Ads to the conclusion that the increase in MMP9 levels in COPD get GM6001 patients is the result of an increase in neutrophil number and not due to an increase in MMP9 release.Besides MMP8 and MMP9, PE is needed to generate PGP from whole collagen; the MMPs cleave whole collagen in fragments of 30 to 100 amino acids in length, after which PE specifically cleaves PGP from these smaller fragments [9]. Recently, it was published that neutrophils contain PE [15], which is confirmed in this study. PE activity was measured in lysates of PMNs. Incubation of PMNs with CSE or CJ-023423 site N-ac-PGP did not affect intracellular PE activity, which suggests that PE is constitutively active. Although PE activity could be measured in the supernatant of CSE or N-ac-PGP incubated PMNs, these levels were very low. We hypothesize that cigarette smoking causes a locally restricted lung inflammation where necrotic neutrophils or neutrophils undergoing NETosis release PE to the exterior, which contributes to PGP generation. This can be substantiated with data from figures 1 and 5; incubating PMNs for 16 hours with CSE resulted in a decrease in cell viability, PE release and subsequent generation of N-ac-PGP from whole collagen. It is possible that other cells besides neutrophils play a role in collagen destruction by supplying PE. Figure 7 shows that also pulmonary alveolar macrophages express PE. Neutrophils and macrophages present in lung tissue of current smokers and COPD patients with GOLD stage II and IV highly expressed PE, while the number of inflammatory cells and consequently the PE expression was decreased in the lung tissue of ex-smokers. The next step was to investigate the effect of CSE on the breakdown of whole collagen into collagen fragment N-ac-PGP by human neutrophils. The multistep pathway of collagen breakdown has been studied in a murine model of cigarette smoke-induced lung emphysema in our group by Braber et al. [29]. There it was demonstrated that all relevant components (neutrophils, MMP8, MMP9 and PE) involved in this pathway to generate (N-ac-)PGP from collagen were upregulated in the airways exposed to cigarette smoke, suggesting that activation of cells by cigarette smoke leads to the release of proteases and extracellular matrix breakdown. Although this murine model showed that (N-ac-)PGP is formed after cigarette smoke exposure in the airways, here we demonstrate using in vitro techniques that upon stimulation with CSE the human neutrophil is able to breakdown collagen into N-ac-PGPCollagen Breakdown Leads to Chronic InflammationFigure 8. The basal PE activity of PMNs from COPD patients is a 25-fold higher when compared to healthy donors. (A) 105 freshly isolated PMNs from healthy donors (block dots, n = 5) and COPD patients (white squares, n = 7) were stimulated for 9 hours with cigarette smoke extract (CSE; OD 0.03?.24). A CXCL8 ELISA was performed on the supernatants. PMNs from COPD patients tend to produce higher amounts of CXCL8 after CSE incubation (p = 0.0560, t-test CSE OD 0.12 donor vs. COPD). Individual data are shown, horizontal bars represent mean values. (B) The PE activity was measured in lysates of unstimulated PMNs (106 cells) using Z-Gly-Pro-AMC as a substrate. The basal PE activation of PMNs from COPD patients (white squares, n = 7) is significantly higher than the PE activity of PMNs from healthy donors (block dots, n = 3) (* p,0.05 Mann-Whitney). (C ) Localization of PE in the human lung. Representative photomicrographs of an immun.Ads to the conclusion that the increase in MMP9 levels in COPD patients is the result of an increase in neutrophil number and not due to an increase in MMP9 release.Besides MMP8 and MMP9, PE is needed to generate PGP from whole collagen; the MMPs cleave whole collagen in fragments of 30 to 100 amino acids in length, after which PE specifically cleaves PGP from these smaller fragments [9]. Recently, it was published that neutrophils contain PE [15], which is confirmed in this study. PE activity was measured in lysates of PMNs. Incubation of PMNs with CSE or N-ac-PGP did not affect intracellular PE activity, which suggests that PE is constitutively active. Although PE activity could be measured in the supernatant of CSE or N-ac-PGP incubated PMNs, these levels were very low. We hypothesize that cigarette smoking causes a locally restricted lung inflammation where necrotic neutrophils or neutrophils undergoing NETosis release PE to the exterior, which contributes to PGP generation. This can be substantiated with data from figures 1 and 5; incubating PMNs for 16 hours with CSE resulted in a decrease in cell viability, PE release and subsequent generation of N-ac-PGP from whole collagen. It is possible that other cells besides neutrophils play a role in collagen destruction by supplying PE. Figure 7 shows that also pulmonary alveolar macrophages express PE. Neutrophils and macrophages present in lung tissue of current smokers and COPD patients with GOLD stage II and IV highly expressed PE, while the number of inflammatory cells and consequently the PE expression was decreased in the lung tissue of ex-smokers. The next step was to investigate the effect of CSE on the breakdown of whole collagen into collagen fragment N-ac-PGP by human neutrophils. The multistep pathway of collagen breakdown has been studied in a murine model of cigarette smoke-induced lung emphysema in our group by Braber et al. [29]. There it was demonstrated that all relevant components (neutrophils, MMP8, MMP9 and PE) involved in this pathway to generate (N-ac-)PGP from collagen were upregulated in the airways exposed to cigarette smoke, suggesting that activation of cells by cigarette smoke leads to the release of proteases and extracellular matrix breakdown. Although this murine model showed that (N-ac-)PGP is formed after cigarette smoke exposure in the airways, here we demonstrate using in vitro techniques that upon stimulation with CSE the human neutrophil is able to breakdown collagen into N-ac-PGPCollagen Breakdown Leads to Chronic InflammationFigure 8. The basal PE activity of PMNs from COPD patients is a 25-fold higher when compared to healthy donors. (A) 105 freshly isolated PMNs from healthy donors (block dots, n = 5) and COPD patients (white squares, n = 7) were stimulated for 9 hours with cigarette smoke extract (CSE; OD 0.03?.24). A CXCL8 ELISA was performed on the supernatants. PMNs from COPD patients tend to produce higher amounts of CXCL8 after CSE incubation (p = 0.0560, t-test CSE OD 0.12 donor vs. COPD). Individual data are shown, horizontal bars represent mean values. (B) The PE activity was measured in lysates of unstimulated PMNs (106 cells) using Z-Gly-Pro-AMC as a substrate. The basal PE activation of PMNs from COPD patients (white squares, n = 7) is significantly higher than the PE activity of PMNs from healthy donors (block dots, n = 3) (* p,0.05 Mann-Whitney). (C ) Localization of PE in the human lung. Representative photomicrographs of an immun.
