Elevated pre-activation basal level of nuclear NF-κB in native macrophages accelerates LPS-induced translocation of cytosolic NF-κB into the cell nucleus.

Signaling via Toll-like receptor 4 (TLR4) in macrophages constitutes an essential part of the innate immune response to bacterial infections. Detailed and quantified descriptions of TLR4 signal transduction would help to understand and exploit the first-line response of innate immune defense. To date, most mathematical modelling studies were performed on transformed cell lines. However, properties of primary macrophages differ significantly. We therefore studied TLR4-dependent activation of NF-κB transcription factor in bone marrow-derived and peritoneal primary macrophages. We demonstrate that the kinetics of NF-κB phosphorylation and nuclear translocation induced by a wide range of bacterial lipopolysaccharide (LPS) concentrations in primary macrophages is much faster than previously reported for macrophage cell lines. We used a comprehensive combination of experiments and mathematical modeling to understand the mechanisms of this rapid response. We found that elevated basal NF-κB in the nuclei of primary macrophages is a mechanism increasing native macrophage sensitivity and response speed to the infection. Such pre-activated state of macrophages accelerates the NF-κB translocation kinetics in response to low agonist concentrations. These findings enabled us to refine and construct a new model combining both NF-κB phosphorylation and translocation processes and predict the existence of a negative feedback loop inactivating phosphorylated NF-κB.

Calpain-controlled detachment of major glycoproteins from the cytoskeleton regulates adhesive properties of activated phosphatidylserine-positive platelets.

In resting platelets, adhesive membrane glycoproteins are attached to the cytoskeleton. On strong activation, phosphatidylserine(PS)-positive and -negative platelet subpopulations are formed. Platelet activation is accompanied by cytoskeletal rearrangement, although the glycoprotein attachment status in these two subpopulations is not clear. We developed a new, flow cytometry-based, single-cell approach to investigate attachment of membrane glycoproteins to the cytoskeleton in cell subpopulations. In PS-negative platelets, adhesive glycoproteins integrin αIIbß3, glycoprotein Ib and, as shown for the first time, P-selectin were associated with the cytoskeleton. In contrast, this attachment was disrupted in PS-positive platelets; it was retained to some extent only in the small convex regions or 'caps'. It correlated with the degradation of talin and filamin observed only in PS-positive platelets. Calpain inhibitors essentially prevented the disruption of membrane glycoprotein attachment in PS-positive platelets, as well as talin and filamin degradation. With the suggestion that detachment of glycoproteins from the cytoskeleton may affect platelet adhesive properties, we investigated the ability of PS-positive platelets to resist shear-induced breakaway from the immobilized fibrinogen. Shear rates of 500/s caused PS-positive platelet breakaway, but their adhesion stability increased more than 10-fold after pretreatment of the platelets with calpain inhibitor. In contrast, the ability of PS-positive platelets to adhere to immobilized von Willebrand's factor at 100/s was low, but this was not affected by the preincubation of platelets with a calpain inhibitor. Our data suggest that calpain-controlled detachment of membrane glycoproteins is a new mechanism that is responsible for the loss of ability of the procoagulant platelets to resist detachment from thrombi by high shear stress.