Problems in identifying microbial-derived neutrophil activators, focusing on Helicobacter pylori

We are encouraged by Bylund and Dahlgren's response to our previous letter and are pleased with this general discussion regarding the problems associated with assaying the pro-inflammatory activities of bacterial virulence factors. We hope that other researchers in the field will take particular note of their comments, which, although focused on human neutrophils, can also be extended to other inflammatory cells. Bylund and Dahlgren point out that: (1) a single bacterial factor might not fully activate the response of neutrophils; (2) different factors can act synergistically in activating neutrophils; and (3) different factors activate the plasma membrane-located NADPH oxidase, which releases oxygen radicals into the medium, and/or the endosome–phagolysosome-located NADPH oxidase, which releases oxygen radicals inside the cell. They mention the different assays available to detect the radicals released at the two cell sites.Each bacterium raises different problems depending on its anatomical location with respect to the inflammatory cells; its capacity for tissue invasion; the extent to which it is phagocytosed; and its ability to release bacterial components [e.g. lipopolysaccharide (LPS), peptides and proteins] into the surrounding medium. In the particular case of Helicobacter pylori, this bacterium is not invasive and resides mainly in the mucus layer and on the apical domain of the epithelial lining. Thus, the release of pro-inflammatory components is believed to be highly relevant to the pathogenesis of H. pylori-associated gastroduodenal diseases. A prominent role could be played by oxygen radicals released from activated neutrophils, which might act as such or as novel molecules generated by their chemical reactions with various molecules present in the local environment. Another peculiarity of H. pylori is that its LPS is poorly active. For these reasons, we did not include LPS in our assay, which used cytochrome c to detect oxygen radicals mainly released into the medium. In addition, we used amounts of a formylated peptide and reaction times that, in our hands, provided maximal activation to prove that even the effect of such saturating amounts of agonist acting for a prolonged period of time were undetectable in the presence of the complex mixture of components present in an H. pylori extract. We believe that initial experiments to detect pro-inflammatory activities can be carried out using crude bacterial extracts. Research should then proceed with the determination of the activity of single purified components, followed by the study of the synergistic effects exhibited by the various purified virulence factors, as Bylund and Dahlgren discussed.