Characterization of the Regulation and Functional Consequences of p21ras Activation in Neutrophils by Antineutrophil Cytoplasm Antibodies

Abstract

Antineutrophil cytoplasm antibodies (ANCA) are implicated in the pathogenesis of systemic vasculitis. ANCA are directed against antigens expressed on the surface of cytokine-primed neutrophils. It was shown previously that whole IgG ANCA and its fraction antigen binding [F(ab) 2 ] fragment can activate the GTPase p21 ras . This study shows a functional involvement of this molecule in the ANCA activation of neutrophils by inhibiting the production of superoxide with farnesylthiosalicylic acid. Using the ras activation assay, farnesylthiosalicylic acid inhibits p21 ras binding to its substrate at comparable concentrations to those seen for superoxide inhibition. It is also shown that activation of p21 ras by ANCA is transient, peaking at 5 to 10 min and returning to baseline by 30 min. The use of ras isoform-specific antibodies in Western blots established, for the first time, that Harvey-ras is not present in human neutrophils, but both Kirsten-ras (K-ras) and Neuronal-ras are. Stimulation with ANCA is able to differentially activate K-ras without effects on neuronal-ras. The activation of p21 ras by ANCA and its F(ab) 2 is prevented by inhibition of both Src kinases and phosphatidylinositol-3-kinase, indicating a cooperative role for both molecules in the G protein pathway activated by ANCA F(ab) 2 upstream of p21 ras . It is concluded that ANCA selectively activates K-ras during induction of a respiratory burst via pathways involving multiple upstream kinases.

Anti-Neutrophil Cytoplasmic Antibody Pathogenesis in Small-Vessel Vasculitis

“…40 Indeed, transient enhanced RAS p21 protein activator activity is reported to precede a rise in superoxide production. 41 ANCA-mediated neutrophil activation disrupts apoptosis by delaying apoptotic neutrophil surface phosphatidylserine expression, which is necessary for macrophage-mediated cell removal. Because of their delayed clearance, neutrophils then undergo secondary necrosis with subsequent release of inflammatory mediators, amplifying the process.…”

“…In the article by Kain et al, 25 although neither epitope was homologous to MPO or PR3, the P 41-49 epitope was 100% homologous to amino acids 72 to 80 of the mature form of FimH, an adhesin located at the tip of type 1 fimbriae that is essential for the attachment of Gram-negative pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis to host epithelia. Reciprocal inhibition experiments showed that autoantibodies that recognized human LAMP-2, specifically its P [41][42][43][44][45][46][47][48][49] epitope, cross-reacted with FimH. Furthermore, rats immunized with recombinant FimH fusion protein developed antibodies to FimH that cross-reacted with human LAMP-2.…”

“…In addition, autoantibodies from these patients bound the region of human LAMP-2 that contained the cross-reactive P [41][42][43][44][45][46][47][48][49] epitope.…”

Anti-Neutrophil Cytoplasm-Associated Glomerulonephritis

“…p21ras, a molecule that is a branch point in multiple signaling cascades, controls a number of cellular processes in the neutrophil, including the respiratory burst. It was shown recently that both intact ANCA IgG and ANCA F(abЈ) 2 fragments activate only one of the two isoforms of p21ras present in the neutrophil, Kirsten-ras, during initiation of a neutrophil respiratory burst (41). This potentially could permit specific inhibition of the ANCA-activated signaling pathways without complete inhibition of all p21ras functions.…”

Vasculitis asociadas a anticuerpos anti-citoplasma de neutrófilos: avances en patogenia y tratamiento

“…Esta interacción induce la cascada respiratoria 23 . A nivel intracelular, los ANCA desencadenan la inducción de múltiples vías 24 : proteína G y GTPasa (a través de fragmento Fab) 25 y tirosina quinasas (a través del receptor Fc) 26 , que convergen en la GTPasa p21 RAS , molécula que participa en muchos procesos celulares del neutrófilo, incluyendo la activación respiratoria. Se ha demostrado cambios en la expresión genética que reflejan la actividad de la enfermedad en vivo 27 .…”