SUMMARYNeutrophil chemotaxis has been shown to be regulated by two different signalling pathways that allow strong chemoattractants, such as bacterial-derived formylated peptides, to dominate over endogenous attractants, such as interleukin-8 (IL-8). Here we show that triggering of the formyl peptide receptor (FPR) with f-Met-Leu-Phe (fMLF) substantially reduced the neutrophil superoxide production induced by activation of the CXC receptors with IL-8. When the order of agonists was reversed, the cells were primed in their response to fMLF, suggesting that the signalling hierarchy between strong, so-called end-type (i.e. fMLF) and weak or intermediatetype (i.e. IL-8) chemoattractants, is also operating during activation of the NADPH-oxidase. The same result was obtained when fMLF was replaced with the hexapeptide, WKYMVM, specific for the formyl peptide-like receptor 1 (FPRL1). There were additional differences between the agonist receptor pairs fMLF/FPR, WKYMVM/FPRL1 and IL-8/CXCR. In contrast to FPR and FPRL1, no reserve pool of CXCR was present in subcellular granules and it was impossible to prime the oxidative response transduced through CXCR by the addition of priming agents such as tumour necrosis factor-a and platelet-activating factor. Moreover, the cytoskeleton-disrupting substance, cytochalasin B, had no effect either on IL-8-triggered oxidase activation or on CXCR reactivation. A pertussis toxin-sensitive G-protein is involved in signalling mediated through both FPR and CXCR, and the signalling cascades include a transient intracellular calcium increase, as well as downstream p38 MAPK and phosphoinositide 3-kinase activation. The data presented in this study provide support for two different signalling pathways to the neutrophil NADPH-oxidase, used by ligand binding to FPR/FPRL1 or CXCR, respectively.

Mature human neutrophils contain small amounts of interleukin‐8 [CXC chemokine ligand 8 (CXCL‐8)], which upon proinflammatory activation, increases significantly. It has been suggested that the CXCL‐8 content of resting human neutrophils is stored in the secretory vesicles. Here, we have used a fractionation technique, which allows isolation of these vesicles, and we find that CXCL‐8 neither colocalizes with the secretory vesicles nor with markers of any of the classical neutrophil granules. To increase resolution in the system, we induced CXCL‐8 production by lipopolysaccharide. After 8 h of stimulation, CXCL‐8 was visualized within the cell using immunoelectron microscopy. The images revealed CXCL‐8‐containing stuctures resembling neutrophil granules, and these were distinct from all known neutrophil organelles, as shown by double immunostaining. Further, the CXCL‐8 organelle was present in nonstimulated neutrophil cytoplasts, entities lacking all other known granules and secretory vesicles. Upon fractionation of the cytoplasts, CXCL‐8 was found to partly cofractionate with calnexin, a marker for endoplasmic reticulum (ER). Thus, part of CXCL‐8 may be localized to the ER or ER‐like structures in the neutrophil.

Detergent-resistant membrane domains (DRMs) are present in the membranes of azurophil granules in human neutrophils (Feuk-Lagerstedt et al., J. Leukoc. Biol. 2002, 72, 970). Using a proteomic approach, we have now identified 106 proteins in a DRM preparation from these granule membranes. Among these proteins were the lipid raft structural proteins flotillin-1 and -2, cytoskeletal proteins such as actin, vimentin and tubulin, and membrane fusion promoting proteins like annexins and dysferlin. Our results suggest that the azurophil granule membrane, in similarity to the plasma membrane, is an elaborate structure that takes part in intracellular signaling and functions other than the mere delivery of bactericidal effector molecules to the phagosome.

Background: Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions.

No abstract