Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids.

Adhesion molecules known to be important for neutrophil recruitment in many other organs are not involved in recruitment of neutrophils into the sinusoids of the liver. The prevailing view is that neutrophils become physically trapped in inflamed liver sinusoids. In this study, we used a biopanning approach to identify hyaluronan (HA) as disproportionately expressed in the liver versus other organs under both basal and inflammatory conditions. Spinning disk intravital microscopy revealed that constitutive HA expression was restricted to liver sinusoids. Blocking CD44-HA interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, with no effect on rolling or adhesion in postsinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesion in sinusoids, yet neutrophil CD44 avidity for HA did not increase significantly in endotoxemia. Instead, activation of CD44-HA engagement via qualitative modification of HA was demonstrated by a dramatic induction of serum-derived HA-associated protein in sinusoids in response to lipopolysaccharide (LPS). LPS-induced hepatic injury was significantly reduced by blocking CD44-HA interactions. Administration of anti-CD44 antibody 4 hours after LPS rapidly detached adherent neutrophils in sinusoids and improved sinusoidal perfusion in endotoxemic mice, revealing CD44 as a potential therapeutic target in systemic inflammatory responses involving the liver.

Targeting adhesion molecules as a potential mechanism of action for intravenous immunoglobulin.

BACKGROUND: Intravenous immunoglobulin (IVIg) therapy has been shown to have therapeutic benefit in more than 50 inflammatory and immune-related diseases; however, the potential benefit of IVIg in cardiovascular disease is more limited, in part because our understanding of the mechanisms underlying the effects of IVIg in innate immunity is incomplete. METHODS AND RESULTS: In this study, a systematic assessment of the role of IVIg in leukocyte recruitment was completed with an in vitro flow-chamber system and in vivo intravital microscopy in a feline ischemia-reperfusion model system. IVIg treatment of blood resulted in a profound decrease in recruitment of either immobilized P-selectin or E-selectin due to direct effects of IVIg on the leukocyte (not substratum). Similar results were observed on endothelium treated with histamine, which induces P-selectin-dependent rolling and beta2-integrin-dependent adhesion. IVIg reduced P-selectin glycoprotein ligand-1 (PSGL-1) antibody binding to PSGL-1 on leukocytes. Use of a beta2-integrin-dependent static assay to bypass selectin-dependent recruitment revealed some inhibitory effectiveness (60%), which suggests that the majority of the effects of IVIg were due to selectin inhibition, with some inhibition of integrin function. In vivo intravital microscopy revealed a potent inhibitory effect of IVIg on P-selectin-dependent rolling and beta2-integrin-dependent adhesion that led to reduced leukocyte recruitment and vascular dysfunction in postischemic microvessels. CONCLUSIONS: Our data demonstrate that IVIg has direct inhibitory effects on leukocyte recruitment in vitro and in vivo through inhibition of selectin and integrin function.

IVIg therapy in brain inflammation: etiology-dependent differential effects on leucocyte recruitment.

Several studies have reported beneficial effects of intravenous immunoglobulin (IVIg) in diseases of the neuroaxis. However, IVIg effects on leucocyte recruitment, a hallmark feature of autoimmunity and acute inflammation, remain largely unexplored. Using intravital microscopy, we studied the effects of IVIg on leucocyte recruitment in experimental autoimmune encephalomyelitis, a model of multiple sclerosis. In IVIg-treated mice, a significant decrease in recruitment (rolling and adhesion) was observed prior to and following disease onset, and this was concomitant with improved clinical score. Since much of the recruitment is dependent upon alpha4-integrin (ligand for VCAM-1) we used an in vitro flow chamber system and demonstrated a 60% decrease in alpha4-integrin-dependent leucocyte adhesion to immobilized VCAM-1. Finally, we used leucocytes from multiple sclerosis patients and demonstrated that IVIg treatment decreased recruitment by 60% on human endothelium. However, when we visualized the role of IVIg in a second model of brain inflammation, cerebral ischaemia-reperfusion, IVIg actually promoted the formation of platelet-leucocyte aggregates in post-ischaemic cerebral vessels. In conclusion, we report a new mechanism of action of IVIg through interference of alpha4-integrin-dependent leucocyte recruitment in both an animal model and human multiple sclerosis. We also report that IVIg will not be beneficial in all types of pro-adhesive states and may in fact be detrimental in a situation such as stroke.

Human fractalkine mediates leukocyte adhesion but not capture under physiological shear conditions; a mechanism for selective monocyte recruitment.

Fractalkine is a unique chemokine possessing a long mucin-like stalk and a transmembrane region that has been proposed to act as an adhesion molecule. We investigated the ability of fractalkine to recruit leukocytes from whole blood, using an immobilized fractalkine fusion protein in the parallel-plate flow-chamber assay. Significant adhesion of leukocytes to fractalkine peaked at 2 dynes/cm(2) but was minimal at 10 dynes/cm(2). In contrast, VCAM-1 could recruit cells from whole blood at 10 dynes/cm(2). Co-immobilization of fractalkine and VCAM-1 at 10 dynes/cm(2) resulted in a twofold increase in adherent cells compared with VCAM-1 alone, suggesting that fractalkine can mediate adhesion at high shear if combined with a molecule that can mediate leukocyte tethering. Pretreatment of blood with pertussis toxin eliminated this increase in adhesion, implicating intracellular signaling in fractalkine-mediated mechanisms of adhesion to co-immobilized fractalkine/VCAM-1. Analysis of the cell types recruited to fractalkine alone at low shear, or to fractalkine and VCAM-1 at 10 dynes/cm(2), revealed that monocytes were recruited to fractalkine with the highest specificity. In conclusion, fractalkine is unlikely to act alone at shear forces found in most vascular beds where it most likely co-operates with tethering molecules, e.g. VCAM-1, in the recruitment of monocytes.