Cholecystokinin blockade alters the systemic immune response in rats with acute pancreatitis

Abstract

Acute pancreatitis (AP) is characterized by initial pancreatic injury resulting from the activation of digestive enzymes and, later, widespread inflammation to distant organs. The aim of this study was to study whether the time-course of inflammatory events during AP induced by bile-pancreatic duct obstruction (BPDO) varies after lowering the acinar enzyme content by L364,718 (0.1 mg/kg/day) administration over 7 days before inducing AP. Flow cytometric immunophenotyping was used to analyse the following at different AP stages: distribution of major circulating leucocyte subsets, activation state of circulating neutrophils and monocytes as reflected by CD11b expression and tumour necrosis factor-alpha (TNF-alpha) production and the contribution of T-cell-derived pro-(TNF-alpha) and anti-(IL-10) inflammatory mediators. TNF-alpha plasma levels and neutrophil infiltration in pancreas and lung were also measured. At early BPDO times, L364,718 treatment partially inhibited leukocytosis and increase in peripheral blood neutrophils and monocytes as well as TNF-alpha expression by monocytes. However, from 6 h onwards after BPDO, L364,718 treatment was unable to prevent either pancreatic and lung neutrophil infiltration or the release of TNF-alpha from activated monocytes. By its action on circulating lymphocytes, L364,718 treatment enhanced the severity of the inflammatory response induced by BPDO. Peripheral blood lymphocytes were recruited from earlier BPDO times, and 12 h after BPDO, T cells displayed a significantly higher reserve of TNF-alpha able to be released under stimulation but lower functional reserve of interleukin-10 (IL-10) than observed in untreated rats. It is concluded that lowering the acinar enzyme content through L364,718 treatment prevents earlier systemic immune events in BPDO-induced AP. However, at the point of maximal injury, the inflammatory response became pronounced, largely due to the role played by activated T lymphocytes.

Figure 1

Absolute number of cells (total leucocytes, neutrophils, monocytes, total lymphocytes, CD4⁺ and CD8⁺ T cells) per µl of peripheral blood (PB) found in controls (open bars) and in rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). There were five animals in each group. Values are expressed as mean ± SEM. Analysis of variance (anova) followed by Dunnett's test showed significant differences vs. control rats (♦P < 0.05, ♦♦P < 0.01, ♦♦♦P < 0.001). Application of Student's t-test to L364,718-pretreated and nontreated BPDO rats showed significant differences (P < 0.05, P < 0.01, P < 0.001).

Figure 2

Expression of CD11b on neutrophils and monocytes in controls (open bars) and in rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). Results are expressed as mean fluorescence intensity (MFI; relative linear units). There were five animals in each group. Values are expressed as mean ± SEM. Analysis of variance (anova) followed by Dunnett's test showed significant differences vs. control rats (♦P < 0.05, ♦♦P < 0.01). Application of Student's t-test to L364,718-pretreated and nontreated BPDO rats showed significant differences (P < 0.01).

Figure 3

Pancreatic and lung myeloperoxidase (MPO) activity in controls (open bars) and in rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). There were five animals in each group. Values are expressed as mean ± SEM. Analysis of variance (anova) followed by Dunnett's test showed significant differences vs. control rats (♦P < 0.05, ♦♦P < 0.01).

Figure 4

Spontaneous and lipopolysaccharide (LPS)-induced production of tumour necrosis factor-α (TNF-α) by peripheral blood (PB) monocytes from controls (open bars) and from rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). For nonstimulated samples, results are expressed as mean fluorescence intensity (MFI; relative linear units), and for stimulated specimens, results are expressed as the percentages over spontaneous production. There were five animals in each group. Values are expressed as mean ± SEM. Analysis of variance (anova) followed by Dunnett's test showed significant differences vs. control rats (♦P < 0.05, ♦♦P < 0.01). Application of Student's t-test to L364,718-pretreated and nontreated BPDO rats showed significant differences (P < 0.05).

Figure 5

Spontaneous and phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced production of tumour necrosis factor-α (TNF-α) by CD4⁺ and CD8⁺ T lymphocytes from controls (open bars) and from rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). For nonstimulated samples, results are expressed as mean fluorescence intensity (MFI; relative linear units), and for stimulated samples, results are expressed as percentage over spontaneous production. There were five animals in each group. Values are expressed as mean ± SEM. Analysis of variance (anova) followed by Dunnett's test showed significant differences vs. control rats (♦P < 0.05, ♦♦P < 0.01, ♦♦♦P < 0.001). Application of Student's t-test to L364,718-pretreated and nontreated BPDO rats showed significant differences (P < 0.05, P < 0.01).

Figure 6

Plasma tumour necrosis factor-α (TNF-α) levels in controls (open bars) and in rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). There were five animals in each group. Values are expressed as mean ± SEM. Results of Dunnett's test showed significant differences vs. control rats (♦P < 0.05, ♦♦P < 0.01).

Figure 7

Spontaneous and phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced production of interleukin-10 (IL-10) by CD4⁺ and CD8⁺ T lymphocytes from controls (open bars) and from rats with bile-pancreatic duct obstruction (BPDO) for different time periods: nontreated (black bars) or previously treated with L364,718 (0.1 mg/kg/day) for 7 days (hatched bars). For nonstimulated samples, results are expressed as mean fluorescence intensity (MFI; relative linear units), and for stimulated samples, results are expressed as percentage over spontaneous production. There were five animals in each group. Values are expressed as mean ± SEM. Results of Dunnett's test showed significant differences vs. control rats (♦P < 0.05).