Complement Component 5 Mediates Development of Fibrosis, via Activation of Stellate Cells, in 2 Mouse Models of Chronic Pancreatitis

Abstract

Background & aims: Little is known about the pathogenic mechanisms of chronic pancreatitis. We investigated the roles of complement component 5 (C5) in pancreatic fibrogenesis in mice and patients.

Methods: Chronic pancreatitis was induced by ligation of the midpancreatic duct, followed by a single supramaximal intraperitoneal injection of cerulein, in C57Bl6 (control) and C5-deficient mice. Some mice were given injections of 2 different antagonists of the receptor for C5a over 21 days. In a separate model, mice were given injections of cerulein for 10 weeks to induce chronic pancreatitis. Direct effects of C5 were studied in cultured primary cells. We performed genotype analysis for the single-nucleotide polymorphisms rs 17611 and rs 2300929 in C5 in patients with pancreatitis and healthy individuals (controls). Blood cells from 976 subjects were analyzed by transcriptional profiling.

Results: During the initial phase of pancreatitis, levels of pancreatic damage were similar between C5-deficient and control mice. During later stages of pancreatitis, C5-deficient mice and mice given injections of C5a-receptor antagonists developed significantly less pancreatic fibrosis than control mice. Primary pancreatic stellate cells were activated in vitro by C5a. There were no differences in the rs 2300929 SNP between subjects with or without pancreatitis, but the minor allele rs17611 was associated with a significant increase in levels of C5 in whole blood.

Conclusions: In mice, loss of C5 or injection of a C5a-receptor antagonist significantly reduced the level of fibrosis of chronic pancreatitis, but this was not a consequence of milder disease in early stages of pancreatitis. C5 might be a therapeutic target for chronic pancreatitis.

Keywords: Complement System; Pancreatic Stellate Cells; Transcriptome Analysis; αSMA.

Figure 1

Characterization of combined duct ligation and supramaximal secretagogue stimulation to induce chronic pancreatitis in mice. Chronic pancreatitis was studied in C57BL6 mice using a combination of duct ligation and supramaximal secretagogue stimulation. In the mouse pancreas the common bile duct passes through the duodenal lobe of the pancreas and the side branches from the pancreatic head drain into the bile duct before reaching the papilla. (A) We therefore ligated the pancreatic duct at the junction between the duodenal, splenic, and gastric lobe. (B) The loss of exocrine tissue was shown by reduced amylase staining in the pancreas 14 days after duct ligation. (C) Fatty tissue replacement developed in the affected part of the pancreas, (D) some stellate cells show a positive oil red staining in small fat droplets. (E) Serum amylase level peaked at day 3. Loss of exocrine tissue was observed only in the disease-affected part, whereas overall exocrine function measured as fecal elastase activity was preserved. Quantification of necrosis on H&E staining and fibrosis in Masson–Goldner staining showed a necrosis-fibrosis sequence (n = 5–8).

Figure 2

Morphologic characterization of the duct ligation model of chronic pancreatitis. The histologic characterization of pancreatic specimens in the duct ligation model of chronic pancreatitis showed typical features of chronic pancreatitis. H&E as well as Masson–Goldner staining showed a large increase in fibrosis. Immunohistochemical staining of collagen I and αSMA showed activation of PSCs and increased fibrogenesis with production of extracellular matrix proteins at 14 and 21 days after duct ligation. Pancreatic regeneration is marked by proliferating Ki67-positive acinar cells and tubular complexes 14 and 21 days after surgery. The transmigration of the innate immune cells was shown by Mac-3 staining for macrophages and MPO staining for neutrophils. Enlarged neural structures also were a common aspect of chronic pancreatitis, labeled here with protein gene product 9.5, suggesting neural sprouting. Insulin staining showed intact β-cells in islands of Langerhans (n = 5–8).

Figure 3

Course of chronic pancreatitis and extent of fibrogenesis in C5-/- and C5+/+ in the duct ligation model. (A) The systemic inflammatory reaction after induction of pancreatitis was illustrated by MPO activity measurements in lung tissue. Inflammation was confirmed by H&E staining of the lung. (B) There was no difference in serum amylase levels between animal strains. H&E staining of pancreatic tissue showed severe acute pancreatitis with large areas of necrosis 3 days after induction of pancreatitis. Twenty-one days after surgery necrotic areas were replaced by fibrotic tissue in C5+/+ but not in C5-/- animals. (C) The quantification of fibrosis showed a significant difference with regard to fibrogenesis between C5+/+ and C5-/- mice. (D) The expression of αSMA in the pancreas was reduced in C5-/- animals after 21 days. (E and F) In the more specific labeling for the extracellular matrix protein collagen I as well as αSMA C5-/- animals showed a less pronounced expression compared with C5+/+ mice (n = 5–10). Asterisks indicate significant differences with P < .05.

Figure 4

Extent of fibrogenesis in the course of chronic pancreatitis in C5-/- and C5+/+ animals in the model of repetitive cerulein stimulation. Masson–Goldner staining of pancreatic tissue was analyzed by ImageJ software (National Institutes of Health). (A–C) C5-deleted animals showed significantly less fibrosis in Masson–Goldner staining as well as αSMA and collagen I expression. (D) The percentage of Ki67-positive cells was reduced significantly in C5-/- animals, whereas (E) serum amylase and lipase activity were unaffected by C5 deletion (n = 5–7). Asterisks indicate significant differences with P < .05.

Figure 5

C5a-receptor expression on pancreatic stellate cells in chronic pancreatitis. (A and B) On immunofluorescence and Western blot analysis PSCs expressed CD88, the receptor of C5a as well as αSMA in pancreatic tissue of animals with chronic pancreatitis. Fluorescence staining indicated expression of CD88 on pancreatic stellate cells as well as on immune cells. (C) Human tissue samples of chronic pancreatitis patients showed a strong signal for αSMA and C5a (CD88) (n = 5–7). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

C5a activates PSCs in vitro, which leads to extracellular matrix deposition. Pancreatic stellate cells were isolated from C57Bl6 mice. (A) The expression of C5a receptor (CD88) on stellate cells was analyzed by immunofluorescence staining of isolated PSCs. (B and C) Reverse-transcription polymerase chain reaction as well as immunoblotting of PSC lysates confirmed positive immunofluorescence staining. (C) Proliferation shown by proliferating cell nuclear antigen (PCNA) Western blot was not affected by C5a stimulation. Murine PSCs express the C5a receptor and respond to recombinant mouse C5a with up-regulation of αSMA. (D) Four independent experiments were analyzed by Western blot and the ratio of αSMA/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) optical density (OD) was calculated. (E) PSCs treated with C5a showed up-regulation of αSMA and fibronectin messenger RNA (mRNA) on real-time polymerase chain reaction analysis. TGFβ-stimulated and untreated cells acted as controls, a C5a-receptor antagonist was used to block stimulation (n = 4).

Figure 7

Treatment with C5a inhibitors attenuates fibrosis in the duct ligation model. Treatment with C5a-receptor antagonist and a peptide inhibitor of C5a was initiated 4 days after duct ligation and effects on fibrosis were evaluated after 14 days. (A and B) Both treatments lead to a significant reduction of Masson–Goldner, collagen I, and αSMA staining on histologic evaluation. (C) Western blot analysis for αSMA from pancreatic lysates confirmed this finding for the C5a-receptor antagonist and showed a similar trend that was not statistically significant for the peptide inhibitor (n = 6–9).

Supplementary Figure 1

Quantification of fibrosis by ImageJ software. ImageJ is used for the quantification of fibrosis from Masson–Goldner trichrome staining or haematoxylin-3,3′-diaminobenzidine (H-DAB) staining or a respective protein such as αSMA or collagen I.

Supplementary Figure 2

Pancreatic duct ligation without cerulein hyperstimulation and open field analysis in animals with chronic pancreatitis (CP). (A) Pancreatic duct ligation without supramaximal cerulein stimulation served as control for the characterization of the model. In duct ligation alone we observed less extended areas of necrosis (H&E staining), less fibrosis (trichrome staining, collagen I), a less-pronounced inflammatory infiltrate, but atrophy of the gland over time. Morphometric quantification of fibrosis on Masson–Goldner trichrome staining and collagen I shows a significant increase in extracellular matrix in duct-ligated, cerulein-stimulated animals compared with ligation without cerulein stimulations. Interestingly, open field analysis to measure pancreatic pain showed a significant decrease in traveled distance in the duct ligation/hyperstimulation model in comparison with repetitive cerulein application. (B) Voluntary movement and the distance traveled by animals in the duct ligation/hyperstimulation. More than 5 animals were used for each experiment and all experiments were performed in triplicate. All experiments were performed independently on 3 or more occasions. Asterisks indicate significant differences with a P value less than .05.

Supplementary Figure 3

Direct comparison of the duct ligation/hyperstimulation model to repetitive supraphysiological cerulein injections with regard to fibrogenesis. (A and B) Morphometric quantification of fibrosis on Masson–Goldner trichrome staining, collagen I, and αSMA staining showed a highly significant difference of fibrosis between the duct ligation model at day 21 and after 10 weeks of repetitive cerulein stimulation. More than 5 animals were used for each experiment and all experiments were performed in triplicate. All experiments were performed independently on 3 or more occasions. Asterisks indicate significant differences with a P value less than .05.

Supplementary Figure 4

Further characterization of the duct ligation/hyperstimulation model in C57Bl6 mice. (A) H&E staining of the lungs of animals with chronic pancreatitis upon concomitant duct ligation and supramaximal secretagogue stimulation showed a strong systemic inflammatory reaction with thickening of the alveolar wall and hyperemia. H&E staining of the liver showed extended areas of necrosis at 3 and 7 days after surgery, which resolved at later time points, ruling out mechanical cholestasis as a confounding factor. Staining of cytokeratin 19 (CK19) illustrated strong expression of intact but dilated pancreatic ducts over the time course of chronic pancreatitis. (B) Time course of serum lipase activity and IL6 serum levels in chronic pancreatitis in the duct ligation model. (C) Staining of insulin over the time course of chronic pancreatitis in mice showed unchanged numbers of insulin-producing cells and, after a dip in the early necrotizing disease phase, a preserved endocrine function in the duct-ligated mouse model. Quantification of the M1 and M2 populations of macrophages showed a predominance of the proinflammatory M1 macrophages in the early disease course, peaking at day 3 in pancreatic tissue. (D) Thereafter, a steady increase in M2 macrophages, which are considered responsible for extracellular matrix deposition in the later course of chronic pancreatitis, was observed. Asterisks indicate significant differences with P < .05.

Supplementary Figure 5

C5 in cerulein-induced pancreatitis. Acute pancreatitis was induced by supramaximal cerulein administration (50 μg/kg/body weight) in C5+/+ and C5-/- animals. (A) Serum amylase and lipase activity showed an increase after induction of pancreatitis, but amylase and lipase activities were not found to be different between the 2 animal strains. (B) Representation of H&E stainings at 0, 1, and 8 hours of repetitive cerulein IP injection in C5+/+ compared with C5-/- animals; 0h represents untreated control. H&E staining of the pancreas showed an equal amount of infiltrating cells as well as equal levels of tissue damage characterized by areas of necrosis or edema. (C) Myeloperoxidase activity in pancreatic tissue homogenate was found to be increased over time but did not differ between C5+/+ and C5-/- animals. (D) Serum levels of the proinflammatory cytokine IL6 or the chemokine monocyte chemoattractant protein (MCP)-1 were not different between C5+/+ and C5-/- mice. More than 5 animals were used for each experiment and all experiments were performed in triplicate. All experiments were performed independently on 3 or more occasions. Asterisks indicate significant differences with a P value less than .05.

Supplementary Figure 6

Expression level of C5 in whole blood corresponding to the SNPs rs17611 and rs2300929. (A) Shown are the residual (ie, after adjustment for technical effects and potential confounders, and overall mean-centered) mean log₂-transformed gene expression levels corresponding to gene-specific messenger RNA level in whole blood from a cis-expression of quantitative trait loci analysis of 976 volunteers from the Study of Health in Pomerania–Trend cohort and 95% confidence intervals (y-axis) per genotype group (x-axis) of C5 adjusted for the first 50 eigenvectors with respect to rs17611 and rs2300929 for SHIP–TREND. We detected a significant association of rs17611_A with increased C5 expression measured in whole blood with a P value of 6.87 × 10^-41.

Supplementary Figure 7

Full-length Western blot for C5a receptor on pancreatic cells. Western blots of different pancreatic cells and tissues were preformed to analyze the expression of C5a receptor (CD88). The expected size of CD88 is approximately 49 kilodaltons. Isolated macrophages served as positive control. Freshly isolated PSCs (3 days of culture) show an expression of CD88 in Western blot. Long-time culture of PSCs (6 days) leads to activation of cells and loss of CD88 expression. Duct cells, similar to isolated acinar cells, do not express CD88, in contrast to whole pancreatic tissue, which shows a clear expression of CD88.

Supplementary Figure 8

Enlarged immunofluorescent staining of C5a receptor (CD88) and αSMA shows expression of CD88 in different populations of cells but not in acinar cells. In addition, CD88/αSMA double-positive cells can be stained in chronic pancreatitis tissue of mice, identifying them as PSCs.