TNFα release from peripheral blood leukocytes depends on a CRM1-mediated nuclear export

Department of Biology, St. John's University, NY 11439, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 01/2007; 351(2):354-60. DOI: 10.1016/j.bbrc.2006.10.045
Source: PubMed


Tumor necrosis factor-alpha (TNFalpha) is a potent pro-inflammatory cytokine that plays a major role in the pathogenesis of acute and chronic inflammatory disorders such as septic shock and arthritis, respectively. Leukocytes stimulated with inflammatory signals such as lipopolysaccharide (LPS) are the predominant producers of TNFalpha, and thus control of TNFalpha release from stimulated leukocytes represents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein export, inhibits TNFalpha release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immunofluorescence confocal microscopy and immunoblotting analysis indicate that TNFalpha is localized in the nucleus of human neutrophils and mononuclear cells. This study demonstrates that the cellular release of TNFalpha from stimulated leukocytes is mediated by the CRM1-dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFalpha could thus provide a novel therapeutic approach for disorders involving excessive TNFalpha release.

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    • "LPS stimulation results in substantial production of TNF, a potent pro-inflammatory cytokine with cytotoxic and pyrogenic effects. Neutrophils stimulated by LPS have the ability to release TNF, potentially through granule stores (118, 120). However, the mechanisms used to secrete cytokines and chemokines has not been thoroughly investigated in neutrophils although it is evident that SNAREs and other trafficking machinery are required for exocytosis of cytokine and chemokine-carrying granules (110, 111, 121, 122). "
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    ABSTRACT: Controlled nucleocytoplasmic localization regulates activity of NFκB as well as other transcription factors. Analysis of the nucleocytoplasmic protein shuttling has been greatly facilitated by the use of leptomycin B (LMB), an inhibitor of CRM1-dependent nuclear export. The authors have previously shown that LMB inhibits NFκB activity in human neutrophils by increasing the nuclear accumulation of NFκB inhibitor, IκBα. In this chapter, the authors describe a protocol that uses LMB to study the nucleocytoplasmic shuttling of IκBα in human macrophage-like U937 cells, thus inhibiting NFκB activity. This protocol should be readily adaptable to analyze the nucleocytoplasmic shuttling of other proteins in human leukocytes. Key WordsIκBα; leptomycin B–leukocytes–nuclear accumulation–nuclear export–NFκB–transcription factors–Western blotting
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