang II (10-8-10-6mol/L), in the presence or absence of AngII type 1 receptor blocker, losartan, or AngII type 2 receptor blocker, PD123319. MCP-1 mRNA was analyzed by real time PCR and its protein by ELISA. Luciferase-labeled promoter studies evaluated the effect of AngII on the transcription of MCP-1 and nuclear factor-κB (NF-κB). The effect of receptor blockers on the endogenous and AngII-induced activation and nuclear translocation of NF-kB was examined by luminescence assay and immunohistochemistry. Results: AngII significantly increased the expression ofMCP-1mRNA and protein in PDA cells, and induced its promoter activity. Constitutive and AngII-induced MCP-1 mRNA and promoter activity were significantly reduced in the presence of losartan but were unchanged by PD123319. AngII induced the activation and nuclear translocation of NF-kB, an effect that was inhibited by losartan, but not PD 1233319. Blocking NF-kB activation by pyrrolidine dithiocarbamate decreased the AngII-mediated increase in MCP1 mRNA. Conclusions: Our data provide a novel insight into an AngII-initiated signal transduction pathway that regulates MCP-1 as a possible inflammatory mechanism in PDA, and suggest that AngII blockade may regulate chemokine-induced signal transduction to prevent or reduce inflammation in PDA.