Haematopoietic stem cell recruitment to injured murine liver sinusoids depends on (alpha)4(beta)1 integrin/VCAM-1 interactions.

OBJECTIVE: Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These were investigated in vivo following hepatic ischaemia-reperfusion (IR) injury and in vitro using flow-based adhesion assays. DESIGN: Partial IR was induced in anaesthetised WT or PECAM-1(-/-) mice for 90 min. Recruitment of systemically administered HSCs was monitored and effects of function blocking antibodies against alpha(4)beta(1) integrin, CD18, CD44, PECAM-1 or VCAM-1 investigated. The kinetics and molecular events governing adhesion to murine cardiac endothelial cells in vitro were also determined. Effects of conditioned media from IR injured liver on HSC adhesion molecule expression was determined by FACS. RESULTS: Administered HSCs homed predominantly to lungs rather than liver, highlighting a potential therapeutic hurdle. Hepatic HSC recruitment following IR injury was inhibited by anti-alpha(4)beta(1) and anti-VCAM-1 antibodies. A role for alpha(4)beta(1) was also confirmed using flow-based adhesion assays. Incubating HSCs with conditioned media from IR injured liver increased alpha(4)beta(1) expression. CD18, CD44 and PECAM-1 were not involved in recruitment. CONCLUSIONS: This novel study demonstrates that the alpha(4)beta(1)/VCAM-1 pathway mediates HSC recruitment to injured liver. Manipulating this pathway may enhance delivery of HSCs to the liver.

Vitronectin in human hepatic tumours contributes to the recruitment of lymphocytes in an alpha v beta3-independent manner.

The degree of lymphocyte infiltration is a prognostic factor in liver cancer, but to date the mechanisms by which lymphocytes infiltrate into and are retained in hepatic tumours are poorly understood. We hypothesised that the extracellular matrix glycoprotein vitronectin, a major component of the stroma of hepatic tumours, might play a role in the recruitment and retention of tumour-infiltrating lymphocytes (TIL). Thus, we investigated the ability of vitronectin to support migration and adhesion of TIL isolated from hepatocellular carcinoma and colorectal hepatic metastases. Soluble vitronectin-induced dose-dependent migration of TIL in in vitro chemotaxis and haptotaxis assays and vitronectin in tissue sections was able to support TIL adhesion to tumour stroma. Neither adhesion nor migration was inhibited by a function blocking mAb against the major vitronectin receptor alpha v beta3 and we were unable to detect alpha v beta3 on TIL in vitro or in vivo on tumour tissue. However, TIL did express high levels of urokinase-type plasminogen activator receptor (uPAR) and inhibitory antibodies and amiloride both significantly inhibited TIL adhesion to vitronectin and reduced transendothelial migration of lymphocytes across liver endothelium in vitro. Thus, we provide evidence that vitronectin in liver tumours can support the recruitment and retention of effector lymphocytes by an uPAR-dependent mechanism.

Human hepatic sinusoidal endothelial cells can be distinguished by expression of phenotypic markers related to their specialised functions in vivo.

The hepatic sinusoids are lined by a unique population of hepatic sinusoidal endothelial cells (HSEC), which is one of the first hepatic cell populations to come into contact with blood components. However, HSEC are not simply barrier cells that restrict the access of blood-borne compounds to the parenchyma. They are functionally specialised endothelial cells that have complex roles, including not only receptor-mediated clearance of endotoxin, bacteria and other compounds, but also the regulation of inflammation, leukocyte recruitment and host immune responses to pathogens. Thus understanding the differentiation and function of HSEC is critical for the elucidation of liver biology and pathophysiology. This article reviews methods for isolating and studying human hepatic endothelial cell populations using in vitro models. We also discuss the expression and functions of phenotypic markers, such as the presence of fenestrations and expression of VAP-1, Stabilin-1, L-SIGN, which can be used to identify sinusoidal endothelium and to permit discrimination from vascular and lymphatic endothelial cells.

MAdCAM-1 expressed in chronic inflammatory liver disease supports mucosal lymphocyte adhesion to hepatic endothelium (MAdCAM-1 in chronic inflammatory liver disease).

Mucosal addressin cell adhesion molecule (MAdCAM-1) plays a pivotal role in T-lymphocyte homing to the gut. Given the strong association between the autoimmune liver diseases primary sclerosing cholangitis and autoimmune hepatitis and inflammatory bowel disease, we investigated the role of MAdCAM-1 in recruiting mucosal lymphocytes to the liver. MAdCAM-1 was strongly expressed on inflamed portal vein/sinusoidal endothelium in autoimmune mediated liver disease. In modified Stamper-Woodruff assays, MAdCAM-1 on hepatic vessels supported adhesion of alpha4beta7+ lymphocytes (i.e., gut-derived T cells) from patients with inflammatory bowel disease and primary sclerosing cholangitis. This adhesion was inhibited by pretreatment with blocking antibodies to MAdCAM-1, alpha4beta7, or the integrin alpha4 chain indicating that MAdCAM-1 in inflamed liver is functionally active. Circulating lymphocytes from patients with primary sclerosing cholangitis showed rolling adhesion on MAdCAM-1 transfectants in a flow-based adhesion assay that could be blocked by anti-MAdCAM-1 or anti-alpha4beta7 mAbs. These findings indicate that, under certain circumstances, vessels in the human liver support adhesion of alpha4beta7+ mucosal lymphocytes via binding to aberrantly expressed MAdCAM-1 on liver endothelium. This provides a mechanism to explain the hepatic recruitment of mucosal lymphocytes in inflammatory liver disease complicating inflammatory bowel disease.

Adhesion of lymphocytes to hepatic endothelium.

Chronic inflammation occurs when factors that regulate the process of leucocyte recruitment are disrupted, and it is dependent on recruitment, activation, and retention of lymphocytes within tissue microenvironments. The molecular mechanisms that mediate lymphocyte adhesion to vascular endothelial cells have been described by several groups, but the signals involved in the recruitment of lymphocytes via the hepatic circulation have yet to be elucidated fully. This article considers the liver as a model of organ specific lymphocyte recruitment. In this context, the roles of leucocyte and endothelial adhesion molecules and chemokines in lymphocyte recruitment are discussed. The article also reviews the mechanisms that regulate lymphocyte recirculation to the liver under both physiological and pathological conditions and draws parallels with other organs such as the gut and skin.

Association between receptor density, cellular activation, and transformation of adhesive behavior of flowing lymphocytes binding to VCAM-1.

We investigated the ability of purified vascular cell adhesion molecule-1 (VCAM-1), adsorbed on plastic, to capture and immobilize flowing lymphocytes, and the dependence of adhesive behavior on activation of the counter-receptor, alpha 4 beta 1 integrin. This integrin/immunoglobulin interaction bound lymphocytes at a wall shear stress at which the beta 2-integrin family has previously been found ineffective (> 0.1 Pa), and whereas lymphocytes rolled on lower concentrations of VCAM-1 (10 micrograms/ml), they were stationary at high concentrations (100 micrograms/ml). Activation of alpha 4 beta 1 integrin by Mn2+ or by antibody 12G10 or treatment of lymphocytes with phorbol ester caused transformation to stationary adhesion, and increased binding significantly only at the lower concentrations of VCAM-1. We thus hypothesized that formation of a high density of ligand between VCAM-1 and alpha 4 beta 1 integrin actively transformed lymphocyte behavior. This concept was supported by the finding that the proportion of lymphocytes rolling on the higher concentrations of VCAM-1 increased if cells were pretreated with azide to block energy-dependent responses, or if intracellular Ca2+ was chelated. However, not all activation responses were equivalent: only phorbol ester induced marked spreading of immobilized cells, and if pretreatment was prolonged, this agent even reduced the efficiency of initial attachment of flowing lymphocytes. Azide treatment had no effect on transformation to stationary adhesion caused by Mn2+ or activating antibody. Thus, different forms of lymphocyte activation were identifiable: external modification of integrin converted rolling to stationary attachment, did not require ATP, and was reversible; high-density ligand binding induced an energy-dependent signal for conversion from rolling to stationary attachment, but not spreading; and protein kinase C activation promoted stationary attachment and spreading, but not necessarily capture. VCAM-1 is thus a versatile adhesion receptor capable of supporting all stages of leukocyte attachment, i.e. rolling, stationary, and spreading, and of ligand-induced transformation of adhesion, although an additional signal appears necessary to promote lymphocyte spreading and migration.