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author:("neel, Indira")
1.  Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release 
In response to inflammation, neutrophils deiminate histones and externalize chromatin. Neutrophil extracellular traps (NETs) are an innate immune defense mechanism, yet NETs also may aggravate chronic inflammatory and autoimmune disorders. Activation of peptidylarginine deiminase 4 (PAD4) is associated with NET release (NETosis) but the precise mechanisms of PAD4 regulation are unknown. We observed that, in human neutrophils, calcium ionophore induced histone deimination, whereas phorbol myristate acetate (PMA), an activator of protein kinase C (PKC), suppressed ionophore-induced deimination. Conversely, low doses of chelerythrine and sanguinarine, two inhibitors of PKC, reversed PMA inhibition and enhanced ionophore-stimulated deimination. In addition, a peptide inhibitor of PKCα superinduced ionophore activation of PAD4, thus identifying PKCα as the PMA-induced inhibitor of PAD4. At higher doses, chelerythrine, sanguinarine, and structurally unrelated PKC inhibitors blocked histone deimination, suggesting that a different PKC isoform activates histone deimination. We identify PKCζ as activator of PAD4 because a specific peptide inhibitor of this PKC isoform suppressed histone deimination. Confocal microscopy confirmed that, in the presence of PMA, NETosis proceeds without detectable histone deimination, and that ionophore cooperates with PMA to induce more extensive NET release. Broad inhibition of PKC by chelerythrine or specific inhibition of PKCζ suppressed NETosis. Our observations thus reveal an intricate antagonism between PKC isoforms in the regulation of histone deimination, identify a dominant role for PKCα in the repression of histone deimination, and assign essential functions to PKCζ in the activation of PAD4 and the execution of NETosis. The precise balance between opposing PKC isoforms in the regulation of NETosis affirms the idea that NET release underlies specific and vitally important evolutionary selection pressures.
PMCID: PMC3576869  PMID: 23430963
NETosis; PAD4; protein kinase C; deimination; inflammation
2.  Knotting the NETs: Analyzing histone modifications in neutrophil extracellular traps 
Neutrophil extracellular chromatin traps (NETs) are a recently described mechanism of innate immune responses to bacteria and fungi. Evidence indicates that NETs are induced by inflammation, that they contribute to diverse disease pathologies, and that they associate with bactericidal substances. Genomic DNA is released in NETs, leading to a cell death that has been labeled NETosis. Although NETosis clearly differs from apoptosis, the classical form of cell death, recent experiments indicate a connection between NETosis and autophagy. The regulated deployment of NETs may require covalent modification of histones, the basic DNA-binding proteins that organize chromatin in the cell's nucleus and within NETs. Histone modification by peptidylarginine deiminase 4 (PAD4) is necessary for NET release. The functions of additional histone modifications, however, remain to be tested.
PMCID: PMC3446426  PMID: 22524286
3.  Divergent Members of a Single Autoreactive B Cell Clone Retain Specificity for Apoptotic Blebs 
Molecular immunology  2006;44(8):1914-1921.
Specificity for double-stranded DNA can arise due to somatic mutations within one of the branches of an autoreactive B cell clone. However, it is not known whether a different autospecificity predates anti-dsDNA and whether separate offshoots of an expanding B cell clone retain or evolve alternative specificities. We compared 3H9, an anti-dsDNA IgG, to 4H8 and 1A11, antibodies produced by hybridomas representing an alternative branch of the 3H9 B cell clone. All three IgG bound chromatin in ELISA and apoptotic cells in confocal microscopy, yet only 3H9 bound dsDNA, as measured by plasmon resonance. Moreover, we demonstrate that despite the unique specificity of 3H9 for dsDNA, all three clone members exhibited indistinguishable binding to chromatin. The binding to chromatin and apoptotic cells was unaffected by N-linked glycosylation in L chain CDR1, a modification that results from a replacement of serine 26 with asparagine in 4H8 and 1A11. These data provide the first evidence that specificity for nucleosome epitopes on apoptotic cells provides the initial positive stimulus for somatic variants that comprise a B cell clone, including those that subsequently acquire specificity for dsDNA. Conversely, selection of autoreactive B cells for binding to apoptotic cells leads to clonal expansion, antibody diversification, and the development of linked sets of anti-nuclear autoantibodies.
PMCID: PMC1812796  PMID: 17084454
Anti-DNA; Autoantibody; Autoimmunity; Apoptosis; B lymphocytes; Somatic Mutations; Systemic Lupus Erythematosus

Results 1-3 (3)