Effect of intratracheal instillation of ultrafine carbon black on proinflammatory cytokine and chemokine release and mRNA expression in lung and lymph nodes of mice.

Our understanding of how ultrafine particles, which are constituents of particulate matter, affect immunological response is poor. To investigate the size-specific effect of ultrafine particles on pulmonary immune responses, translocation to lymph nodes, and chemokine mRNA expressions in lung and lymph nodes, we performed three experiments in 8-week-old male BALB/c mice. In experiment 1, we instilled 25 microg, 125 microg, or 625 microg of 14 nm carbon black (CB) particles intratracheally, once weekly for 4 weeks, and in experiment 2, we instilled 95 nm CB. For detection of total and differential cell counts and cytokine and chemokine protein release, we collected bronchoalveolar lavage (BAL) fluid 24 h after the last instillation of CB. Experiments 1 and 2 showed that 125 microg was the suitable dose for experiment 3, which we then performed on the same schedule and 4 h after the last instillation, we harvested the lung and mediastinal lymph node to detect chemokine mRNA expression by real-time RT-PCR. The total cell count as well as the differential cell counts such as macrophages, lymphocytes, and neutrophils in BAL fluid increased significantly in mice exposed to 14 nm CB in a dose-dependent manner. Release of cytokines such as interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha increased significantly in BAL fluid in mice instilled with 14-nm CB. Macrophage inflammatory protein 1 alpha/CCL-3 protein and mRNA expression were increased significantly in the lungs and lymph nodes of mice given 14 nm CB. Histologically, deposition of CB was observed greater in the mediastinal lymph nodes of mice given 14 nm than in 95 nm CB. These findings indicate that repeated intratracheal instillation of ultrafine carbon black in mice leads to pulmonary inflammation, their translocation to mediastinal lymph nodes and increased chemokine mRNA expression in lung and lymph nodes size-specifically.