Journal: Molecular Biology of the Cell

Article Title: Ligand-specific binding forces of LFA-1 and Mac-1 in neutrophil adhesion and crawling

doi: 10.1091/mbc.E16-12-0827

Figure Lengend Snippet: AFM tests for β2 integrin-ligand bonds. (A) Schematic of AFM setup. A PZT was used to drive the movement of an AFM cantilever. Adhesion events and forced bond rupture signals were collected from a QPD that measures the deflection of a laser beam reflected on the cantilever. (B) AFM functionalization. Recombinant human LFA-1s or Mac-1s were adsorbed onto the AFM tip and treated with TS1/18 mAbs or Mn 2+ to obtain low- or high-affinity conformation integrins, respectively. Soluble ligand (ICAM-1, ICAM-2, RAGE, JAM-A, or JAM-C)–IgG Fc chimeras were coated via anti-IgG Fc secondary antibodies precoated on a Petri dish. (C) Typical force–displacement curves. An LFA-1– or Mac-1–captured AFM tip was driven to approach to (from left to right, blue lines), contact, and retract from (from right to left, red lines) a ligand-coated Petri dish. Adhesion was visualized from cantilever deflection and rupture force ( f r ) was measured from the force–displacement curve (middle and lower trajectories). k s is the system spring constant derived from the slope of the adhesive event. (D, E) Binding specificity. The LFA-1– or Mac-1–captured tip was pretreated with isotype control (closed bars) or LFA-1/Mac-1 blocking (open bars) mAbs. Adhesion probabilities were measured between the tip and various ligand-coated Petri dishes with TS1/18 (D) or Mn 2+ (E). All measurements were acquired at a cantilever approach and retraction velocity of 1 μm/s, a contact duration of 50 ms, and a compression force of 200 pN. An anti-IgG Fc secondary antibody–coated substrate was used as a negative control. Data are presented as the mean ± SEM of three or four tips in each case. Significant differences are indicated by *, p < 0.05; **, p < 0.01; ***, p < 0.001 between each ligand and negative control, by # , p < 0.05; ## , p < 0.01; ### , p < 0.001 between each blocking group and isotype control, or by $ , p < 0.05; $$ , p < 0.01; $$$ , p < 0.001 among various ligands. (F) Typical rupture force distributions for the interactions between LFA-1 and ICAM-1 with TS1/18 at the indicated loading rates. Total 251–394 single-bond rupture force data at each loading rate were collected and analyzed using a force bin of 50 pN.

Article Snippet: For example, shear stress regulates leukocyte adhesion , induces activation and cleavage of β2 integrins during PMN migration ( Makino et al. , 2007 ), triggers guanine nucleotide exchange factor (GEF)-H1–dependent spreading and crawling of PMNs ( Fine et al. , 2016 ), and promotes lymphocyte transmigration ( Cinamon et al. , 2001 ).

Techniques: Recombinant, Derivative Assay, Adhesive, Binding Assay, Control, Blocking Assay, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Entamoeba histolytica Induce Signaling via Raf/MEK/ERK for Neutrophil Extracellular Trap (NET) Formation

doi: 10.3389/fcimb.2018.00226

Figure Lengend Snippet: Entamoeba histolytica- induced NET formation is independent on Syk, TAK1, β2 integrins, PI3K, and p38 MAPK. Human neutrophils were left untreated (—), stimulated with 20 nM phorbol 12-myristate 13-acetate (PMA), or with E. histolytica trophozoites. PMN were previously treated with solvent alone (—) or 1 μM iSyk, a Syk inhibitor, or 10 μg/ml of the blocking monoclonal antibody anti-β2 integrins (IB4), or 10 nM LLZ 1640-2 (LLZ), a TAK1 inhibitor, or 50 nM Wortmannin (Wort), a PI3K inhibitor, or 200 nM SB203580 (SB), a p38 MAPK inhibitor. The relative amount of NETs was estimated from SYTOX® Green fluorescence in relative fluorescent units (RFU) at 4 h after stimulation. Data are mean ± SEM of three experiments. Asterisk denotes a condition that was statistically different from control ( p < 0.05).

Article Snippet: The involvement of β2 integrins in NET formation might be more related to the adhesion requirement of neutrophils to form NETs (Brinkmann et al., ) than to a signaling capacity of the integrin.

Techniques: Solvent, Blocking Assay, Fluorescence, Control