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LPS activation of leukocytes attenuates adhesion to endothelial cells under shear stress

Leukocyte adhesion is triggered by upregulation of cell surface adhesion molecules and counteracted by the shear forces of the flowing blood. Since both factors, inflammatory response and flow dynamics are severely altered during endotoxemia, we studied the effects of endotoxin on leukocyte–endothelial interactions under different levels of shear stress in vitro. In order to perform the experiments at precisely adjustable levels of shear stress, we used a parallel plate flow chamber as has been employed in a number of adhesion studies previously. Within this chamber, endothelial cells (HUVEC) could be perfused with neutrophils (PMN) at 0.25–3 dynes/cm2. FACS analysis of adhesion molecules, trypan blue exclusion and PMN migration showed that the cells were not altered during cell separation. To determine the effects of lipopolysaccharide (LPS) on shear-dependent adhesion, we studied HUVEC and PMN in a native state and following activation with LPS at 100 ng/ml and 10 ng/ml. All flow experiments were videotaped and the results were analysed by the paired t-test (P < 0.05). The effects of LPS on leukocyte–endothelial interactions strongly depended on the site of activation. Whereas LPS pretreatment of HUVEC increased PMN adhesion by 5–10 fold, pre-activation of the PMN resulted in a 50% reduction of adherent cells. In addition, after pre-activation of PMN, adhesion became increasingly dependent on shear stress and, thus, inhibition by LPS was most pronounced at a normal, postcapillary shear stress of 2–3 dynes/cm2. As demonstrated by addition of antibodies against selectins, the effect of LPS was due to a functional loss of L-Selectin and P-Selectin mediated interactions. In addition, integrin-mediated adhesion was impaired despite upregulation of CD11b on activated PMN. In summary, our results show that leukocyte–endothelial interactions become increasingly dependent on shear stress as soon as PMN are activated. Once PMN have undergone LPS activation, leukocyte tethering to the endothelium becomes entirely dependent on endothelial E-Selectin. Furthermore, adhesion efficiency decreases despite upregulated expression of CD11b. Since LPS increased the rigidity of PMN, it seems that the increased stiffness attenuated PMN adhesion by hampering PMN flattening and, thereby, reducing the number of available bonds to the endothelium.

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Nohé, B., Schmidt, V., Hientz, A. et al. LPS activation of leukocytes attenuates adhesion to endothelial cells under shear stress. Crit Care 6 (Suppl 1), P105 (2002).

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