- Paper Report
- Open Access
Peptide inhibition of a cytokine response
- Cheryl Smythe1
© Current Science Ltd 2000
- Published: 29 June 2000
- Endotoxic shock
- nuclear import
Activation of genes encoding tumour necrosis factor (TNF)-a, interferon (IFN)-? or interleukin (IL)-1 in response to pro-inflammatory agonists involves the nuclear translocation of SRTFs such as nuclear factor kappa B (NF?B) or nuclear factor of activated T cells (NFAT), and their subsequent interaction with the promoter region of the specific gene. Disruption of the SRTF signalling pathway would suppress the expression of TNF-a and IFN-?, cytokines that are known to play a key role in the systemic inflammatory response syndrome induced by LPS. A linear peptide (SN50) containing a membrane translocation motif and a nuclear localisation sequence (NLS), derived from the p50-NF?B1 subunit of the transcription factor NF?B, was therefore designed and shown to inhibit the nuclear import of NF?B in human monocytic and murine endothelial cells stimulated with LPS or TNF-a. SN50 also inhibited the expression of downstream SRTF-regulated genes such as cycloxygenase and IL-2 in vitro and was shown to interact with a cytoplasmic NLS receptor comprised of the Rch 1/importin (karyopherin)-? heterodimer. To inhibit SRTF mediated inflammatory responses in vivo using a cyclic peptide.
Fluorescence confocal laser scanning microscopy demonstrated intracellular accumulation of the FITC-cSN50 peptide in RAW 264.7 macrophages. The cyclic peptide was 3-10 times more effective than its linear analogue at inhibiting nuclear import of NF?B in LPS-stimulated cells. Nuclear import of activating protein (AP)-1 and NFAT in stimulated Jurkat T cells was also inhibited by the cSN50 peptide. FACS analysis of blood and spleen cells from mice intraperitoneally injected with FITC-cSN50 demonstrated the rapid delivery of the cyclic peptide to these cells. Injection of LPS into animals has previously been shown to lead to an early rise in pro-inflammatory mediators of endotoxic shock such as TNF-α, IFN-? and IL-1. In order to test whether cSN50 would inhibit cytokine gene expression in vivo, peptide-treated and saline-treated control mice were injected with LPS and plasma levels of cytokines were measured. Increases in both TNF-a and IFN-? following LPS treatment were significantly suppressed by the cSN50 peptide. Injection of cSN50 peptide also significantly increased the survival rate in LPS treated mice. LPS control mice and those injected with a mutated control peptide died within 72 h of injection, whereas mice treated with 1.5 mg of the cSN50 peptide, in seven injections taking place from 30 min prior to LPS administration to 12 h after, demonstrated a 90% survival rate over 72 h. Injection of the peptide 30 min after LPS exposure reduced the survival rate to 60%.
A cell permeable cyclic peptide (cSN50) was generated from the linear SN50 peptide by inserting two cysteine residues to form an intrachain disulphide bond. Peptides were either detected indirectly, using a fluorescently labelled antibody against the epitope tag LMP, or they were first fluorescein isothyanate (FITC) labelled and then detected directly using confocal laser scanning microscopy. In vivo detection of FITC-cSN50 in blood and spleen cells was by fluorescence-activated cell sorting (FACS). Nuclear import of NF?B in RAW 264.7 cells was measured by electrophoretic mobility gel shift assay using a radiolabelled ?B probe. Levels of TNF-a and IFN-? were measured by ELISA. LPS injected female C57BL/6 mice were used to model LPS-induced lethal shock. Histological analysis was carried out by haematoxylin and eosin staining of formalin-fixed, paraffin-embedded sections of the liver, spleen, kidneys, and lungs.