Pancreatitis severity in mice with impaired CFTR function but pancreatic sufficiency is mediated via ductal and inflammatory cells-Not acinar cells

Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) are an established risk factor for cystic fibrosis (CF) and chronic pancreatitis. Whereas patients with CF usually develop complete exocrine pancreatic insufficiency, pancreatitis patients with CFTR mutations have mostly preserved exocrine pancreatic function. We therefore used a strain of transgenic mice with significant residual CFTR function (CFTR^tm1HGU ) to induce pancreatitis experimentally by serial caerulein injections. Protease activation and necrosis were investigated in isolated acini, disease severity over 24h, pancreatic function by MRI, isolated duct stimulation and faecal chymotrypsin, and leucocyte function by ex vivo lipopolysaccharide (LPS) stimulation. Pancreatic and lung injury were more severe in CFTR^tm1HGU but intrapancreatic trypsin and serum enzyme activities higher than in wild-type controls only at 8h, a time interval previously attributed to leucocyte infiltration. CCK-induced trypsin activation and necrosis in acini from CFTR^tm1HGU did not differ from controls. Fluid and bicarbonate secretion were greatly impaired, whereas faecal chymotrypsin remained unchanged. LPS stimulation of splenocytes from CFTR^tm1HGU resulted in increased INF-γ and IL-6, but decreased IL-10 secretion. CFTR mutations that preserve residual pancreatic function significantly increase the severity of experimental pancreatitis-mostly via impairing duct cell function and a shift towards a pro-inflammatory phenotype, not by rendering acinar cells more susceptible to pathological stimuli.

Keywords: CFTR; acute pancreatitis; ductal cells; inflammatory cells.

FIGURE 1

CFTR is aberrantly expressed in acinar cells of CFTR^tm1HGU mice, but exocrine pancreatic function is maintained. (A) A transgenic mouse model containing a neomycin cassette and a part of exon 10 of the CFTR gene was used that has residual CFTR expression. (B) Immunofluorescence labelling of CFTR (Clone: M3A7, DLN‐06996, Dianova) in wild‐type and CFTR^tm1HGU mice showed a clear localization of CFTR on the cellular membrane in wild‐types and a prominent labelling in the cytoplasm of CFTR^tm1HGU mice. A second antibody (Clone: A‐3, Santa Cruz Biotechnology) was used for immunohistochemistry to verify the labelling. We confirmed the localization of CFTR at the cell membrane in wild‐type mice, which was absent in CFTR^tm1HGU animals. (C) Comparable stool chymotrypsin activities wild‐type and CFTR^tm1HGU mice indicate that exocrine pancreatic enzyme secretion is not impaired in the transgenic animals

FIGURE 2

Acute pancreatitis has a more severe course in CFTR^tm1HGU mice. (A and B) Acute pancreatitis was induced in wild‐type and CFTR^tm1HGU mouse strains by intraperitoneal injections of caerulein (8 x50 µg/kg bodyweight). Serum amylase and lipase activities were higher in CFTR^tm1HGU mice only at 8h, but not after 1h, a time point directly affected by the pathological stimulation of acinar cells. (C) During acute pancreatitis, trypsin shows a typical biphasic curve of its activity after 1 and 8 h. There was no significant difference in activity between both groups at 1 h. However, at 8 h trypsin activity was increased in the transgenic mice. (D, E) Local infiltration of neutrophils into the pancreas and lungs was determined by measuring MPO activity in tissue homogenates. At 8 and 24 h after onset of the pancreatitis the activity was markedly increased in CFTR^tm1HGU mice

FIGURE 3

Histological damage was increased in CFTR transgenic mice as shown by haematoxylin and eosin stainings (A). The calibration bar represents 100 µm. (A) Quantification of histological changes was performed by a separate evaluation of necrosis, inflammation and pancreatic oedema (B). Summed up, there was a significant increase of pancreatic damage at 8h after induction of pancreatitis in the CFTR transgenic animals, seen by a higher histology score (B). The single parameters showed a clear trend towards an increase but only the quantification of the infiltrating leucocytes reached significance 8h after the onset of the disease (C), and still showed a trend at 24 h. A detailed analysis of infiltrating leucocytes by immunohistochemical staining of CD11b and p67‐phox showed a prominent infiltration of CD11b + macrophages in both mice strains at 8h after induction of pancreatitis that was more predominant in the CFTR^tm1HGU mice (D). In contrast to CD11b + macrophages, p67‐phox‐positive neutrophils could rarely be observed (E). At least five mice were used in every group. The experiments were performed in triplicates. Asterisks indicate significant differences with P <.05

FIGURE 4

Premature intracellular zymogen activation and necrosis are similar in CFTR‐mutant and wild‐type mice. (A) Intracellular elastase activation in CFTR^tm1HGU mice upon supramaximal CCK stimulation was equal to controls. (B) Cellular necrosis measured by inclusion of propidium iodide was not affected by the gene mutation. Data points show means ± SE of at least 5 experiments in each group and each time point

FIGURE 5

Changes in HCO₃ ^‐ and fluid secretion in pancreatic duct cells in CFTR^tm1HGU and wild‐type mice during acute pancreatitis. Intralobular pancreatic ducts were isolated 1 h after the last caerulein injection. The ducts were used for experiments after overnight incubation. (A) Representative intracellular pH (pH_i) traces of pancreatic duct cells, demonstrating the effect of 0.2 mmol/L amiloride and 0.5 mmol/L H₂DIDS administered from the basolateral membrane in standard HCO₃ ^‐/CO₂‐buffered solution. (B) Summarized data for the dpH/dt changes that were calculated by linear regression analysis of pH_i measurements made over the first 60 s after exposure of the transport inhibitors. (C) Wild‐type animals clearly responded to forskolin stimulation by increased fluid secretion, but the CFTR^tm1HGU animals did not. (D) The maximum of fluid secretion was significantly lower in CFTR^tm1HGU compared to wild‐type mice. Pancreatic ducts were prepared for at least from five mice per group and measurements were performed in triplicates. Asterisks indicate significant differences with P <.05

FIGURE 6

Fluid secretion after secretin stimulation in magnetic resonance cholangiopancreatography (MRCP). Fluid volume was markedly reduced in CFTR^tm1HGU compared to wild‐type mice after secretin stimulation (A + B)

FIGURE 7

Pro‐inflammatory alterations of the immune system in isolated and LPS stimulated splenocytes of CTFR‐mutant mice. (A‐C) An elevated secretion of TNFα, INF‐γ and IL6 was found, whereas (D) release of the anti‐inflammatory IL‐10 was lower in the supernatant of splenocytes derived from transgenic animals. Splenocytes were isolated from at least five mice per group and measurements were performed in triplicates. Data points show means ± SE Asterisks indicate significant differences with P <.05