DAILY FINE AND ULTRAFINE PARTICULATE MATTER EXPOSURE AFFECTS INFLAMMATORY AND COAGULATORY MARKERS AMONG SUSCEPTIBLE SUBJECTS.

Current concerns about the health effects of airborne particles are largely based on the results of epidemiological studies, suggesting effects on mortality and morbidity for cardiovascular and respiratory causes at very low levels of particulate matter exposure. The mechanisms behind these effects are still unknown, although some hypothesis have been postulated. The studies support the idea that inhalation of PM can instigate extra-pulmonary effects by the release of pro-inflammatory mediators (eg. cytokines, activated immune cells, platelets), vasculoactive molecules (eg. ET, histamine, microparticles) from lung-based cells; and/or by translocation of PM (UFPs) or particle constituents (organic compounds, metals) into the systemic circulation. Subsequently, these events may contribute to a systemic inflammatory state, which may in turn be capable of activating haemostatic pathways, impairing vascular function, and accelerating atherosclerosis. Which fraction of PM is the most harmful is still controversial, and few studies investigated the role of personal exposure to different fractions, in particular fine and ultrafine particles. Aims. My doctoral thesis aims at characterize the effects of PM exposure on inflammatory and coagulatory indices in susceptible subjects (with chronic heart diseases or chronic respiratory diseases), focused on the role of the fine and ultrafine particles in determining changes in these markers. Materials and methods. 27 healthy individuals (Healthy group), 34 individuals with chronic ischemic heart disease (Heart group), 18 with chronic asthma or COPD (Lung group) underwent a 24-hour exposure/clinical evaluation protocol during their habitual activities, both in the warm season (no heating period) and in the cold season (heating period). Individual exposure to UFPs (0.02–1 µm, Aerodynamic Diameter, D.a), fine and coarse particles number concentration (0.3-0.5; 0.5-1.0; 1.0-2.5; 2.5-5.0; 5.0-10 µm D.a), gravimetric PM0.5, PM1, PM2.5 and PM10 was assessed for each subject, along with measurement of total blood cells count and blood markers of inflammation [TNF-alfa, sRI- and sRII-TNF-alfa, IL-8, IL-10, hs-CRP] and coagulation [fibrinogen, aPTT, INR, D-dimer, vWF, tPA, F1+2, closure time measured with PF 100 Analyzer® (PFA100 C-EPI CT e PFA100 C-ADP CT)]. Since the three groups had different clinical status, each group were analyzed independently using mixed effects models for repeated measurements to evaluate the associations between particles exposure and clinical parameters. Models include time varying factor (PM, temperature and relative humidity) and time-invariant subjects specific characteristics (age, gender, BMI, drug assumption). Pollution effects were expressed as percent changes by interquartile range (IQR) changes of PM ( Chuang K. et al. 2007). Results. The mean age for the overall studied population was 64±10 years at the beginning of the study and the gender was male for the 63% of individuals. The median (25°-75° percentiles) 24h concentration of PM10 during the no-heating period was 35.5 (29.3-51.1) μg/m3 and during the heating period 58.0 (41.7-79.0) μg/m3. For PM2.5, the median concentration (25°-75° percentiles) during the no-heating period was 26.8 (21.4-37.7) μg/m3 and during the heating period 49.8 (33.7-66.3) μg/m3. Comparing the data from the two monitoring periods, the results showed a significant increase for particulate matter concentrations in the heating ignition power-on period. The PM10 percentage variation was 63.4% and for PM2.5 was 85.8%. The three groups of subjects were exposed to similar PM concentration, except for fine particles (PM0.5, FP0.3-1 D.a), that were higher in the Healthy group. The subjects in the three groups provided different values of total leukocytes count, inflammatory parameters and coagulation parameters. Healthy group showed lower values of inflammatory markers than those in Heart and Lung groups, which in particular is characterized by slight higher levels of inflammatory markers and lower levels of an antinflammatory marker (IL-10). Concerning coagulation parameters, Heart group presented longer closure time (PFA-100 CT) than Lung and Healthy groups. The results of the mixed models in the Heart group showed significant increase in monocytes number associated with fine and coarse particles. The monocytes increased of 7.9% (p=0.06) in association with PM10. Erythrocytes number increased of 2.14% (p=0,02) in association with FP0.3-0.5 and of 1,9% (p=0.01) in association with CP5-10. Platelets number increased of 6.77% (p=0.08) in association with PM0.5, and of 5.19% (p=0.08) with CP5-10, although the relation is slight significant. In Healthy group, variations of lymphocytes number were associated with fine and coarse particles. The lymphocytes increased of 8,71% (p=0,01) in association with PM10, and of 5.68% (p=0.004) in association with FP0.3-0.5. In the Lung group there is a negative association with monocytes and lymphocytes with fine and coarse particles and only with fine particles respectively. The number of monocytes decreased of 14,73% (p=0,01) in association with PM10, and decreased of 14.45 % (p=0.02) in association with FP0.3-0.5. The lymphocytes decreased of 11.48% (p=0.04) in association with FP0.3-0.5 . There were negative associations between closure time (PFA-100 C-EPI CT) and fine particles in Healthy group, and between fine and coarse particles in Heart group. In Healthy group, the closure time was shortened of 10% (p=0.06) in association with PM1 and of 6.81 % (p=0.05) with FP0,5-1. In Heart group closure time was shortened of 17.84% (p=0.02) in association with PM1 and of 14.98 % (p=0.004) with FP0,5-1. In Healthy group the t-PA is positive associated with ultrafine, fine and coarse particles. The t-PA varied of 22.47% (p=0.07) in association with PM1 and of 22.38% (p=0.05) with FP0,3-0,5 No changes were present in the Lung Group. No statistical associations were found for cytokines, interleukins, hsPCR and the others parameters. Discussion. Investigated subjects experienced high levels of individual exposure to PM2.5 and PM10. In particular, exposure to PM2.5 exceeded the 24-hour mean of 25 µg/m3 suggested by WHO in both the investigated periods, while the limit suggested for PM10 (i.e 50 µg/m3) was exceeded only in cold season (WHO, 2005). Observed PM10 concentrations were similar to twenty four hours average reported for urban background in Europe (Larssen S. et al. 2005), and PM2.5 concentrations were similar to the previously reported for personal monitoring indoor and outdoor in Milan (Rotko T. et al. 2002). As expected, higher levels of exposure to almost all particles were observed in the cold season. The great contribution of these higher winter levels is mainly due to particles in the accumulation mode (FP0.3-1 µm). In cold season the PM levels are strongly affected by the stability of the atmosphere, by the low degree of air convection (characteristics of the warm season), and also by the heating which is one of the major sources of the particles in the accumulation mode. The different levels of exposure among the three groups were mainly caused by fine particles in the accumulation mode (FP0.3-1 µm) and the Healthy group seemed to be the higher exposed. An analysis of the activity of these subjects showed that they have spent more time outdoor than the subjects of other groups, so they were much exposed to particles from outdoor origin, the particles in the accumulation mode, confirming the outdoor origin of these particles. A comparative analysis of the biological data among the three groups shows that the Healthy group presented strongly different values from the other groups, as expected, confirming a great status of health of these subjects compared with the others. The Heart group showed an impairment of the coagulation parameters in comparison to the others, probably due to the characteristics of the diseases and the assumed anti-clotting therapy. The Lung group seemed to have an impairment of the activation of the antinflammatory markers with a persistent low grade condition of inflammation. The increase of monocytes, erythrocytes and platelets number in Heart group and lymphocytes number in Healthy group in association with fine and coarse particles could suggest an increased bone marrow activity, involving a variety of cell types, as a result of the effects of cytokines and chemokines from the lung that spill over into the circulation and trigger a cascade of inflammatory reaction signals generated in the lung. A leukocytosis associated with the activation of bone marrow activity was demonstrated in human exposed to PM, that reacted mobilizing leucocytes into circulation as a part of systemic exposure (Tan WC. et al. 2000; Sakai M. et al. 2004). On the other hand we observed a negative association between PM and monocytes and lymphocytes number in the Lung group that could be explain by the presence of allergic subjects in this group; the summer PM in Milan is more rich in pollens, endotoxins and biological materials (Camatini M. et al. 2010), so we could hypothesize a pro-allergic effects of no-heating PM levels, an increase of the inflammatory pattern during the no-heating period. The activation of the platelets aggregation capacity, measured as closure time with PF 100 Analyzer, in Heart and Healthy groups in association with fine particles could suggest that particles with little aerodynamic diameter could pass directly from the alveoli to the blood and interact with the platelets, impairing their aggregability. Another possible mechanism of platelets activation might reside in the pulmonary oxidative stress and the activation of subsets of white blood cells, that lead to a systemic lowering of endothelial- and platelet-derived nitrogen oxide and concomitant platelets activation (Brook RD et al 2008). Moreover, the increment of the tissue type plasminogen activator in the Healthy group in association with fine particles could suggest an increased thrombin generation and a reduced fibrinolytic activity. The lack of consistencies in the association with PM and cytokines and interleukins could be explain by the fact that we have measured these factor after about 12 hours of the higher PM exposure levels, missing probably the concentration peaks in the blood (they have very short half-life). While the lack of consistencies in the association with PM and fibrinogen and C reactive protein could be due to the time necessary for the ex-novo synthesis of these proteins in the liver, that requires an induction time of 1-2 days (Seaton A. et al. 1999, Ruckerl R. et al. 2006). The strength of this study is that it is one of the few using individual monitoring of gravimetric and number concentration of particles. The use of fixed site monitoring stations could not be representative of personal exposure resulting in imprecise associations (Delfino RJ. et al. 2008), therefore the individual monitoring is the only way to measure the real exposure of the subjects. A limitation of our study is that we enrolled a small number of subjects with different pathologies and drug therapies, that had a large impact on the biological parameters. Moreover we monitored these subjects only twice, resulting in few data for each subjects. Despite of these limitation, this work supports the hypothesis that exposure to PM results in a systemic inflammatory response, characterized by stimulation of bone marrow activity, that could increase the blood coagulability. It could also support the hypothesis that small particles may translocate form the lung into circulation and directly activate platelets and blood vessels. Together these mechanisms may account for the increase of cardiovascular events associated with episodes of air pollution. Conclusion. The results suggest that PM exposure could contribute to the risk of cardiovascular events, in particular in elderly and subjects with cardiovascular diseases. Since there are evidences linking PM air pollution exposure and cardiovascular mortality and morbidity, may we consider PM as a risk factor for cardiovascular diseases or not? Particulate matter exposure is ubiquitous, it may continuously enhance acute cardiovascular risk among susceptible people worldwide; moreover it may further elicits numerous adverse biological responses that could augment cardiovascular risk over the long term. Therefore, PM could be surely considered as a factor that modify and contribute to cardiovascular mortality and morbidity. Despite the huge amount of studies about health effects of PM exposure, some issues remain open: to define the role of particles with different aerodynamic diameters and their chemical composition; to characterize the contribution of other co-pollutants (ozone, nitrogen dioxide, sulphur dioxide); to assess the importance of regional and intra-city differences in composition and combination of pollutants; to better define the susceptible subjects and define recommendations to help to reduce PM exposure; and finally to define whether there is a safe PM threshold concentration that eliminates both acute and chronic effects in susceptible subjects but also in general population.