Expression of interleukin 8 (IL-8) and substance P in human chronic pancreatitis

Abstract

Background: Changes in substance P content and a relationship between the degree of perineural inflammation and pain has been demonstrated in chronic pancreatitis. Whether a relationship exists between neural alteration and pancreatic inflammation (neurogenic inflammation) is not known.

Aims: In the present study we evaluated gene expression of preprotachykinin A (PPT-A), the gene encoding substance P, and interleukin 8, a proinflammatory and hyperalgesic mediator whose release is co-regulated by substance P.

Patients: Pancreatic tissue specimens obtained from 21 patients (16 male, five female) with chronic pancreatitis and 18 healthy organ donors (nine male, nine female) were analysed.

Methods: Gene expression of PPT-A and interleukin 8 was studied by northern blot analysis. Respective proteins were localised using immunohistochemistry.

Results: Northern blot analysis showed that PTT-A mRNA expression levels were present at comparable levels in normal and chronic pancreatitis tissue samples. In contrast, interleukin 8 mRNA was expressed at very low levels in normal controls but was increased 41-fold (p<0. 001) in chronic pancreatitis tissue samples. Using immunohistochemistry, interleukin 8 protein was localised mainly in immune cells often found around enlarged pancreatic nerves. In addition, in chronic pancreatitis, intense interleukin 8 immunostaining was present in metaplastic ductal cells of the atrophic pancreatic parenchyma. In chronic pancreatitis samples there was a positive relationship between interleukin 8 mRNA levels and the presence of ductal metaplasia (r=0.795; p<0.001) and the inflammation score (r=0.713; p<0.001).

Conclusions: Our data indicate that in chronic pancreatitis, the increase in substance P in enlarged pancreatic nerves is not caused by enhanced intrapancreatic PTT-A mRNA expression, suggesting that the location of substance P synthesis is outside of the pancreas. In addition, localisation of interleukin 8 positive immune cells around pancreatic nerves further supports the existence of neuroimmune interactions as a pathophysiological mechanism in chronic pancreatitis.

Figure 1

Northern blot analysis. Preprotachykinin A (PPT-A) and interleukin 8 (IL-8) mRNA expression in the normal pancreas (first four lanes) and in tissue samples from patients with chronic pancreatitis (CP) (latter four lanes). PPT-A mRNA was detectable in comparable levels in normal and CP tissue samples. In contrast, enhanced levels of IL-8 mRNA were present in CP samples. The filters were rehybridised with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA to verify equivalent RNA loading and RNA transfer. PPT-A mRNA migrates as a 1.2 kb band and IL-8 as a 1.8 kb band.

Figure 2

Densitometric analysis of northern blots. Preprotachykinin A (PPT-A), interleukin 8 (IL-8), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were analysed by laser densitometry in the normal pancreas and in chronic pancreatitis. The ratio of the optical density between PPT-A, IL-8, and the corresponding GAPDH signal was calculated. Bars represent the median with upper and lower quartiles. *p<0.001.

Figure 3

Immunohistochemical analysis. Interleukin 8 (IL-8) immunohistochemical analysis in the normal pancreas (A) and in chronic pancreatitis (CP) (B-D, F, H). In the normal pancreas (A), no IL-8 immunostaining was detectable. In contrast, in CP tissue sections (B-D, F, H), moderate to intense IL-8 immunoreactivity was found, mainly in the inflammatory cells (D, F, arrows) surrounding enlarged pancreatic nerves. In some cases those nerves were substance P immunoreactive (E). Panels (G) and (H) (arrowheads) show staining of consecutive tissue sections of CP for CD-68 (G) and IL-8 (H). The inserts in (G) and (H) show that IL-8 immunoreactive cells (H) are most probably also stained CD-68 positive (G). In addition, in CP tissue samples, moderate to strong IL-8 immunoreactivity was also found in metaplastic ductal cells (B, C, arrows). n=nerve; d=duct. (A) IL-8 immunostaining; (B- D, F, H) IL-8 immunostaining (patients Nos 14, 16, see table 1); (E) substance P immunostaining (patient No 14); (G) CD-68 immunostaining (patient No 16). Original magnification ×200.