TNFa release from peripheral blood leukocytes depends
on a CRM1-mediated nuclear export
Veronika Miskolcia,b, Chandra C. Ghosha, Janet Rollinsa, Carlos Romeroa, Hai-Yen Vua,
Staci Robinsona, Dennis Davidsonb, Ivana Vancurovaa,b,*
aDepartment of Biology, St. John’s University, NY 11439, USA
bDepartment of Pediatrics, Feinstein Institute for Medical Research at the North Shore-Long Island Jewish Health System,
New Hyde Park, NY 11040, USA
Received 4 October 2006
Available online 16 October 2006
Tumor necrosis factor-a (TNFa) 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 TNFa, and thus control of TNFa release from stimulated leukocytes rep-
resents a potential therapeutic target. Here, we report that leptomycin B (LMB), a specific inhibitor of CRM1-dependent nuclear protein
export, inhibits TNFa release from LPS-stimulated human peripheral blood neutrophils and mononuclear cells. In addition, immuno-
fluorescence confocal microscopy and immunoblotting analysis indicate that TNFa is localized in the nucleus of human neutrophils and
mononuclear cells. This study demonstrates that the cellular release of TNFa from stimulated leukocytes is mediated by the CRM1-
dependent nuclear export mechanism. Inhibition of CRM1-dependent cellular release of TNFa could thus provide a novel therapeutic
approach for disorders involving excessive TNFa release.
? 2006 Elsevier Inc. All rights reserved.
Keywords: TNFa; Leptomycin B; Nuclear export; Neutrophils; Mononuclear cells
Release of TNFa by activated neutrophils (polymorpho-
nuclear leukocytes, PMN) and mononuclear cells (PBMC)
represents a crucial step in the inflammatory response that
occurs in acute host defense against sepsis, or in the path-
ogenesis of chronic diseases with significant localized tissue
injury such as rheumatoid arthritis [1–4]. Human TNFa is
initially synthesized as a 233 amino acid, 26 kDa pro-pro-
tein containing a hydrophobic membrane region that is lat-
er cleaved to yield the mature, soluble, and biologically
active 17 kDa cytokine [2–5]. The regulation of TNFa syn-
thesis occurs at both transcriptional and post-transcrip-
tional levels [2–5]. At the transcriptional level, the TNFa
expression is regulated by the transcription factor NFjB
NFjB comprises a family of transcription factors that
serve as crucial regulators of genes involved in host
immune and inflammatory responses, proliferation, and
differentiation [6,7]. In addition to TNFa, NFjB regulates
transcription of other pro-inflammatory cytokines such as
interleukin-8 (IL-8), as well as transcription of anti-apopto-
tic proteins [6,7]. We have previously shown that human
neutrophils differ from other cell types by containing pre-
dominant amounts of the NFjB inhibitor, IjBa, in the
nucleus . In addition, we showed that leptomycin B
(LMB), a specific inhibitor of CRM1-mediated nuclear
protein export, further increases the nuclear accumulation
of IjBa in human neutrophils, thus inhibiting NFjB activ-
ity and inducing neutrophil apoptosis . In contrast to
PMN, in human PBMC, LMB does not affect the NFjB
activity and apoptosis, since in PBMC, IjBa is localized
exclusively in the cytoplasm and LMB does not induce its
0006-291X/$ - see front matter ? 2006 Elsevier Inc. All rights reserved.
*Corresponding author. Fax: +1 718 990 5958.
E-mail address: firstname.lastname@example.org (I. Vancurova).
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