Delivery of biologically active peptides into human polymorphonuclear neutrophils (PMNs) has implications for studying cellular functions and may be therapeutically relevant. The transcription factor nuclear factor-B (NF-B) regulates the expression of multiple genes controlling inflammation, proliferation, and cell survival. PMNs play a crucial role in first-line defense. Targeting NF-B in these cells may promote apoptosis and therefore facilitate resolution of inflammation. We used an 11-amino acid sequence NEMO-binding domain (NBD) that selec- IntroductionThe introduction of biologically active peptides into human polymorphonuclear neutrophils (PMNs) may help in clarifying intracellular signal transduction and ultimately could have therapeutic implications. However, all peptide delivery methods available thus far are inefficient. Previously, peptide transduction domains (PTDs) were identified that shuttle even large proteins in excess of 100 000 Da into mammalian cells in vitro and in vivo. 1 These "Trojan-horse" peptides include the homeodomain of Antennapedia (a Drosophila transcription factor), a short amino acid sequence of HIV-1, and the herpes simplex virus 1 (HSV-1) structural protein VP22. [2][3][4] We explored the use of an 11-amino acid sequence (amino acids 47-57) from the HIV TAT protein (HIV-TAT) in PMNs to target nuclear factor-B (NF-B).NF-B is a transcription factor controlling gene expression during inflammation, immunity, cell proliferation, stress response, and apoptosis. 5-8 NF-B is activated by many agents including cytokines, viral infection, UV radiation, and free radicals. 9 In unstimulated cells, NF-B is sequestered in the cytoplasm by tightly bound inhibitors (IB␣, IB, IB⑀). The inhibitors are phosphorylated and rapidly degraded, allowing NF-B to translocate into the nucleus and activate target genes. IB␣ is phosphorylated on serine residues by the multicomponent IB kinase (IKK) containing 2 catalytic subunits (IKK␣ and IKK) and one regulatory subunit (IKK␥). [10][11][12][13] In contrast to other cell types, the role of NF-B in PMNs is incompletely characterized due to rapid NF-B degradation by proteolytic enzymes, difficulties with PMN transfection, and the lack of specific NF-B inhibitors. Previous studies with pharmacologic compounds, such as pyrrolidine dithiocarbamate (PDTC), SN50, and gliotoxin, have suggested that NF-B is involved in regulating PMN apoptosis. [14][15][16] However, the specificity of these agents has been questioned. [17][18][19] We used a highly specific small peptide to block the interaction of IKK␥ with the IB kinase complex (IKK). 20 We generated a linear 2-domain peptide containing the NEMO-binding domain (NBD) to specifically block NF-B and the TAT-PTD to shuttle NBD into PMNs. Materials and methods MaterialsGranulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 8 (IL-8) were obtained from R&D Systems (Wiesbaden-Nordenstedt, Germany). Lipopolysaccharide (LPS), tumor necrosis factor ␣ (TNF-␣), dexamethasone (DEX), Ficoll-Hypaque, and pr...
The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1 pos /PR3 high neutrophils, but not in the mNB1 neg /PR3 low subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/ CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1 pos /mPR3 high neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when 2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.
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