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.
Antineutrophil cytoplasmic autoantibodies (ANCA) are associated with necrotizing crescentic glomerulonephritis (NCGN) and systemic vasculitis. We examined the role of phosphoinositol 3 kinase-gamma isoform (PI3Kgamma) in ANCA-activated neutrophil functions. Further, we tested whether its inhibition protects a mouse model of ANCA NCGN from developing NCGN. We transplanted bone marrow from wild-type mice or PI3Kgamma-deficient mice into myeloperoxidase-deficient mice immunized with myeloperoxidase. Bone marrow from PI3Kgamma(-/-) mice protected against development of the disease. Similarly, bone marrow transplanted from wild-type mice followed by treatment with the specific PI3Kgamma inhibitor AS605240 also protected these mice against NCGN in this model. AS605240 significantly abrogated myeloperoxidase- or proteinase 3-ANCA-stimulated superoxide production in vitro. Furthermore, ANCA-induced degranulation and GM-CSF-stimulated migration in a transwell assay of isolated human neutrophils were also abrogated by the drug. We found that PI3Kgamma plays a pivotal role in ANCA-induced NCGN and suggest that its specific inhibition may provide a novel treatment target.
Abstract. Antineutrophil cytoplasmic antibodies (ANCA) activate human polymorphonuclear neutrophils (PMN) primed with tumor necrosis factor ␣ (TNF-␣) in vitro. Phosphatidylinositol 3-kinase (PI3-K) and the protein-serine/threonine kinase Akt have been implicated in the control of the phagocyte respiratory burst. The hypothesis that PI3-K controls the ANCA-induced respiratory burst was tested. TNF-␣-primed PMN were stimulated with a monoclonal antibody to myeloperoxidase (MPO) and with PR3-and MPO-ANCA, respectively. Akt activation was assessed with phospho-specific antibodies. Superoxide release was measured with ferricytochrome. ANCA antigen translocation was assessed by fluorescence-activated cell sorter. The effect of TNF-␣ and MPO-ANCA on Akt signaling was studied with immunoprecipitation and glutathione S-transferase pull-down assays. Western blotting revealed rapid transient Akt phosphorylation during TNF-␣ priming and a second phosphorylation after ANCA. PI3-K inhibition by LY294002 blocked both Akt phosphorylation and superoxide generation.
Background: How endothelial cells (ECs) could acquire exogenous neutrophil myeloperoxidase (MPO) is unknown. Results: ECs acquired enzymatically active MPO directly from neutrophils, via 2 integrin-mediated cell-cell contact independent of extracellular MPO release. Conclusion: Neutrophils directly transfer MPO to ECs by cell-cell contact. Significance: Direct delivery of MPO to ECs may contribute to the vascular pathology and dysfunction seen in atherosclerosis and vasculitides.
These findings demonstrate that HMG-CoA reductase inhibitors decrease respiratory burst activity of human PMN in response to ANCA. This effect was independent of mevalonate, but involved inhibition of ERK activation during TNF-alpha priming. Our data suggest that HMG-CoA reductase inhibitors may help limit inflammatory responses.
Microparticles from various cells are generated during inflammation. Platelet-derived microparticles (PMPs) harbor receptors that are not genuinely expressed by neutrophils. We tested whether or not functional glycoprotein IIb/IIIa (GPIIb/ IIIa) receptors can be acquired by neutrophils via PMPs and whether these receptors participate in pro-inflammatory signaling. Surface expression was analyzed by flow cytometry and confocal microscopy. NF-B activation was analyzed by Western blot experiments, electrophoretic mobility shift assays, and reverse transcription-PCR. Cell adhesion and spreading were estimated by myeloperoxidase assay and light microscopy. We found that PMPs transfer GPIIb/IIIa receptors to isolated and whole blood neutrophils via PMPs. We used specific antibodies in granulocyte macrophage colony-stimulating factor-treated neutrophils and observed that acquired GPIIb/IIIa receptors colocalized with 2-integrins and cooperated in NF-B activation. We show that Src and Syk non-receptor tyrosine kinases, as well as the actin cytoskeleton, control NF-B activation. In contrast to NF-B, acquisition of GPIIb/IIIa receptors was not necessary to induce adhesion to fibronectin or phosphatidylinositol 3-kinase/Akt signaling. When granulocyte macrophage colonystimulating factor-stimulated neutrophils were incubated on fibronectin, strong NF-B activation was observed, but only after loading with PMPs. Blocking either 2-integrins or GPIIb/ IIIa receptors abrogated this effect. Therapeutic GPIIb/IIIa inhibitors were similarly effective. The compounds also inhibited NF-B-dependent tumor necrosis factor-␣ mRNA up-regulation. The data implicate GPIIb/IIIa receptors as new therapeutic targets in neutrophil-induced inflammation.
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