Pancreatic stellate cells respond to inflammatory cytokines: potential role in chronic pancreatitis

Abstract

Background: It is now generally accepted that chronic pancreatic injury and fibrosis may result from repeated episodes of acute pancreatic necroinflammation (the necrosis-fibrosis sequence). Recent studies suggest that pancreatic stellate cells (PSCs), when activated, may play an important role in the development of pancreatic fibrosis. Factors that may influence PSC activation during pancreatic necroinflammation include cytokines known to be important in the pathogenesis of acute pancreatitis, such as tumour necrosis factor alpha (TNF-alpha), and the interleukins 1, 6, and 10 (IL-1, IL-6, and IL-10).

Aim: To determine the effects of these cytokines on PSC activation, as assessed by cell proliferation, alpha smooth muscle actin (alpha-SMA) expression, and collagen synthesis.

Methods: Cultured rat PSCs were incubated with cytokines for 24 hours. Cell proliferation was assessed by measuring (3)H thymidine incorporation into cellular DNA, alpha-SMA expression by western blotting, and collagen synthesis by incorporation of (14)C proline into collagenase sensitive protein. mRNA levels for procollagen alpha(1)(1) in PSCs were determined by northern and dot blotting methods.

Results: Expression of alpha-SMA by PSCs was increased on exposure to each of the cytokines used in the study. Stellate cell proliferation was stimulated by TNF-alpha but inhibited by IL-6, while IL-1 and IL-10 had no effect on PSC proliferation. Collagen synthesis by PSCs was stimulated by TNF-alpha and IL-10, inhibited in response to IL-6, and unaltered by IL-1. Changes in collagen protein synthesis in response to TNF-alpha, IL-10, and IL-6 were not regulated at the mRNA level in the cells.

Conclusion: This study has demonstrated that PSCs have the capacity to respond to cytokines known to be upregulated during acute pancreatitis. Persistent activation of PSCs by cytokines during acute pancreatitis may be a factor involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis.

Figure 1

Effect of cytokines on α-smooth muscle actin (α-SMA) expression by pancreatic stellate cells (PSCs). Data are expressed as per cent of control (mean (SEM); n=5 cell preparations). (A) Tumour necrosis factor α (TNF-α, at a concentration of 10 U/ml) significantly increased α-SMA expression in PSCs. (B) Interleukin (IL)-1 (0.001, 0.01, and 0.1 ng/ml) significantly increased α-SMA expression in PSCs. (C) IL-6 caused a biphasic increase in α-SMA expression in PSCs, with peaks at 0.001 ng/ml and 10 ng/ml. (D) IL-10 (10 ng/ml) also increased expression of α-SMA in PSCs. MW, molecular weight.

Figure 2

Effect of cytokines on DNA synthesis in pancreatic stellate cells (PSCs). Data are expressed as per cent of control (mean (SEM); n=5 separate cell preparations). (A) Tumour necrosis factor α (TNF-α) significantly increased DNA synthesis in PSCs to a maximum of 144 (13)% of control levels. (B) Interleukin (IL)-1 had no effect on DNA synthesis in PSCs. (C) IL-6, at a concentration of 10 ng/ml, inhibited DNA synthesis in PSCs, as demonstrated by a decrease in ³H thymidine incorporation to 72 (14)% of control. (D) IL-10 had no effect on DNA synthesis in PSCs.

Figure 3

Effect of cytokines on collagen synthesis by pancreatic stellate cells (PSCs). Data are expressed as per cent of control (mean (SEM); n=5 separate cell preparations). (A) Tumour necrosis factor α (TNF-α, 10 U/ml) significantly increased collagen synthesis. (B) PSCs incubated with interleukin (IL)-1 showed no change in collagen synthesis. (C) PSCs incubated with 10 ng/ml IL-6 exhibited a decrease in collagen synthesis to 49.7 (7.8)% of control levels. (D) IL-10, at a concentration of 50 ng/ml, stimulated collagen synthesis by PSCs to 203 (40)% of control levels.

Figure 4

Representative northern blot showing procollagen-α₁(1) and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA expression in pancreatic stellate cells treated with tumour necrosis factor α (TNF-α).