Domenico Viterbo

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 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.

Comparison of His and GST tagged versions of recombinant pancreatitis associated protein 2 in modulation of inflammatory responses

Objective and designPancreatitis associated proteins (PAP) are highly upregulated in acute pancreatitis and other inflammatory states and have been shown to possess immunomodulatory properties. However, continued study of PAP has been hampered by the ability to effectively isolate appropriate amounts of protein from pancreatic juice or efficient generation of recombinant proteins. Here we describe two different methods for generating recombinant PAP2 protein (rPAP2), using either His or GST tagged bacterial methodology with comparison of function.MethodsHis or GST tagged rPAP2 were generated, cultured with clonal (NR8383) macrophages and compared with respect to inflammatory cytokine expression (IL-1α, IL-1β, IL-6, and TNF-α) and bacterial (E. coli) agglutination. Significance was determined by student’s t test (P<0.05).ResultsPAP2His treatment induced a 3.6, 2.8, 13.0, 3.5 fold induction of IL-1α, IL-1β, TNF-α and IL-6, respectively; similar cytokine expression changes were observed with PAP2GST treatment (3.9, 2.6, 12.2, and 3.0 fold induction of IL-1α, IL-1β, TNF-α and IL-6, respectively) (P<0.05). Further, incubation with recombinant PAP2 led to a time dependent increase in bacterial aggregates which was absent in controls.ConclusionsThese data demonstrate that both methods maintain functional immunomodulatory integrity for PAP2 and provide the ability to generate sufficient quantities to further study structure and function.