Neutrophil String Formation: Hydrodynamic Thresholding and Cellular Deformation during Cell Collisions

Neutrophils unexpectedly display flow-enhanced adhesion (hydrodynamic thresholding) to L-selectin in rolling or aggregation assays. We report that the primary collision efficiency (ε) of flowing neutrophils with preadhered neutrophils on intercellular adhesion molecule-1 (ICAM-1) or fibrinogen also displayed a maximum of ε ~ 0.4–0.45 at a wall shear rate of 100 s-1, an example of thresholding. Primary collision lifetime with no detectable bonding decreased from 130 to 10 ms as wall shear rate increased from 30 to 300 s-1, whereas collision lifetimes with bonding decreased from 300 to 100 ms over this shear range using preadhered neutrophils on ICAM-1, with similar results for fibrinogen. Antibodies against L-selectin, but not against CD11a, CD11b, or CD18, reduced ε at 100 s-1 by \u3e85%. High resolution imaging detected large scale deformation of the flowing neutrophil during the collision at 100 s-1 with the apparent contact area increasing up to ~40 μm2. We observed the formation of long linear string assemblies of neutrophils downstream of neutrophils preadhered to ICAM-1, but not fibrinogen, with a maximum in string formation at 100 s-1. Secondary capture events to the ICAM-1 or fibrinogen coated surfaces after primary collisions were infrequent and short lived, typically lasting from 500 to 3500 ms. Between 5 and 20% of neutrophil interactions with ICAM-1 substrate converted to firm arrest (\u3e3500 ms) and greatly exceeded that observed for fibrinogen, thus defining the root cause of poor string formation on fibrinogen at all shear rates. Additionally, neutrophils mobilized calcium after incorporation into strings. Static adhesion also caused calcium mobilization, as did the subsequent onset of flow. To our knowledge, this is the first report of 1), hydrodynamic thresholding in neutrophil string formation; 2), string formation on ICAM-1 but not on fibrinogen; 3), large cellular deformation due to collisions at a venous shear rate; and 4), mechanosensing through neutrophil β2-integrin/adhesion. The increased contact area during deformation was likely responsible for the hydrodynamic threshold observed in the primary collision efficiency since no increase in primary collision lifetime was detected as shear forces were increased (for either surface coating)

Calcium Flux in Neutrophils Synchronizes β2 Integrin Adhesive and Signaling Events that Guide Inflammatory Recruitment

Intracellular calcium flux is an early step in the signaling cascade that bridges ligation of selectin and chemokine receptors to activation of adhesive and motile functions during recruitment on inflamed endothelium. Calcium flux was imaged in real time and provided a means of correlating signaling events in neutrophils rolling on E-selectin and stimulated by chemokine in a microfluidic chamber. Integrin dependent neutrophil arrest was triggered by E-selectin tethering and ligation of IL-8 seconds before a rapid rise in intracellular calcium, which was followed by the onset of pseudopod formation. Calcium flux on rolling neutrophils increased in a shear dependent manner, and served to link integrin adhesion and signaling of cytoskeletally driven cell polarization. Abolishing calcium influx through membrane expressed store operated calcium channels inhibited activation of high affinity β2 integrin and subsequent cell arrest. We conclude that calcium influx at the plasma membrane integrates chemotactic and adhesive signals, and functions to synchronize signaling of neutrophil arrest and migration in a shear stress dependent manner

Cross-protection experiments with parasitoids in the genus Microplitis (Hymenoptera: Braconidae) suggest a high level of specificity in their associated bracoviruses

The immunological and developmental effects of bracoviruses (BVs) from three parasitoids in the genus Microplitis (Braconidae: Microgastrinae) were compared in the hosts Pseudoplusia includens and Heliothis virescens (Lepidoptera: Noctuidae). Southern blotting experiments indicated that viral DNAs from Microplitis demolitor bracovirus (MdBV) cross-hybridized with viral DNAs from Microplitis croceipes bracovirus (McBV) and Microplitis mediator bracovirus (MmBV) under conditions of high stringency. Injection of calyx fluid plus venom from each parasitoid species dose-dependently delayed development of P. includens and H. virescens. Each virus also inhibited pupation of P. includens but not H. virescens. In situ hybridization experiments indicated that MdBV and McBV persistently infect hemocytes in both hosts while MmBV persistently infects hemocytes in P. includens but not H. virescens. While MdBV infection induced a loss of adhesion by most plasmatocytes, McBV and MmBV infection induced a loss of adhesion in less than 50% of cells. Cross-protection experiments indicated that calyx fluid plus venom from one species usually protected progeny of another species from encapsulation but did not always promote successful development [KEYWORDS: Polydnavirus; Parasite; Encapsulation; Hemocytes; Development