Monocytic cells are involved in the pathogenesis of immune and inflammatory disorders of the lung. A characteristic feature of alveolar macrophages obtained from asbestosis patients is the release of proinflammatory cytokines. We have shown previously that asbestos-induced p38 MAP kinase activity and TNF-a gene expression are mediated by H₂O₂. Since kinase activity is tightly regulated by phosphatases, we ask if MKP-1, which is a dual-specificity phosphatase that can be inactivated by H₂O₂-induced oxidation, played a role in regulating p38 activity in alveolar macrophages compared to blood monocytes. We hypothesized that MKP-1, in part, regulates p38 activity in alveolar macrophages due to inadequate H₂O₂ generation in response to asbestos. We found that MKP-1 was constitutively expressed in alveolar macrophages, and its level increased with asbestos stimulation. In contrast, blood monocytes had minimal MKP-1 protein expression. When protein translation was inhibited with cyclohexamide or MKP-1 was inhibited with triptolide or NaVO₄, p38 activity was recovered in alveolar macrophages. In addition, when MKP-1 was immunoprecipitated from whole-cell lysates, an activated p38a remained phosphorylated when exposed to alveolar macrophage lysates, unlike when MKP-1 was present. To determine if MKP-1 oxidation was different in these cells, lysates from blood monocytes and alveolar macrophages were subjected to immunoprecipitation for MKP-1, which was then labeled with the thiol reactive fluorescent dye F5M. We found that MKP-1 in blood monocytes was oxidized at a significantly higher level than in alveolar macrophages. Blood monocytes stimulated with asbestos released TNF-a, while alveolar macrophages did not. These data suggest that MKP-1, through increased expression and lack of oxidation, regulates p38 MAP kinase activity and TNF-a gene expression in alveolar macrophages stimulated with crocidolite asbestos.

VA MERIT and ALA Career Investigator Award.