We studied how the rheological properties of blood influenced capture and rolling adhesion of leukocytes as well as their margination in the bloodstream. When citrated, fluorescently labeled blood was perfused through glass capillaries coated with P-selectin, leukocytes formed numerous rolling attachments. The number of attached leukocytes increased as the hematocrit was increased between 10% and 30% and was essentially constant from 30% to 50%. In EDTA-treated blood, adhesion was absent, and the flux of marginated cells varied little with increasing hematocrit. However, the velocity of marginated leukocytes increased monotonically, whereas the volumetric flow rate was constant, implying that the flow velocity profile became blunted and wall shear rate increased. Thus increasing hematocrit promoted attachment for a given total flow rate, without increasing margination, even though wall shear rate and blood viscosity increased. Blood was diluted to 20% hematocrit with plasma, 40-kDa dextran (to reduce red blood cell aggregation), or 500-kDa dextran (to enhance aggregation). Increasing aggregation correlated with increasing leukocyte adhesion and with more slow-flowing leukocytes near the wall. Thus flowing erythrocytes promote leukocyte adhesion, either by causing margination of leukocytes or by initiating and stabilizing attachments that follow.

Use of whole blood in adhesion assays allows analysis of the rheological and haematological factors that may influence adhesion, and avoids the need for isolation procedures that may modify the properties of leucocytes. We have adapted an in vitro flow model to allow videomicroscopy of leucocytes fluorescently labelled with rhodamine 6G (20 microg/ml) in anticoagulated whole blood. Blood was perfused at a range of wall shear rates (35-280/s) through a vertical glass capillary with a rectangular cross-section (microslide) that had been coated with P-selectin (10 microg/ml). Nearly all adherent cells were rolling in blood that had been anticoagulated with buffered citrate, but 40-50% became immobilized when heparin or thrombin inhibitor (PPACK) were used. The efficiency of leucocyte adhesion decreased steadily during 1-4 h of blood storage. Smaller fluorescent cells (lymphocytes) adhered less efficiently than larger cells (granulocytes) and rolled faster. Adhesion decreased monotonically with increasing wall shear rate or stress, but the velocity of rolling varied little. Among healthy volunteer donors, adhesion correlated with blood leucocyte count, but did not vary significantly with natural variation in haematocrit, blood viscosity or red cell aggregation. In conclusion, we have characterized adhesion of leucocytes in flowing whole blood, identified key experimental variables and demonstrated that physical environmental factors can markedly influence adhesive behaviour.

No abstract

Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.

Changes in the rheological properties of neutrophils may influence flow in microvessels that are cooled below normal body temperature. We investigated the effects of temperature on the mechanical and adhesive properties of human neutrophils by measuring transit times for individual cells flowing through 8-microm-pores in filters, and adhesion to P-selectin for cells perfused over a monolayer of activated platelets. Pore transit time increased as temperature was decreased from 37 degrees C to 0 degrees C. Upon rapid cooling, there was an instantaneous increase attributable to changes in aqueous viscosity. Interestingly, at 10 degrees C specifically, there was an additional increase in transit time, which was abolished by the inhibitor of actin polymerization, cytochalasin B. This meant that by 15 min, transit time at 10 degrees C was greater than at 0 degrees C. Most adherent cells on P-selectin were rolling, rather than stationary, at 10, 26 or 37 degrees C. The velocity of rolling slowed with decreasing temperature. The total number of adherent cells decreased with increasing wall shear rate, but for a given shear rate there was relatively little effect of temperature on attachment. However, when adhesion at 10, 26 or 37 degrees C was compared at equal shear stress (taking into account fluid viscosity), adhesion was greatest at 10 degrees C. Measurements of immunofluorescence showed that exposure to 10 degrees C gradually increased expression of beta2-integrin CD11b/CD18, but this did not cause transformation to stationary adhesion with time in the flow assay. Thus, neutrophils show an anomalous rheological response around 10 degrees C, which may impair local microcirculation in the cold. On rewarming, "activated" cells might inhibit recovery or become released into the systemic circulation.