Exposure of human neutrophils to cigarette smoke or N-acetyl proline-glycine-proline results in CXCL8 and matrix metalloprotease 8 and 9 release

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by progressive lung tissue destruction, leading to the formation of collagen fragments, such as N-acetylated Proline-Glycine-Proline (PGP). Cigarette smoke is one of the major risk factors for the development of COPD. PGP, due to its chemotactic properties, could be a key player in the continuous lung inflammation as seen in COPD and cystic fibrosis (CF). The production of PGP is suggested to be dependent on the actions of MMP8 and 9 with a final cleavage step catalyzed by prolylendopeptidase (PE). In this study, this proteolytic cascade was investigated. Method: Human neutrophils (n=5) were incubated with cigarette smoke extract (CSE; OD 0-0.12) or PGP (10-4-3×10-3M) during 9 and 16 hours. Subsequently, MMP8 and CXCL8 were measured by ELISA. MMP9 was determined by Western blot while PE was detected in human fresh neutrophils by immunofluorescence microscopy. Results: CSE and PGP both induced dose dependent chemotaxis of freshly isolated neutrophils in vitro. Furthermore, neutrophils released MMP8, MMP9 and CXCL8 after stimulation for 9 or 16 hours with CSE and PGP. Moreover, it was demonstrated that PE is present in neutrophils. Conclusion: In conclusion, our data show that CSE and PGP activate neutrophils to release MMP8, MMP9 and CXCL8. Moreover, PE can be detected in neutrophils. Interestingly, this increased MMP8, MMP9 and PE release may be responsible for the specific generation of PGP from lung collagen, leading to further lung destruction. Proposed proteolytic cascade of neutrophil collagen breakdown, leading to further tissue breakdown. (Figure presented) Cigarette smoke stimulates the neutrophil to release mediators, ultimately leading to the release of PGP from collagen and a prolonged neutrophil influx. Interestingly, PGP stimulates the neutrophil to MMP and PE release, thereby inducing collagen breakdown and self-synthesis. Moreover, this tripeptide can stimulate the neutrophil to release CXCL8, which leads to a self-perpetuating situation where neutrophils attract more neutrophils.