Functional Characterization of Pancreatitis Associated Protein 2 In Acute Pancreatitis and Its Effect on Macrophages.

Abstract

Severe acute pancreatitis is characterized by extensive pancreatic necrosis, a systemic inflammatory response, multi-organ failure, and a 40% mortality rate. Pancreatitis-associated proteins (PAP) are members of the pancreatic regeneration (Reg) family that are minimally expressed in normal pancreas but strongly induced in acute pancreatitis. We have previously demonstrated gene knock down of PAP isoforms correlate with worsening of pancreatitis severity. However the effect of PAP on the immune system is not well understood. In the present studies, antibody neutralization of Reg/PAP in an animal model of pancreatitis demonstrated worsening of pancreatitis severity as evidenced by increased inflammation and necrosis. To better understand the effect of PAP on the immune system we investigated the effect of PAP2 on macrophages. Culture of macrophages with PAP demonstrated increased expression of inflammatory cytokines including IL-1α, IL-1β, IL-6, and TNFα, and correlated with increased agglutination in a dose dependant manner. The effects were similar with two different versions of recombinant PAP2. The proinflammatory effects of PAP2 were decreased with addition of serum suggesting a putative binding protein, the effects of which are can be saturated. The active component of the PAP2 molecule was further investigated by truncation and mutational analysis. The observed proinflammatory effects of PAP2 are thought to reside in the C terminus of the molecule and are contingent on its disulfide bonds as mutagenesis of cysteine residues inhibited its effect. Furthermore PAP2 likely operates through the NF kappa B system since inhibition of this pathway obviated its effects and culturing with PAP2 activated this pathway as demonstrated by translocation of the active components into the nucleus. These data are the first to demonstrate the function and characterization of PAP and substantiate an immunomodulatory effect of PAP2 in the setting of acute pancreatitis.