Nox2 Regulates Platelet Activation and NET Formation in the Lung.

The mortality rate of patients with critical illness has decreased significantly over the past two decades, but the rate of decline has slowed recently, with organ dysfunction as a major driver of morbidity and mortality. Among patients with the systemic inflammatory response syndrome (SIRS), acute lung injury is a common component with serious morbidity. Previous studies in our laboratory using a murine model of SIRS demonstrated a key role for NADPH oxidase 2 (Nox2)-derived reactive oxygen species in the resolution of inflammation. Nox2-deficient (gp91phox-/y) mice develop profound lung injury secondary to SIRS and fail to resolve inflammation. Alveolar macrophages from gp91phox-/y mice express greater levels of chemotactic and pro-inflammatory factors at baseline providing evidence that Nox2 in alveolar macrophages is critical for homeostasis. Based on the lung pathology with increased thrombosis in gp91phox-/y mice, and the known role of platelets in the inflammatory process, we hypothesized that Nox2 represses platelet activation. In the mouse model, we found that platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) and CXCL7 were increased in the bronchoalveolar fluid of gp91phox-/y mice at baseline and 24 h post intraperitoneal zymosan-induced SIRS consistent with platelet activation. Activated platelets interact with leukocytes via P-selectin glycoprotein ligand 1 (PSGL-1). Within 2 h of SIRS induction, alveolar neutrophil PSGL-1 expression was higher in gp91phox-/y mice. Platelet-neutrophil interactions were decreased in the peripheral blood of gp91phox-/y mice consistent with movement of activated platelets to the lung of mice lacking Nox2. Based on the severe lung pathology and the role of platelets in the formation of neutrophil extracellular traps (NETs), we evaluated NET production. In contrast to previous studies demonstrating Nox2-dependent NET formation, staining of lung sections from mice 24 h post zymosan injection revealed a large number of citrullinated histone 3 (H3CIT) and myeloperoxidase positive cells consistent with NET formation in gp91phox-/y mice that was virtually absent in WT mice. In addition, H3CIT protein expression and PAD4 activity were higher in the lung of gp91phox-/y mice post SIRS induction. These results suggest that Nox2 plays a critical role in maintaining homeostasis by regulating platelet activation and NET formation in the lung.

Effects of Transient Exposure to High Shear on Neutrophil Rolling Behavior.

INTRODUCTION: Neutrophils display an array of behaviors ranging from rolling and migration to phagocytosis and granule secretion. Several of these behaviors are modulated by the local shear conditions. In the normal circulation, neutrophils experience shear rates from approximately 10-2,000 s-1. However, neutrophils are also exposed to pathological shear levels in natural conditions such as severe stenosis and arteriosclerosis, as well as in blood-contacting devices such as ventricular assist devices (VADs) and hemodialysis machines. The effects of transiently (< 1 sec) exposing neutrophils to abnormally high shear rates (>3,000 s-1) are not well understood. METHODS: We developed a set of microfluidic devices capable of exposing neutrophils to high shear rates for short durations (100-400 msec). Suspensions of isolated neutrophils were perfused through the devices and their rolling velocities on P-selectin were analyzed before and after shear exposure. RESULTS: We observed a significant increase in neutrophil rolling velocities on P-selectin coated regions following transient high shear exposure. The magnitude of the rolling velocity increase was dependent upon the duration of high shear exposure and became statistically significant for exposure times of 310 msec or longer. When polystyrene beads coated with a glycosulfopeptide that mimics the binding region of P-selectin glycoprotein ligand-1 (PSGL-1) were perfused through the devices, no change between the pre-shear and post-shear rolling velocities was observed. CONCLUSIONS: These results suggest that high shear levels alter normal neutrophil rolling behavior and are important for understanding neutrophil biology in high shear conditions, as well as for improving medical device performance.

Constricted microfluidic devices to study the effects of transient high shear exposure on platelets.

Due to the critical roles that platelets play in thrombosis during many biological and pathological events, altered platelet function may be a key contributor to altered hemostasis, leading to both thrombotic and hemorrhagic complications. Platelet adhesion at arterial shear rates occurs through binding to von Willebrand Factor via the glycoprotein (GP) GPIb receptor. GPIb binding can induce platelet activation distinguishable by P-selectin (CD62P) surface expression and αIIbß3 activation, resulting in platelet aggregation and formation of the primary hemostatic plug to stop bleeding. Previous studies have used cone and plate viscometers to examine pathologic blood flow conditions, applied shear rates that are relatively low, and examined exposure times that are orders of magnitude longer compared to conditions present in ventricular assist devices, mechanical heart valves, or pathologic states such as stenotic arteries. Here, we evaluate the effect of short exposure to high shear on granule release and receptor shedding utilizing a constricted microfluidic device in conjunction with flow cytometry and enzyme-linked immunosorbent assay. In this study, platelets were first perfused through microfluidic channels capable of producing shear rates of 80 000-100 000 s-1 for exposure times of 0-73 ms. We investigated platelet activation by measuring the expression level of CD62P (soluble and surface expressed), platelet factor 4 (PF4), and beta-thromboglobulin (ßTG). In addition, we measured potential platelet receptor shedding of GPVI and GPIb using flow cytometry. The results showed that a single pass to high shear with short exposure times (milliseconds) had no effect on the levels of CD62P, GPVI and GPIb, or on the release of alpha granule content (PF4, ßTG, and sP-selectin).

Effects of shear on P-selectin deposition in microfluidic channels.

Traditional leukocyte adhesion assays have provided significant insight into the mechanisms of leukocyte rolling in part through the use of homogeneously coated surfaces. These assays typically involve protein coating of glass coverslips or plastic petri dishes applied via a static drop of protein solution. With this approach, it is difficult to spatially control the location of proteins to fabricate surface-bound protein gradients that mimic in vivo situations. Microfluidic patterning of proteins with microfluidic devices has become a popular technique due to the ability to spatially pattern proteins on a cellular scale. Despite the advantages of microfluidic patterning, few studies have systematically investigated the effects of perfusion time, protein concentration, and perfusion shear stress on protein deposition. Herein, we demonstrated the fabrication of both line and step gradients of P-selectin on glass substrates that support cell rolling and adhesion assays. Investigation of the flow conditions during the microfluidic patterning led to several significant findings. We observed that the protein deposition time of 5 min was sufficient to deposit adequate P-selectin to support neutrophil rolling. We demonstrated that the amount of membrane P-selectin (mP-selectin) or recombinant P-selectin (rP-selectin) deposited showed a dependence on the perfusion shear stress between 4.0 and 32.0 dyn/cm(2), while similar studies with fibronectin or fibrinogen showed no shear stress dependence. Finally, we also created step changes in surface adherent protein concentration of P-selectin to characterize leukocyte-rolling behavior in response to sudden changes in ligand density.