Ohistological staining for PE (brown color, DAB staining) inCollagen Breakdown Leads to Chronic Inflammationlung tissue of (C) a current smoker, (D) ex-smoker, (E) COPD patient with GOLD stage II and (F) a COPD patient with GOLD stage IV. Magnification, 6200. doi:10.1371/journal.pone.0055612.gfragments. Our results confirm the findings of O’Reilly et al, who found that in vitro human neutrophils were capable to generate PGP from whole collagen after LPS exposure [15]. Neutrophils contain all necessary components for PGP generation and in this report we demonstrated that simultaneous incubation of these cells with CSE and collagen leads to PGP generation. Although N-ac-PGP levels were measurable after a 16 hour incubation period, non-acetylated PGP could not be detected in these supernatants. This can be explained by the fact that cigarette smoke itself is responsible for N-terminally acetylating PGP, thereby enhancing its chemotactic capacity [17]. Louhelainen et al. [26] and Miller et al. [30] showed that smoking cessation improved lung function although elevated neutrophil counts and the protease burden in the airways continued for months. An explanation for the elevated neutrophil influx and protease levels after smoke cessation is that the continued neutrophil chemotaxis and activation is mediated via Nac-PGP. In this study we demonstrate that this chemotactic tripeptide can activate neutrophils to release CXCL8 that will lead to an increase in neutrophilic migration. In addition, N-ac-PGP also induced the release of MMP8 and MMP9 from neutrophils, which will result in more collagen breakdown and formation of Nac-PGP. It was recently published that N-ac-PGP can induce the release of MMP9, which is confirmed in this study. It was indicated that extracellular matrix-derived N-ac-PGP could result in a feed-forward cycle by releasing MMP-9 from activated PMNs through the ligation of CXCR1 and CXCR2 and subsequent activation of the ERK1/2 MAPK [31]. Tissue destruction is a hallmark of COPD. Since PE is essential in the collagen breakdown process, we measured the basal intracellular PE activity in PMNs from COPD patients. Interestingly, the basal PE activity of PMNs from COPD patients was remarkably Naringin higher than in PMNs from healthy donors, which suggests that PE can play an important role in lung collagen breakdown leading to the development of COPD. This phenomenon is not limited to COPD; Gaggar et 18325633 al. also observed that the PE activity was elevated in cystic fibrosis samples when compared with normal controls [9]. Moreover, here we suggest that the PMNs from COPD patients are activated to a greater extend, since the CXCL8 levels released by these PMNs appeared to be higher than from PMNs from healthy controls. Finally, to our knowledge, this is the first in vitro study that indicates that neutrophils activated by cigarette smoke can destruct collagen into N-ac-PGP and that this collagen fragment can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. From this study but also from other studies on collagen breakdown in COPD mice models [8,29], we can conclude that LIMKI3 different treatment interventions are possible to tackle the ongoing inflammation observed in COPD. MMP inhibitors might be a valuable drug target; in addition to suppressing the accelerated extracellular matrix turnover, the generation of chemotactic N-acPGP can be counteracted. Since we.Ohistological staining for PE (brown color, DAB staining) inCollagen Breakdown Leads to Chronic Inflammationlung tissue of (C) a current smoker, (D) ex-smoker, (E) COPD patient with GOLD stage II and (F) a COPD patient with GOLD stage IV. Magnification, 6200. doi:10.1371/journal.pone.0055612.gfragments. Our results confirm the findings of O’Reilly et al, who found that in vitro human neutrophils were capable to generate PGP from whole collagen after LPS exposure [15]. Neutrophils contain all necessary components for PGP generation and in this report we demonstrated that simultaneous incubation of these cells with CSE and collagen leads to PGP generation. Although N-ac-PGP levels were measurable after a 16 hour incubation period, non-acetylated PGP could not be detected in these supernatants. This can be explained by the fact that cigarette smoke itself is responsible for N-terminally acetylating PGP, thereby enhancing its chemotactic capacity [17]. Louhelainen et al. [26] and Miller et al. [30] showed that smoking cessation improved lung function although elevated neutrophil counts and the protease burden in the airways continued for months. An explanation for the elevated neutrophil influx and protease levels after smoke cessation is that the continued neutrophil chemotaxis and activation is mediated via Nac-PGP. In this study we demonstrate that this chemotactic tripeptide can activate neutrophils to release CXCL8 that will lead to an increase in neutrophilic migration. In addition, N-ac-PGP also induced the release of MMP8 and MMP9 from neutrophils, which will result in more collagen breakdown and formation of Nac-PGP. It was recently published that N-ac-PGP can induce the release of MMP9, which is confirmed in this study. It was indicated that extracellular matrix-derived N-ac-PGP could result in a feed-forward cycle by releasing MMP-9 from activated PMNs through the ligation of CXCR1 and CXCR2 and subsequent activation of the ERK1/2 MAPK [31]. Tissue destruction is a hallmark of COPD. Since PE is essential in the collagen breakdown process, we measured the basal intracellular PE activity in PMNs from COPD patients. Interestingly, the basal PE activity of PMNs from COPD patients was remarkably higher than in PMNs from healthy donors, which suggests that PE can play an important role in lung collagen breakdown leading to the development of COPD. This phenomenon is not limited to COPD; Gaggar et 18325633 al. also observed that the PE activity was elevated in cystic fibrosis samples when compared with normal controls [9]. Moreover, here we suggest that the PMNs from COPD patients are activated to a greater extend, since the CXCL8 levels released by these PMNs appeared to be higher than from PMNs from healthy controls. Finally, to our knowledge, this is the first in vitro study that indicates that neutrophils activated by cigarette smoke can destruct collagen into N-ac-PGP and that this collagen fragment can activate neutrophils, which may lead in vivo to a self-propagating cycle of neutrophil infiltration, chronic inflammation and lung emphysema. From this study but also from other studies on collagen breakdown in COPD mice models [8,29], we can conclude that different treatment interventions are possible to tackle the ongoing inflammation observed in COPD. MMP inhibitors might be a valuable drug target; in addition to suppressing the accelerated extracellular matrix turnover, the generation of chemotactic N-acPGP can be counteracted. Since we.
