Proinflammatory genes expression in granulocytes activated by native proteinase-binding fragments of anti-proteinase 3 IgG.
The classical pathway of neutrophils activation due to cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA) involves specific antigen binding to proteinase-3 and activation of the immunoglobulin G receptors by the constant fragment of the antibody. A requirement for this double signaling was suggested also because proteinase-3 is presented within a complex of NB-1 glycoprotein lacking transmembrane domain. An integrin Mac-1 receptor was postulated to cooperate in neutrophil stimulation by anti-proteinase 3 (anti-PR3). A characteristic profile of transcriptional activation of neutrophils by c-ANCA was described by us previously. We ascertained mRNA expression of neutrophils following stimulation with antigen-binding fragments of native anti-PR3 IgG. Expression of targeted transcripts was compared with our previous results, in which intact anti-PR3 IgG was used. Human neutrophils were isolated from healthy volunteers negative for ANCAs. Antigen-binding fragments of human anti-PR3 were prepared from sera of patients with granulomatosis with polyangiitis. We analyzed reactive oxygen species production and abundance of mRNA of 151 genes by quantitative real time-PCR in neutrophils stimulated with anti-PR3 IgG F(ab)(2). We observed a consistent upregulation of 17 genes (CYSLTR1, HPGD, IL1R1, IL1RL1, MAPK1, MAPK8, NR3C1, PLA2G7, PTGDR, CD302, DNAJB1, F2R, F2RL1, IER3, RAC1, RPL41, PTGER3), whereas other 9 genes were up-regulated only in some donors. No reactive oxygen species production was observed in neutrophils stimulated with anti-PR3 F(ab)(2). Stimulation of neutrophils with F(ab)(2) of anti-PR3 autoantibodies activated cells to a lesser extent than intact IgG. However, several cellular pathways were up-regulated, involving calcium and phosphatidylinositol 3-kinase AKT, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK) signaling. Interestingly, binding of F(ab)(2) to the PR-3 present on the surface of neutrophil is sufficient for lipid mediators and G-protein pathways activation. Specific F(ab)(2) antibodies against PR-3 seems not a good candidate for decoy therapy of granulomatosis with polyangiitis.
Division of Molecular Biology and Clinical Genetics, Department of Medicine, Jagiellonian University Medical College, Cracow, Poland.
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M
Surmiak
Division of Molecular Biology and Clinical Genetics, Department of Medicine, Jagiellonian University Medical College, Cracow, Poland.
M
M
Kaczor
Division of Molecular Biology and Clinical Genetics, Department of Medicine, Jagiellonian University Medical College, Cracow, Poland. nfsanak@cyf-kr.edu.pl.
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M
Sanak
D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology*; D001323:Autoantibodies / Q000494:pharmacology; D015870:Gene Expression / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000494:pharmacology*; D066298:In Vitro Techniques; D007249:Inflammation / Q000235:genetics*; D008262:Macrophage Activation / Q000187:drug effects; D053146:Myeloblastin / Q000276:immunology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D000888:Antibodies, Anti-Idiotypic; D001323:Autoantibodies; D007128:Immunoglobulin Fragments; D017382:Reactive Oxygen Species; C048844:anti-IgG; D053146:Myeloblastin