Distinct functionality of neutrophils in multiple sclerosis and neuromyelitis optica

Background: In contrast to multiple sclerosis (MS), lesions in neuromyelitis optica (NMO) frequently contain neutrophils. However, the phenotypic profile of neutrophils in these two distinct pathologies remains unknown. Objective: Our aim is to better understand the potential contribution of neutrophils to NMO and MS pathology. Methods: We performed the first functional analysis of blood neutrophils in NMO and MS, including evaluation of neutrophil immune response (fMLP receptor, TLR2), chemotaxis and migration (CXCR1, CD62L, CD43), regulation of complement (CD46, CD55, CD59), respiratory burst, phagocytosis and degranulation. Results: Compared with healthy controls (HC), neutrophils in NMO and MS show an activated phenotype characterized by an increased surface expression of TLR2 and fMLP receptor. However, contrary to MS neutrophils, NMO neutrophils show reduced adhesion and migratory capacity as well as decreased reduced production of reactive oxygen species (respiratory burst) and degranulation. Conclusion: Although NMO and MS neutrophils display an activated phenotype in comparison with HC, NMO neutrophils show a compromised functionality when compared with MS patients. These results suggest a distinct functional profile of neutrophils in MS and NMO.

p21-Activated Kinases Regulate Directional Migration and Cytoskeletal Organization in Human Neutrophils

Abstract 834 Neutrophil chemotaxis is controlled by coordinated processes of directional sensing, polarization and motility. This study was designed to characterize the role of p21-activated kinases (PAKs) during the chemotaxis of human primary neutrophils. PAKs are known as effectors of the Rho GTPases Rac and Cdc42. It has been shown that PAK1 and PAK2 are strongly activated downstream of the f-Met-Leu-Phe (fMLP) receptor via Rac (Huang et al., MCB 1998). PAK1 is known to localize in lamellipodia at the leading edge of human neutrophils (Dharmawardhane et al., JLB 1999) and mediate persistent directional migration via Cdc42 in a neutrophil-like cell line (Li et al., Cell 2003). However, little is known about the specific role of PAK isoforms in spatial/temporal regulation of cytoskeletal dynamics in human neutrophils. Our data show that human neutrophils express PAK1, 2 and 4. Under an fMLP gradient, human neutrophils developed morphological polarity with a distinct leading edge and rear, and migrated up the fMLP gradient at the speed of 7.5 ± 0.56 μm/min. Inhibition of Rac or PI3K impaired directionality but did not significantly affect migration speed of chemotaxing neutrophils (6.3 ± 0.56 μm/min or 6.2 ± 0.85 μm/min, respectively). In contrast, neutrophils treated with the PAK inhibitor, PF3758309 (PF), displayed random migration, less polarization and reduced motility (3.1 ± 0.21 μm/min). These results suggest that PAK regulates neutrophil chemotaxis independently of the Rac-PI3K axis. The presence of PF did not abrogate intracellular Ca2+mobilization in fMLP-driven chemotactic condition. Instead, the decreased migratory ability by PAK inhibition was associated with multiple Ca2+ spikes. Immunofluorescence imaging shows that PAK2 but not PAK1, was phosphorylated and translocated from cytosol to actin-rich leading edge in the proximity to GTP-bound Rac within 3 min of fMLP stimulation. Notably, PF treatment resulted in partial neutrophil spreading and actin/myosin II translocation in the absence of extracellular stimuli, suggesting that basal level of PAK phosphorylation may be required for cytoskeletal integrity of resting neutrophils. Neutrophils pretreated with PF displayed less activation and translocation of PAK2 and Rac. In summary, our data demonstrate for the first time the distinct roles of PAK isoforms in human neutrophil morphological polarity and directional migration and suggest that PAK2 is activated downstream of fMLP receptor through Rho-family small GTPases. Disclosures: No relevant conflicts of interest to declare.

Knockdown of Peroxiredoxin VI in K562 Cells Stably Transfected with Oxidase Components Reduces NADPH Oxidase Activity in Response to PMA and fMLP.

Introduction: An intact respiratory burst is critical to the microbicidal activity of neutrophils. The respiratory burst is initiated by assembly and activation of the NADPH oxidase complex. We recently identified a 29 kDa neutrophil protein which binds p67phox, is identified as peroxiredoxin VI by sequence and activity, translocates to the plasma membrane of neutrophils during activation and enhances superoxide anion (O2−) production in a cell-free assay exhibiting saturable kinetics. The activity of p29 peroxiredoxin (Prx) requires cysteine residues of the protein. The present studies demonstrate the requirement of p29 Prx for optimal oxidase activity in intact cells. Methods: K562 cells stably transfected with p47phox, p60phox, gp91phox and the low affinity fMLP receptor and expressing p22phox, p40phox and Rac were grown in tissue culture at 37°C in 5% CO2 with RPMI and 10% fetal calf serum. Three siRNA molecules (Q, A1, A2) as well as non-interfering RNA (Nsi) used as a control were obtained commercially. Transfection of K562 cells was completed with Nucleofector technology (Amaxa Biosystems, Gaithersburg, MD) using Solution L and protocol T020. Transfection was >90% by expression of a GFP plasmid; viability after the procedure was >90%. O2− was measured after stimulation with PMA (200 ng/ml) as SOD inhibitable cytochrome c reduction or in response to fMLP (1 μM) by the addition of Diogenes (National Diagnostics) and detection of chemiluminescence in a luminometer (FB12, Berthod Detection Systems, Pforzheim, Germany) as relative light units. Cell lysates were made with the addition of 10% Triton X-100 and stored at −70°C. Proteins (5–10μg of cell lysate) were separated on a 10% SDS-PAGE and blotted onto nitrocellulose. Detection of specific proteins was completed using polyclonal antibodies to p29 Prx and to other phox proteins and actin and secondary antibodies with a chemiluminescent technique. Results: Transfection of the K561 cells with Q, A1, and A2 resulted in a knockdown of p29 Prx (25–40%) compared to Nsi. Optimal knockdown occurred in the presence of 2μM siRNA, at 48 hours after transfection. No changes in actin, p67phox, p47phox, p40phox, p22phox or gp91phox were documented with nucleofection, siRNAs or Nsi. In 5 separate experiments, knockdown of p29 Prx resulted in a decrease (p<0.05) in O2− production (PMA) or O2− associated chemiluminescence (fMLP), see Figure (columns and bars represent mean ± SEM). Conclusion: O2− production in response to PMA or fMLP was reduced in K562 cells with a knockdown of p29. These results extend previous studies and demonstrate the requirement of p29 Prx, peroxiredoxin VI, for optimal NADPH oxidase activity within intact cells. Figure Figure

Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor

Actin assembly at the leading edge of migrating cells depends on the availability of high-affinity free barbed ends (FBE) that drive actin filament elongation and subsequent membrane protrusion. We investigated the specific mechanisms through which the Rac1 and Rac2 small guanosine triphosphatases (GTPases) generate free barbed ends in neutrophils. Using neutrophils lacking either Rac1 or Rac2 and a neutrophil permeabilization model that maintains receptor signaling to the actin cytoskeleton, we assessed the mechanisms through which these two small GTPases mediate FBE generation downstream of the formyl-methionyl-leucyl-phenylalanine receptor. We demonstrate here that uncapping of existing barbed ends is mediated through Rac1, whereas cofilin- and ARP2/3-mediated FBE generation are regulated through Rac2. This unique combination of experimental tools has allowed us to identify the relative roles of uncapping (15%), cofilin severing (10%), and ARP2/3 de novo nucleation (75%) in FBE generation and the respective roles played by Rac1 and Rac2 in mediating actin dynamics.

N-Formyl peptide receptor subtypes in human neutrophils activate l-plastin phosphorylation through different signal transduction intermediates

We investigated the coupling of the fMLP (N-formyl-l-methionyl-l-leucyl-l-phenylalanine; ‘chemotactic peptide’) receptor with phosphorylation of the actin-binding protein l-plastin in neutrophils. Using two-dimensional IEF (isoelectric focusing)/PAGE and MALDI–TOF (matrix-assisted laser desorption ionization–time-of-flight)-MS, l-plastin was identified as a major phosphoprotein in fMLP-stimulated neutrophils whose phosphorylation was dependent on phosphoinositide 3-kinase, PLD (phospholipase D) and PKC (protein kinase C) activity. Two fMLP receptor subtypes were identified in neutrophils, characterized by a distinct sensitivity to fMLP and antagonistic peptides. Both receptor subtypes induced the phosphorylation of l-plastin. l-plastin phosphorylation induced by low-affinity fMLP receptors involves an action of phosphoinositide 3-kinase, PLD and PKC isotypes. In contrast, none of these intermediates are utilized by high-affinity fMLP receptors in the phosphorylation of l-plastin. However, the PKC inhibitor Ro-31-8220 inhibits l-plastin phosphorylation induced by the high-affinity fMLP receptor. Thus, an as yet unknown Ro-31-8220-sensitive kinase regulates l-plastin phosphorylation in response to the high-affinity fMLP receptor. The results suggest a model in which receptor subtypes induce a similar endpoint event through different signal-transduction intermediates. This may be relevant in the context of cell migration in which one receptor subpopulation may become desensitized in a concentration gradient of chemoattractant.