Elastase, a serine proteinase released by activated human neutrophils, can degrade a wide variety of biomacromolecules including elastin, and is considered a marker of inflammatory diseases. As the logical strategy to protect tissue is to inhibit excessive elastase activity, experimental and clinical researches have concentrated on trying to find efficient elastase inhibitors. As thymol, one of the major components of thyme oil with a phenolic structure, has been credited with a series of pharmacological properties, that include antimicrobial and antioxidant effects, the aim of this study was to explore whether it can also interfere with the release of elastase by human neutrophils stimulated with the synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). After the neutrophils were incubated with increasing amounts of thymol (2.5, 5, 10, 20 µg/ml), elastase release was initiated by fMLP and measured using MeO-Suc-Ala-Ala-Pro-Val-MCA. The results showed that thymol inhibited fMLP-induced elastase release in a concentration-dependent manner, with the effects of 10 and 20 µg/ml being statistically significant. The behavior of cytosolic calcium mobilization revealed by fura-2 closely resembled that of elastase, thus suggesting that they may be related. The hydrophobic nature of thymol means that it can approach ion channel proteins through the lipid phase of the membrane, alter the local environment of calcium channels and thus inhibit capacitative calcium entry. In brief, thymol inactivates calcium channels machinery, thus triggering a corresponding reduction in elastase. The antibacterial and antimycotic activity of thymol is already well known, but our findings that it inhibits elastase extend our knowledge of the anti-inflammatory activity of this interesting molecule that is already credited with antioxidant activity. These two latter characteristics make thymol a molecule that can have helpful effects in controlling the inflammatory processes present in many infections.

The aim of this study was to investigate the effect on the induction of interleukin-8 of particulate matter (PM) from fir and beech pellets burnt in domestic appliances on two human cells lines, namely the lung epithelial cell line A549 and the promyelocytic cell line THP-1. The effects of PM2.5 obtained from combustion of beech and fir pellets were compared to reference diesel exhaust particulates (DEP). In parallel, wood smoke PM-induced genotoxicity and oxidative stress were also investigated in A549 cells. Cells were treated for different times (3-72 h) with increasing concentrations of PM2.5 obtained from sequential combustions of fir and beech pellets or reference DEP. Cell viability was assessed by lactate dehydrogenase leakage, and the release of interleukin-8 or CXCL8 (IL-8) was measured to evaluate the pro-inflammatory effect. Oxidative stress was evaluated by the 5(6)-carboxy-2',7'dichlorofluorescein diacetate (DCFH-DA) assay and DNA damage by the alkaline comet assay and micronucleus frequency by flow cytometry. Both A549 and THP-1 cells responded in a dose- and time-related manner to wood smoke PM2.5 with IL-8 release, particles obtained from late combustions being the most active. THP-1 cells were more sensitive than A549 cells. On a mass base, similar effects were observed for both fir and beech PM2.5. However, the combustion of beech pellets generated approximately three times more PM2.5 than fir pellets. Regarding the mechanism of PM2.5 uptake, in both THP-1 and A549 cells, cytochalasin D prevented PM2.5-induced IL-8 mRNA expression and cytokine release, indicating a key role for actin polymerization in particles uptake and that the production of IL-8 correlated with particle phagocytosis. As signal transduction pathway involvement, in both THP-1 and A549 cells, PM2.5-induced IL-8 release could be completely blocked by the selective inhibitor SB203580, indicating a role of p38 MAPK activation. PM2.5 from both fir and beech pellets also induced modest DNA lesions dose related, measured as strand breaks, whereas no increase in the number of micronucleus was observed. Similar effects were observed with DEP, arguing against less dangerous effects of wood smoke particles than other categories of combustion-derived particles in the same size range. Overall, results suggest that combustion conditions can significantly affect the characteristics of particles and the consequent toxicity, and that different woods can generate different amounts of PM2.5.

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