Inflammatory activation of neutrophils by Helicobacter pylori; a mechanism insensitive to pertussis toxin

Abstract

Chronic active gastritis of the antral mucosa is a characteristic feature of infection with Helicobacter pylori and interactions between bacterial components and inflammatory cells are believed to play an important pathogenic role. Neutrophils stimulated with H. pylori sonicate were demonstrated to release L-selectin (CD62L) expressed on the cellular surface, with a subsequent up-regulation of the beta2-integrins CD11b and CD11c, both in a dose- and time-dependent manner, reaching maximum levels after 45-60 min of stimulation. No changes were observed for the CD11a receptor upon stimulation. The activating properties of H. pylori sonicates on neutrophils were heat-labile and susceptible to protease attack, indicating the protein nature of the activating factor. After size fractionation, the major neutrophil-inducing activity was detected in the high molecular weight fraction exhibiting urease activity. Pertussis toxin was unable to inhibit neutrophil activation by the H. pylori protein(s). We conclude that proteins from H. pylori have a potent inflammatory effect on the surface membrane molecules CD62L, CD11b and CD11c essential for transendothelial migration of neutrophils to areas of inflammation. The neutrophil-activating protein(s) act via a pertussis toxin-insensitive mechanism.

Fig. 1

Kinetics of up-regulation of CD11b (a), CD11c (b) and shedding of CD62L (c) on neutrophils stimulated with Helicobacter pylori sonicate. The results are expressed as mean fluorescence intensity (MFI) after substracting the values for unstimulated control cells at each time point ± s.d. (a,b) or as a ratio of MFI of stimulated cells/unstimulated cells at each time point (c). The results represents mean of four experiments with neutrophils from H. pylori-seronegative donors. ▪, 28i6 at 100 μg/ml; □, NCTC 11638 at 100 μg/ml; •, fMLP 10⁻⁸ mol/l; Δ, PMA 10 ng/ml.

Fig. 2

Up-regulation of adherence molecules on neutrophils stimulated with Helicobacter pylori sonicate fractions at a concentration of 100 μg/ml for 30 min. The results for up-regulation of CD11b are expressed as mean fluorescence intensity (MFI) after substracting the values for unstimulated control cells, whereas results for shedding of CD62L are expressed as a ratio of the MFI of stimulated cells/unstimulated control cells multiplied by 100. The results represent mean of three experiments performed in duplicate with sonicate fractions from two separate column preparations. □, CD62L; ▪, CD11b.

Fig. 3

SDS–PAGE in an 18% Tris-trisine gel with crude sonicate from Helicobacter pylori NCTC 11638 and with fractions I–VI obtained after separation on a Sephacryl S-200 H column. Molecular weight standards are shown on the left lane.

Fig. 4

(a) Time-resolved chemiluminescence of neutrophils induced by sonicate proteins from Helicobacter pylori strain NCTC 11638 at 100 μg/ml. ▪, Control cells; □, pertussis toxin (PT)-treated neutrophils; ○, fMLP 10⁻⁸ mol/l PT-treated neutrophils. Representative of four experiments. (b) Time-resolved fluorimetry emission measured at 490 nm. The curves represent changes for stimulated neutrophils after substracting values of unstimulated control cells, measured in arbitrary units. (i) fMLP 10⁻⁸ mol/l PT-untreated cells; (ii) H. pylori strain NCTC 11638 at 100 μg/ml PT-untreated cells; (iii) fMLP 10⁻⁸ mol/l PT-treated cells; (iv) H. pylori strain NCTC 11638 at 100 μg/ml PT-treated cells. Representative of four experiments.