The glucocorticoid-induced TNF receptor family-related protein (GITR) is critical to the development of acute pancreatitis in mice

Abstract

Background and purpose: Pancreatitis represents a life-threatening inflammatory condition where leucocytes, cytokines and vascular endothelium contribute to the development of the inflammatory disease. The glucocorticoid-induced tumour necrosis factor (TNF) receptor family-related protein (GITR) is a costimulatory molecule for T lymphocytes, modulates innate and adaptive immune system and has been found to participate in a variety of immune responses and inflammatory processes. Our purpose was to verify whether inhibition of GITR triggering results in a better outcome in experimental pancreatitis.

Experimental approach: In male GITR knock-out (GITR(-/-)) and GITR(+/+) mice on Sv129 background, acute pancreatitis was induced after i.p. administration of cerulein. Other experimental groups of GITR(+/+) mice were also treated with different doses of Fc-GITR fusion protein (up to 6.25 µg·mouse⁻¹), given by implanted mini-osmotic pump. Clinical score and pro-inflammatory parameters were evaluated.

Key results: A less acute pancreatitis was found in GITR(-/-) mice than in GITR(+/+) mice, with marked differences in oedema, neutrophil infiltration, pancreatic dysfunction and injury. Co-treatment of GITR(+/+) mice with cerulein and Fc-GITR fusion protein (6.25 µg·mouse⁻¹) decreased the inflammatory response and tissue injury, compared with treatment with cerulein alone. Inhibition of GITR triggering was found to modulate activation of nuclear factor κB as well as the production of TNF-α, interleukin-1β, inducible nitric oxide synthase, nitrotyrosine, poly-ADP-ribose, intercellular adhesion molecule-1 and P-selectin.

Conclusions and implications: The GITR-GITR ligand system is crucial to the development of acute pancreatitis in mice. Our results also suggest that the Fc-GITR fusion protein could be useful in the treatment of acute pancreatitis.

Figure 1

Expression of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) in the pancreas following cerulein treatment. Histological staining for GITR was absent in pancreatic sections from saline-treated (sham) GITR^+/+ (A) and GITR^−/− (B) or cerulein-treated GITR^−/− mice (D). Only the sections from cerulein-treated GITR^+/+ pancreas were positively stained for GITR (C, C1 and C2, at higher magnifications). Histological results shown are representative of two experiments performed in different experimental days.

Figure 2

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on cerulein-induced enzymic activity. Twenty-four hours after cerulein injection, GITR^−/− mice and GITR^+/+ mice, treated with Fc-GITR (at the different specified doses, by mini-osmotic pump), show lower levels of serum amylase (A) and lipase (B) in comparison with cerulein-treated GITR^+/+ mice. Data from saline-treated (sham) mice are also reported. One representative experiment is shown. Data, are the mean ± SE of 10 mice each group. *P < 0.01 versus sham; #P < 0.01 versus cerulein-treated GITR^+/+ mice.

Figure 3

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on cerulein-induced injury of the pancreas. Haemotoxylin and eosin stain (A–E panels): sections of pancreas from saline-treated (sham) GITR^+/+ mice (A and B) show the normal architecture of the pancreas. On the contrary, sections of pancreas from GITR^+/+ mice, show that 24 h after cerulein injection, marked inflammatory changes are present (C). Sections of pancreas from cerulein-treated GITR^−/− and GITR^+/+ mice co-treated with cerulein and Fc-GITR (6.25 µg·mouse⁻¹, by mini-osmotic pump) show less pathological changes in the tissues (D and E respectively). Silver impregnation (F–H panels): in sham-treated mice (F) there was a normal presence of reticular and nervous fibres as well as connective tissues, while a significant alteration of the same tissues associated with a collagen reduction was observed in the pancreatic tissues of GITR^+/+ mice (G). In the sections of pancreas from cerulein-injected GITR^−/− mice, a significantly reduced alteration of reticular and nervous fibres as well as connective tissues was observed (H). Oedema following cerulein treatment is shown in panel I. Figure is representative of one out of three independent experiments.

Figure 4

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on cerulein-induced nuclear factor κB (NF-κB) activation in pancreas. Panels A: basal levels of inhibitor of kappa B-α (IκB-α) were detected by Western blot analysis in the homogenates of pancreatic tissues from saline-treated (sham) animals. IκB-α expression is low 24 h after cerulein administration, while it was not reduced in GITR^−/− mice. β-actin was used as internal control. Panels B: levels of NF-κB p-65 subunit protein in the nuclear fractions of the pancreatic tissues were significantly increased by cerulein-administration compared to those in the sham-treated mice. The levels of NF-κB p-65 protein in the nuclear fractions of pancreatic tissues from GITR^−/− mice were similar to that observed in sham-treated animals. Laminin B1 was used as internal control. Results shown in the lower half of panels A and B are expressed as mean ± SEM of the normalized densitometric analysis of five independent experiments. *P < 0.01 versus sham, #P < 0.01 versus cerulein-treated GITR^+/+ mice.ND, not detectable.

Figure 5

Effect of glucocorticoid-induced tumour necrosis factor (TNF) receptor family-related protein (GITR) inhibition on plasma and pancreatic tumour necrosis factor-α (TNF-α) and interleukin (IL)-1β levels. Twenty-four hours after cerulein injection an increase of plasma and pancreatic TNF-α (A and B, respectively) and IL-1β (C and D, respectively) levels was found in GITR^+/+ mice when compared with GITR^−/− and GITR^+/+ mice that had been co-treated with Fc-GITR (6.25 µg/mouse of Fc-GITR, by mini-osmotic pump). No cytokine production was found in sham-operated mice. One representative experiment out of three is shown. Data are expressed as mean ± SE of 10 mice each group. *P < 0.01 versus sham; #P < 0.01 versus cerulein-treated GITR^+/+ mice.

Figure 6

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and P-selectin. Twenty-four hours after cerulein injection, immunohistochemical localization of ICAM-1 and P-selectin in pancreas of GITR^+/+ mice showed a positive staining for both ICAM-1 (B) and P-selectin (F), when compared with sham-operated mice (A and E respectively). There was no detectable immunostaining for ICAM-1 and P-selectin in pancreas of cerulein-treated GITR^−/− mice (C and G, respectively) and cerulein/Fc-GITR (6.25 µg/mouse of Fc-GITR, by mini-osmotic pump) co-treated GITR^+/+ mice (D and H respectively). Photographs are representative of one out of three independent experiments (including five mice in each group). Panel I: myeloperoxidase activity in pancreatic samples of cerulein-treated GITR^+/+ mice was significantly increased compared to sham-treated mice. Cerulein-treated GITR^−/− mice and cerulein/Fc-GITR (6.25 µg·mouse⁻¹ of Fc-GITR, by mini-osmotic pump) co-treated GITR^+/+ mice showed a significantly decrease of MPO activity in pancreas (C). One representative experiment out of three is shown. Data are expressed as mean ± SE of 10 mice each group. *P < 0.01 versus sham; #P < 0.01 versus cerulein-treated GITR^+/+ mice.

Figure 7

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on inducible nitric oxide synthase (iNOS) expression, nitrosative stress and poly ADP-ribose polymerase activation. Twenty-four hours after cerulein injection, immunohistochemical localization of iNOS (A–D) nitrotyrosine (E–H) and poly ADP-ribose (PAR) (I–N) in the pancreatic tissue of GITR^+/+ mice showed a positive iNOS (B) nitrotyrosine (F) and PAR (L) staining, when compared with sham operated mice (A, E and I respectively). After cerulein administration, in the pancreatic tissue of GITR^−/− mice and Fc-GITR co-treated (6.25 µg·mouse⁻¹, by mini-osmotic pump) GITR^+/+ mice, there was no detectable immunostaining for iNOS (C and D, respectively), nitrotyrosine (G and H, respectively) and PAR (M and N respectively). One representative experiment out of three is shown.

Figure 8

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on apoptosis and S100 protein expression in pancreatic tissue. Twenty-four hours after cerulein administration, Terminal deoxynucleotidyltransferase-mediated UTP nick-end labelling (TUNEL) staining (A–E) showed a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments in pancreatic tissue of GITR^+/+ mice (B, see cell count E), when compared with sham animals (A, see cell count E). In the pancreatic tissue of GITR^−/− mice (C, see cell count E) and Fc-GITR co-treated (6.25 µg·mouse⁻¹, by mini-osmotic pump) GITR^+/+ mice (D, see cell count E), the number of dark brown cells was significantly reduced. One representative experiment out of three is shown. Data are expressed as number of cells per field (n= 10) and as mean ± SE. *P < 0.01 versus Sham; #P < 0.01 versus cerulein-group. ND, not detectable. Immunohistochemical localization of S100 protein (F–I) in the pancreatic tissue of GITR^+/+ mice 24 h after cerulein injection showed a positive staining (G), when compared with sham operated mice (F). No detectable immunostaining in the pancreatic tissue of GITR^−/− mice (H) and Fc-GITR-treated (6.25 µg·mouse⁻¹, by mini-osmotic pump) GITR^+/+ mice (I) was found. One representative experiment out of three is shown.

Figure 9

Effect of glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR) inhibition on pancreatic expression of Bcl-2 family members. Bax and Bcl-2 expression levels were detected in pancreatic samples 24 h after cerulein injection by Western blot. A significant increase of Bax expression was detected in GITR^+/+ pancreatic tissue, whereas Bax levels were substantially reduced in GITR^−/− mice (A). Bcl-2 expression was not observed in pancreas of GITR^+/+ mice, while was more evident in the pancreatic tissue from cerulein-treated GITR^−/− mice (F). Results shown in the lower half of panels A and F are expressed as mean ± SEM of the normalized densitometric analysis of five independent experiments. *P < 0.01 versus cerulein-treated GITR^+/+ mice. Bax and Bcl-2 expression in pancreas was also evaluated by immunohistochemical analysis. Twenty-four hours after cerulein injection, positive Bax staining was found in pancreas of GITR^+/+ mice (C). No positive staining for Bax was detected in pancreas of sham-treated GITR^+/+ mice (B) cerulein-treated GITR^−/− mice (D) and cerulein/Fc-GITR co-treated (6.25 µg·mouse⁻¹ of Fc-GITR, by mini-osmotic pump) GITR^+/+ mice (E). No positive staining for Bcl-2 was observed in pancreas of cerulein-treated GITR^+/+ mice (G). A positive staining for Bcl-2 was observed in pancreas from sham-treated GITR^+/+ mice (F), cerulein-treated GITR^−/− mice (H) and cerulein/Fc-GITR (6.25 µg·mouse⁻¹ of Fc-GITR, by mini-osmotic pump) co-treated GITR^+/+ mice (I). One representative experiment out of three is shown.