Human recombinant MCP-1 (0.1 or 0.9 ng/ml) and LPS (10 μg/ml) were dissolved in RPMI culture medium. The concentrations of MPC-1 were based on levels found in the supernatant of ex vivo vehicle or HQ-exposed tracheal tissue (0.1 or 0.9 ng/ml,

respectively). The bottom wells of Palbociclib clinical trial the Boyden chamber were filled with RPMI culture medium or MCP-1 and LPS solution. The THP-1 cells (1 × 105 cells/ml) were placed in the top wells. The filters were stained after an incubation period of 24 h (37 °C; 5% CO2) and THP-1 migration within the filter was determined under light microscopy. The distance was measured from the top of the filter to the furthest plane still containing two cells using 40× objectives, according to the methods of Mello et al. (1992) and Zigmond and Hirsch (1973). Duplicate wells were tested for each variable and five fields were counted and averaged per filter. Means and standard errors of the mean (s.e.m.) of all data presented herein were compared using Student’s t-test or ANOVA. Tukey’s multiple comparisons were used to determine the significance of differences calculated between the values for the experimental conditions. GraphPad Prism 5.0 software (San Diego, CA, USA) was used. Differences

were considered significant at P < 0.05. Hydroquinone exposure did not alter the number of circulating cells (Table 1) or the number of AM macrophages in the BALF in the absence of inflammation caused by LPS inhalation (data not shown). Eight hours after the beginning of the inflammatory process the number of circulating mononuclear Androgen Receptor antagonist cells was equally increased in vehicle and HQ-exposed animals (Table 1). On the other hand, the influx of mononuclear cells into BALF was markedly reduced in the HQ-exposed mice (Fig. 1A). It is worth noting that LPS was an effective stimulus as the number of cells in the BALF of vehicle-exposed animals was significantly increased after inflammation. The dotted line indicates the basal number of cells present in the BALF (Fig. 1A). According to cell identification on the basis of surface

markers, the majority of mononuclear cells present in the BALF after LPS stimulation were macrophages, and their numbers were reduced PtdIns(3,4)P2 in HQ-exposed mice (Fig. 1B and C). Leukocyte traffic depends on a highly coordinated process involving the sequential expression of adhesion molecules. Therefore, the possibility that HQ exposure could impair mononuclear cell adhesion molecules expression was investigated. The data obtained showed that in vivo HQ exposure did not modify the expression of the adhesion molecules l-selectin, β2-integrin, β3-integrin and PECAM-1 in circulating mononuclear cells under either non-stimulated ( Fig. 2A) or LPS-stimulated conditions ( Fig. 2B). Screening of the chemotactic chemical mediators in BALF was performed and the results obtained showed that the levels of MCP-1 in the BALF of HQ-exposed animals were reduced in comparison to those of vehicle-exposed mice (Fig. 3).