Effects of different leukocyte subpopulations and flow conditions on leukocyte accumulation during reperfusion

Background/Aims: The study examined the interdependent effects of shear stress and different leukocyte subpopulations on endothelial cell activation and cell interactions during low flow and reperfusion. Methods: Human umbilical venous endothelial cells were perfused with either neutrophils or monocytes at different shear stress (2-0.25 dyn/cm 2) and adhesion was quantified by microscopy. Effects of adherent neutrophils and monocytes on endothelial cell adhesion molecule expression were analyzed by flow cytometry after 4-hour static coincubation. After coincubation, the cocultures were reperfused with labeled neutrophils at 2 dyn/cm 2 and their adhesion was quantified selectively. For the control, endothelium monocultures with and without lipopolysaccharide activation were used. Results: At 2 dyn/cm 2, adhesion did not exceed baseline levels on nonactivated endothelium. Decreasing shear stress to 0.25 dyn/cm 2 largely increased the adhesion of both leukocyte subpopulations, similar to the effect of lipopolysaccharide at 2 dyn/cm 2. However, only adherent monocytes increased adhesion molecule expression, whereas neutrophils had no effect. As a functional consequence, adherent monocytes largely increased neutrophil adhesion during reperfusion, whereas adherent neutrophils did not. Conclusion: Compromised shear stress is an autonomous trigger of leukocyte adhesion even in the absence of additional activators. Exceeding this immediate effect, adherent monocytes induce further endothelial activation and enhance further neutrophil adhesion during reperfusion. Copyrigh

Gu-4 Suppresses Affinity and Avidity Modulation of CD11b and Improves the Outcome of Mice with Endotoxemia and Sepsis

BACKGROUND: Systemic leukocyte activation and disseminated leukocyte adhesion will impair the microcirculation and cause severe decrements in tissue perfusion and organ function in the process of severe sepsis. Gu-4, a lactosyl derivative, could selectively target CD11b to exert therapeutic effect in a rat model of severe burn shock. Here, we addressed whether Gu-4 could render protective effects on septic animals. METHODOLOGY/PRINCIPAL FINDINGS: On a murine model of endotoxemia induced by lipopolysaccharide (LPS), we found that the median effective dose (ED50) of Gu-4 was 0.929 mg/kg. In vivo treatment of Gu-4 after LPS challenge prominently attenuated LPS-induced lung injury and decreased lactic acid level in lung tissue. Using the ED50 of Gu-4, we also demonstrated that Gu-4 treatment significantly improved the survival rate of animals underwent sepsis induced by cecal ligation and puncture. By adhesion and transwell migration assays, we found that Gu-4 treatment inhibited the adhesion and transendothelial migration of LPS-stimulated THP-1 cells. By flow cytometry and microscopy, we demonstrated that Gu-4 treatment inhibited the exposure of active I-domain and the cluster formation of CD11b on the LPS-stimulated polymorphonuclear leukocytes. Western blot analyses further revealed that Gu-4 treatment markedly inhibited the activation of spleen tyrosine kinase in LPS-stimulated THP-1 cells. CONCLUSIONS/SIGNIFICANCE: Gu-4 improves the survival of mice underwent endotoxemia and sepsis, our in vitro investigations indicate that the possible underlying mechanism might involve the modulations of the affinity and avidity of CD11b on the leukocyte. Our findings shed light on the potential use of Gu-4, an interacting compound to CD11b, in the treatment of sepsis and septic shock