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16 TUMOR NECROSIS FACTOR a EXPRESSION IS REGULATED BY DIFFERENTIAL MKP-1 OXIDATION IN BLOOD MONOCYTES COMPARED TO ALVEOLAR MACROPHAGES.
  1. L. A. Tephly,
  2. A. B. Carter
  1. University of Iowa Carver College of Medicine and Iowa City Veterans Administration Medical Center, Iowa City, IA

Abstract

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.

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