A PM-mediated murine model of pulmonary inflammation has been well established [12] . We investigated the role of ROS and calpain in PM-induced pulmonary inflammation by examining protein leakage, white blood cell infiltration (inflammatory leukocytes), and the release of proinflammatory cytokines into BAL fluids ( Figure 5 ). Pre-administration of NAC (150 mg/kg) or calpeptin (1 mg/kg) 1 hr before PM challenge (10 mg/kg, 24 hr) significantly attenuated PM-induced protein leakage into BAL fluids (~50% reduction, Figure 5A ). PM challenge (10 mg/kg, 24 hr) resulted in an increase in inflammatory leukocytes, e.g. neutrophils and eosinophils [12, 15] , in BAL fluids ( Figure 5B ). Pre-administration of NAC (150 mg/kg) or calpeptin (1 mg/kg) attenuated PMinduced inflammatory leukocyte infiltration. Furthermore, NAC (150 mg/kg) or calpeptin (1 mg/kg) attenuated the release of PM-induced proinflammatory cytokines IL-6 and THF-α into BAL (~50% inhibition, Figure 5C-D) . These results suggest that ROS scavenging by NAC or Figure 2 Activated calpain is required for PM-mediated ZO-1 degradation and EC barrier disruption. (A) Human lung microvascular ECs grown in 60-mm dishes to approximately 95% confluence were treated with ALLN (30 μM), calpeptin (CALP, 10 μM), NAC (5 mM), or BAPTA-AM (50 μM) for 1 hr, and then challenged with PM (100 μg/ml) for 1 hr. Cell lysates were subjected to calpain activity assay by using the calpain activity kit (Calbiochem). Calpain activity was normalized to protein concentration and expressed as fold changed compared to control. *p < 0.05 compared to control. **p < 0.05 compared to PM only group. (B) ECs grown on ECIS gold electrodes were treated with ALLN (30 μM), calpeptin (CALP, 10 μM) for 1 hr, and then challenged with PM (100 μg/ml). Changes in TER were measured with ECIS. *p < 0.05 compared to PM only group. (C) ECs grown on 6-well plates were treated with ALLN (30 μM), calpeptin (CALP, 10 μM), or BAPTA-AM (50 μM) for 1 hr, and challenged with PM (100 μg/ml) for 1-6 hr. Cell lysates were analyzed by Western blotting with antibody to ZO-1. Changes in levels of ZO-1 are expressed as fold changes and normalized to β-actin. Shown are representative blots from three independent experiments. *p < 0.05 compared to PM-1 hr group. **p < 0.05 compared to PM-6 hr group. calpain activity inhibition by calpeptin leads to multiple protective effects including enhancement of lung endothelial barrier function, reduction of inflammatory cell infiltration, and prevention of proinflammatory cytokine release in the lungs of PM-challenged mice. We next examined tight junction ZO-1 levels in the PM-exposed lung. PM challenge (10 mg/kg, 24 hr) induced a significant reduction of ZO-1 protein levels in the murine lung, while pre-administration of NAC (150 mg/kg) or calpeptin (1 mg/kg) 1 hr before PM challenge attenuated PMinduced ZO-1 loss from lung tissues. Taken together, these data suggest a crucial role for the ROS-calpain-ZO-1 signaling pathway in the regulation of EC barrier disregulation in PM-mediated pulmonary inflammation.