Human neutrophils stimulated with collagen breakdown product PGP results in chemotaxis, instant calcium influx and CXCL8 release

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by the progressive development of irreversible airflow limitation. Neutrophils contribute to lung tissue destruction leading to the formation of collagen fragments such as N-acetylated Proline-Glycine-Proline (PGP), which is mediated through the chemokine receptors CXCR1/2. In this study, we propose a new mechanism for PGP in neutrophilic inflammation related to COPD. Methods: Chemotaxis was performed with PGP or PGG (control peptide) on freshly isolated human neutrophils for one hour after which the chemotactic index was measured. Moreover, the ability of PGP or PGG to induce an instant calcium influx in neutrophils, a measurement for cell activation, was examined. Furthermore, neutrophils were incubated with medium, PGP or PGG for one hour and accordingly CXCL8 production was assessed by ELISA. Finally, human neutrophils were pre-incubated with or without α-CXCL8 for one hour, after which chemotaxis was performed with or without PGP for one hour. Results: PGP induced dose dependently chemotaxis of neutrophils, which is associated with a calcium influx in the cell. Furthermore, neutrophils were able to produce CXCL8 after one hour of stimulation by PGP. To determine if PGP was solely responsible for the migration and activation of neutrophils in vitro and not the released CXCL8 upon neutrophil stimulation, cells were incubated with α-CXCL8. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of PGP, whereas the effects of CXCL8 were attenuated. Conclusion: In this report, we propose a new mechanism for PGP in neutrophilic inflammation related to COPD. PGP is chemotactic for the neutrophil and induces intracellular calcium mobilization and CXCL8 release. However, the observed chemotactic and calcium effects are independent of CXCL8 in vitro. Our data suggest that PGP stimulates the neutrophils at the side of inflammation to release CXCL8, presumably leading to a self-perpetuating situation where neutrophils attract more neutrophils mediated by PGP and CXCL8 synergistically, provoking further lung destruction.