Severe pancreatitis with exocrine destruction and increased islet neogenesis in mice with suppressor of cytokine signaling-1 deficiency

Abstract

Mice with suppressor of cytokine signaling-1 (SOCS-1) deficiency die within 3 weeks of birth from a multiorgan inflammatory disease. Increased systemic levels and sensitivity of cells to the inflammatory cytokines interferon-gamma and tumor necrosis factor may contribute to the disease. Hepatitis and liver failure are thought to be the cause of the neonatal lethality in these mice. Here, we show that the pancreata of SOCS-1(-/-) mice are also severely affected by inflammation, displaying extensive edema and infiltration by T cells and macrophages. Acinar cells in particular were atrophied and reduced in their zymogen content. The expression of inflammatory markers, including class I major histocompatibility complex and inducible nitric oxide synthase, were increased in the SOCS-1(-/-) pancreas. Although there was generalized up-regulation of class I major histocompatibility complex, inducible nitric oxide synthase expression was more prominent on exocrine tissues. There appeared to be preferential damage and apoptosis of exocrine over endocrine components. Unexpectedly, increased islet neogenesis, possibly from proliferating ductal cells, was observed in the pancreas of SOCS-1(-/-) mice. This is reminiscent of the pancreatitis and islet neogenesis that occur in mice that transgenically overexpress interferon-gamma and/or tumor necrosis factor. This study suggests that in addition to liver failure, the pancreatitis may also be an important contributor to the neonatal lethality in SOCS-1(-/-) mice.

Figure 1

Histopathology of SOCS-1̃ pancreata. A: Pancreas from a littermate control (SOCS-1^+/+) with an endocrine islet (i), exocrine acini (ii), and a blood vessel indicated (iii). B: SOCS-1̃ pancreata typically displayed interstitial (i), interlobular (ii), and subcapsular edema (iii). Note the hyperplastic fibrous supporting tissue (arrow). C: Numerous extravasating cells (long arrow) and shrunken acini (short arrow) were observed such as those indicated in this severely inflamed SOCS-1^−/− pancreatic lobule. Note the loss of eosin staining of acinar cells, which is indicative of reduced zymogen content. D: Islets in a SOCS-1̃ pancreas frequently displayed shape irregularities, such as closeness to ductules (arrows) and a blurring of islet boundary. E–J: Pancreatic sections were stained for the hematopoietic markers CD4, CD8, B220, F4/80, and GR1 by immunohistochemistry to determine the identity of infiltrating cells. The relative frequencies of different hematopoietic populations are indicated in E. Examples of SOCS-1̃ pancreas sections stained for hematopoietic markers are shown in F–J. F: Numerous CD4⁺ T cells were found, particularly surrounding vessels (arrow). G: CD8⁺ T cells were similarly prominent, particularly surrounding vessels (arrow). H: B220⁺ B cells were only found occasionally (arrows). I: F4/80⁺ macrophages were frequently found within the fibrous septa between lobules (short arrows) and near blood vessels (long arrow). J: Very few GR1⁺ neutrophils were found in the parenchyma of SOCS-1̃ pancreata (short arrow). Most were found within blood vessels (long arrow). All pancreatic sections shown are from 10- to 14-day-old mice. Original magnifications: ×200 (A, C, D); ×100 (B); ×400 (E–J).

Figure 2

Increased circulating levels of inflammatory cytokines in SOCS-1^−/− mice. Serum from moribund SOCS-1^−/− mice and littermate controls were analyzed for cytokine levels by ELISA.

Figure 3

Expression of inflammatory markers in the pancreas of SOCS-1^−/− mice. SOCS-1^+/+ (A) and SOCS-1̃ (B) pancreata were analyzed for class I MHC expression by immunohistochemistry. Note the high level of class I MHC expression on inflammatory cells (arrow), stroma, acini, and islets (dashed boundary). SOCS-1^+/+ (C) and SOCS-1̃ (D) pancreata were analyzed for iNOS expression by immunofluorescence (red). The same sections were also stained for insulin (green), and are shown in the insets. SOCS-1^+/− (E) and SOCS-1̃ (F) pancreata were analyzed for β-galactosidase by X-gal staining (blue). Note the different staining patterns evident in SOCS-1 pancreata, punctate stainting in islets (dashed boundary) compared with diffuse staining in exocrine cells. SOCS-1^+/+ (G) and SOCS-1̃ (H) pancreata were analyzed apoptosis by TUNEL fluorescence staining (red). Note the increased frequency of TUNEL⁺ cells in SOCS-1̃ pancreata. I–K: SOCS-1̃ pancreata were co-stained for the pan-leukocyte marker CD45, amylase, or insulin (green) to determine the identity of apoptotic cells. The arrows indicate examples of double-staining cells. Most apoptotic cells in SOCS-1̃ pancreata were infiltrating CD45⁺ (I) cells or acinar (amylase⁺) cells (J). K: Some apoptotic cells in SOCS-1̃ pancreata were found to be insulin⁺. Note that TUNEL stains apoptotic nuclei while anti-CD45, amylase, and insulin stain the cytoplasm of cells, and therefore does not completely co-localize. Original magnifications: ×200 (A–E); ×400 (F); ×600 (G–K).

Figure 4

Perturbed endocrine islet morphology in the pancreas of SOCS-1̃ mice. SOCS-1^+/+ (A, C, E) and SOCS-1^−/− (B, D, F) pancreata were stained for insulin (A, B), glucagon (C, D), or somatostatin (E, F) expression by immunohistochemistry. One islet in A displays punctate insulin staining typical of a newly formed islet (arrow). B: Note the abundance of insulin staining single cells or cell clusters (arrows) in SOCS-1^−/− pancreata. Original magnifications, ×100.

Figure 5

The pancreas of SOCS-1̃ mice display features of increased islet neogenesis. A: Insulin-expressing ductal cells (detected by immunohistochemistry) were frequently found in SOCS-1^−/− pancreata. A mitotic figure is seen in a nearby ductal cell (arrow). B: Numerous insulin-staining single cells or cell clusters were present in SOCS-1̃ pancreata. C: SOCS-1^−/− pancreata display a significant increase in the frequency of insulin-staining cells within ductal epithelia. Shown are the means ± SD. Statistical significance, P < 0.0001 (unpaired t-test). D: Mitotic figures (arrows) were frequently found in epithelium of intralobular ducts of SOCS-1̃ pancreata. SOCS-1^+/+ (E) and SOCS-1̃ (F) pancreata were analyzed with proliferating cells by measuring the incorporation of BrdU (red immunofluorescence) after an intraperitoneal injection of BrdU at 100 μg g/g body weight. Note the increased frequency of BrdU⁺ cells in SOCS-1̃ pancreata. G–L: SOCS-1̃ pancreata were co-stained for insulin, CD45, or the ductal cell marker cytokeratin 18 (green) to determine the identity of proliferating cells. The arrows indicate examples of double-staining cells. G: Only a few β cells were found to be BrdU⁺. Most proliferating cells were CD45⁺ (I) or cytokeratin 18⁺ (K) ductal cells. H, J, and L: As a negative control, uninjected mice were also analyzed for BrdU staining. M: Few cytokeratin 18⁺ ductal cells were BrdU⁺ in SOCS-1^+/+ pancreata. Note that anti-BrdU stains nuclei while anti-CD45, insulin, and cytokeratin 18 stain the cytoplasm of cells, and therefore does not completely co-localize. Original magnifications: ×1000 (A, D); ×200 (B, E, F); ×600 (G–L).