The  -galactoside binding immunomodulatory lectin galectin-3 reverses the desensitized state induced in neutrophils by the chemotactic peptide f-Met-Leu-Phe: role of reactive oxygen species generated by the NADPH-oxidase and inactivation of the agonist

Abstract

Neutrophils interacting with a chemoattractant gradually become nonresponsive to further stimulation by the same agonist, a process known as desensitization. Receptor desensitization is a highly regulated process that involves different mechanisms depending on which receptor-ligand pair that is studied. Galectin-3, a member of a large family of beta-galactoside-binding lectins, has been suggested to be a regulator of the inflammatory process, augmenting or directly triggering the neutrophil functional repertoire. We show here that the desensitized state of neutrophils interacting with the chemotactic peptide fMLF is broken by galectin-3 and that this is achieved through an oxygen radical-mediated inactivation of the chemoattractant. The effect was inhibited by the competitor lactose and required the affinity of galectin-3 for N-acetyllactosamine, a saccharide typically found on cell surface glycoproteins. The latter was shown using a galectin-3 mutant that lacked N-acetyllactosamine binding activity, and this protein was not active. The mechanism behind the inactivation of the chemoattractant was found to depend on the ability of galectin-3 to induce a neutrophil generation/secretion of reactive oxygen species which in combined action with myeloperoxidase inactivated the peptides.

Stable formyl peptide receptor agonists that activate the neutrophil NADPH-oxidase identified through screening of a compound library

“…Many FPR agonists can trigger their own inactivation when interacting with neutrophils (reported here for fMLF and WKYMVM) and the activity of the MPO-hydrogen peroxide system is the basis for the loss of biological activity [14,44]. Agents sensitive to this type of inactivation is not restricted to chemotactic factors [45,46], but the three most potent agonists in our ROS production assay (4, 5 and, 10), were all stable in relation to the radicals generated.…”

“…We know from earlier studies that the inactivation is dependent on the granule enzyme MPO [14], but no MPO secretion was induced by any of the compounds used (data not shown), suggesting that the basal level MPO is sufficient. Moreover, the neutrophil activity induced by a cell free supernatant, originating from a mixture of cells, WKYMVM (an FPR2 agonist) and, compound 5 (primarily an FPR1 agonist; see below) was totally inhibited by 14 cyclosporine H (an FPR1 antagonist), showing that when one sensitive and one stable agonist are added to the cells simultaneously, only one of them "survives".…”

“…The release of myeloperoxidase (MPO), an azurophil granule marker of importance for inactivation of chemoattractants [14], was determined as described earlier [27] Page 10 of 35 A c c e p t e d M a n u s c r i p t 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 A c c e p t e d M a n u s c r i p …”

“…Even though the EC 50 value for activation of the oxidase with compound Neutrophil derived reactive oxygen species are highly reactive not only with bacterial proteins during bacterial clearance process, they could also oxidize and inactive small molecules very rapidly. Accordingly, we recently showed that the FPR agonists fMLF and WKYMVM are rapidly inactivated by ROS producing neutrophils in a process that is dependent on the azurophil granule protein MPO [14,28]. To determine the sensitivity of the new compounds for neutrophil derived oxygen radicals, we added the compounds 4, 5, or 10 at high concentrations (10µM) to neutrophils.…”

“…The eicosanoid is easily oxidized and by that biologically inactivated [11][12][13], and this is true also for many of the peptide agonists [14].…”