Complement Receptor Mac-1 Is an Adaptor for NB1 (CD177)-mediated PR3-ANCA Neutrophil Activation

Abstract

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.

Anti–Human Neutrophil Antigen-3a Induced Transfusion-Related Acute Lung Injury in Mice by Direct Disturbance of Lung Endothelial Cells

“…To confirm these findings, neutrophils ROS production were re-evaluated by the use of the ferricytochrome-c reduction-based method. 24 As shown in Figure 4B, similar results were obtained. Again, the treatment of neutrophils with anti-HNA-3a IgG induced no significant ROS production, neither in quiescent nor in formyl-methionyl-leucyl phenylalanine post-treated neutrophils.…”

Extracellular protein disulfide isomerase regulates ligand-binding activity of αMβ2 integrin and neutrophil recruitment during vascular inflammation

“…24,25 Although most proteins were detected in nonraft fractions, we found that the majority of PDI is localized in lipid rafts irrespective of fMLF stimulation ( Figure 5E, supplemental Figure 3A-B). Using lipid raft (flotilin-1) and nonraft (Cdc42) markers, we confirmed that aMb2 integrin translocates to lipid rafts upon fMLF stimulation.…”

Endothelium-Neutrophil Interactions in ANCA-Associated Diseases

“…135 to specifically trigger degranulation and extracellular release of superoxide from the CD177 + neutrophil subset by a mechanism involving b2-integrins. 59,60 The requirement of cytochalasin b in these observations implies a reorganization of the actin cytoskeleton similar to that induced by neutrophil adhesion and spreading. It is thus tempting to speculate that anti-PR3 ANCA preferentially promotes the release of oxidants and enzymes by mPR3 + /CD177 + neutrophils adherent through b2-integrins to endothelial cells.…”

“…130 The same signals induce the association of b2-integrins, NADPH-oxidase components, and FcgRII with the cytoskeleton, the latter being increased in the presence of anti-PR3 or anti-MPO antibodies. 16,131 Upon TNF-a priming, PR3 forms a complex with NB1 and with CD11b/CD18, all three proteins being localized in cholesterol-rich domains (lipid rafts), 60,132 together with signaling molecules, cross-linked FcgRII, 133,134 and NADPH-oxidase membrane components. 135 to specifically trigger degranulation and extracellular release of superoxide from the CD177 + neutrophil subset by a mechanism involving b2-integrins.…”