Yaw-Chyn Lim

MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions

Monocytes contribute to the development of atherosclerotic lesions in mouse models. The chemoattractant proteins (chemokines), monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8), are found in human atheroma, and mice lacking receptors for these chemokines are less susceptible to atherosclerosis and have fewer monocytes in vascular lesions. Although MCP-1 has a powerful effect on monocytes, IL-8 is thought to act predominantly on neutrophils and it is unclear howit could recruit monocytes. Here we investigate the ability of chemokines to control the interaction of monocytes under flow conditions with vascular endothelium that has been transduced to express specific leukocyte-adherence receptors. We find that MCP-1 and IL-8 can each rapidly cause rolling monocytes to adhere firmly onto monolayers expressing E-selectin, whereas related chemokines do not. These effects do not correlate with either the induction of a calcium transient or chemotaxis. We conclude that chemokines are important modulators of monocyte–endothelial interactions under flow conditions. Moreover, our finding that IL-8 is a powerful trigger for firm adhesion of monocytes to vascular endothelium reveals an unexpected role for this chemokine in monocyte recruitment.

Neutrophils from MMP‐9‐ or neutrophil elastase‐deficient mice show no defect in transendothelial migration under flow in vitro

Recent evidence has suggested a role for neutrophil proteases during certain inflammatory responses. We demonstrated previously that neutrophil proteases can degrade components of the adherens junctions during neutrophil‐endothelial adhesion. We tested the hypothesis that degradation of VE‐cadherin at lateral junctions by elastase or MMP‐9 facilitates neutrophil transendothelial migration. Neutrophils from MMP‐9 or elastase null mice and strain‐matched control mice expressed high levels of LFA‐1, Mac‐1, and L‐selectin on their cell surface. Under flow conditions, wild‐type and deficient neutrophils rolled, arrested, and transmigrated activated murine endothelium. There was no difference in the total numbers of interacting neutrophils or in the percentage of transmigrated cells. In addition, deficient neutrophils remained capable of degrading murine endothelial VE‐cadherin. These results indicate that although neutrophil proteases may play a role in the acute inflammatory response, neutrophil elastase or MMP‐9 is not essential for neutrophil transendothelial migration in this murine system.

Heterogeneity of Endothelial Cells from Different Organ Sites in T-Cell Subset Recruitment

Chemokines and adhesion molecules play a critical role in the recruitment of leukocytes into specific organ sites. Little is known, however, regarding the repertoire of chemokines and adhesion molecules expressed within different vascular beds. In this study, we compare adhesion molecule expression, chemokine induction, and T-cell subset-endothelial interactions under defined flow conditions on resting and tumor necrosis factor (TNF)-alpha-activated murine lung endothelial cells (MLECs) and heart endothelial cells (MHECs). Our study revealed that only MHECs exhibited high constitutive VCAM-1 expression. Exposure to TNF-alpha up-regulated adhesion molecule expression and chemokine production in both MLECs and MHECs. However, high levels of Regulated on Activation Normal T cell Expressed And Secreted (RANTES) expression were detected only in TNF-alpha-activated MHECs. TNF-alpha-stimulated MLECs and MHECs both supported T-helper cell interactions under defined flow conditions. Most T cells instantaneously arrested on MHECs but exhibited a rolling phenotype on MLECs. Blocking studies revealed that T-cell arrest on MHECs was mediated by constitutive VCAM-1 and TNF-alpha-induced RANTES. These findings are consistent with the hypothesis that functional heterogeneity of endothelial cells from different sites exists and some of it is retained in vitro. Furthermore, these results provide an insight into the molecular mechanisms that may mediate T-helper cell recruitment to these organs.

IL-12, STAT4-Dependent Up-Regulation of CD4+ T Cell Core 2 β-1,6-n-Acetylglucosaminyltransferase, an Enzyme Essential for Biosynthesis of P-Selectin Ligands

TCR activation of naive T cells in the presence of IL-12 drives polarization toward a Th1 phenotype and synthesis of P- and E-selectin ligands. Fucosyltransferase VII (Fuc-T VII) and core 2 β-1,6-N-acetylglucosaminyltransferase (C2GnT) are critical for biosynthesis of selectin ligands. P-selectin glycoprotein ligand-1 is the best characterized ligand for P-selectin and also binds E-selectin. The contributions of TCR and cytokine signaling pathways to up-regulate Fuc-T VII and C2GnT during biosynthesis of E- and P-selectin ligands, such as P-selectin glycoprotein ligand 1, are unknown. IL-12 signals via the STAT4 pathway. Here, naive DO11.10 TCR transgenic and STAT4−/− TCR transgenic CD4+ T cells were stimulated with Ag and IL-12 (Th1 condition), IL-4 (Th2), or neutralizing anti-IL-4 mAb only (Th0). The levels of Fuc-T VII and C2GnT mRNA in these cells were compared with their adhesive interactions with P- and E-selectin in vitro under flow. The data show IL-12/STAT4 signaling is necessary for induction of C2GnT, but not Fuc-TVII mRNA, and that STAT4−/− Th1 cells do not traffic normally to sites of inflammation in vivo, do not interact with P-selectin, and exhibit a partial reduction of E-selectin interactions under shear stress in vitro. Ag-specific TCR activation in CD4+ T cells was sufficient to trigger induction of Fuc-TVII, but not C2GnT, mRNA and expression of E-selectin, but not P-selectin, ligands. Thus, Fuc-T VII and C2GnT are regulated by different signals during Th cell differentiation, and both cytokine and TCR signals are necessary for the expression of E- and P-selectin ligands.

Isolation and culture of murine heart and lung endothelial cells for in vitro model systems.

The inflammatory response is a critical component of host defense. An important goal of our group has been to understand the endothelial-dependent mechanisms that mediate leukocyte recruitment during an inflammatory response. In this chapter, we present a detailed method for the isolation and in vitro culture of murine vascular endothelial cells from the lung and heart. The endothelial cells are of high purity (85-99%) and retain certain of their functional differences, including constitutive and cytokine inducible adhesion molecule expression and chemokine production. Such endothelial cells, therefore, can be used for various in vitro models including leukocyte adhesion assay or in depth biochemical analyses.

α4β1-Integrin Activation Is Necessary for High-Efficiency T-Cell Subset Interactions with VCAM-1 under Flow

Objective: The purpose of this study was to examine the relationship between α4β1‐integrin state of activation on CD4+ T‐cell subsets and their adhesive interaction to VCAM‐1 under flow.

β-Galactoside α2,3-sialyltransferase-I gene expression during Th2 but not Th1 differentiation: implications for core2-glycan formation on cell surface proteins

Biosynthesis of core2 O‐glycans on T cell surface glycoproteins is essential for their interactions with selectins expressed by activated endothelium, and may also regulate susceptibility to apoptosis. β‐Galactoside α2,3‐sialyltransferase‐I (ST3Gal‐I) is a major inhibitor of core2 O‐glycan formation on CD43 and CD45 in naive T cells. Here we show that ST3Gal‐I mRNA is extensivelyexpressed during Th2, but not Th1 differentiation. Consistent with this, developing Th1 cells display the non‐sialylated core1 galactose (Gal1–3GalNAc‐Ser/Thr) and strongly react with peanut agglutinin, while Th2 cells do not. These Th subset differences are also associated with greater expression of the high molecular glycoform of CD43 on the surface of Th1 cells compared with Th2 cells, andsimilar differences in surface expression of sialyl Lewis‐X. We suggest that lack of ST3Gal‐I expression in Th1 cells allows the formation of surface core2 O‐glycans and supports their interactionswith endothelial selectins.

Wall shear stress: the missing step for T cell transmigration?

Our current understanding of lymphocyte migration across the endothelium includes four steps: attachment, rolling, arrest and diapedesis. New evidence suggests the involvement of another step, chemorheotaxis.

Current In Vitro Models of Leukocyte Migration

A large component of airway inflammatory disease is the recruitment of activated leukocytes (primarily eosinophils and T lymphocytes) from the lung vasculature into the bronchial walls resulting in lung edema. Ultimately, many of the infiltrating leukocytes progress across the airway epithelium into respiratory bronchioles, compromising lung capacity (1,2). In the case of an infection, such as pneumonia, leukocytes (primarily neutrophils and monocyte/macrophages) are recruited to alveolar air spaces reducing the capacity for gaseous exchange. In both cases, resident leukocytes then release further factors that promote additional leukocyte recruitment. During an inflammatory response in the peripheral microvasculature leukocyte recruitment takes place predominantly in the postcapillary venules via the multistep adhesion cascade (reviewed in 3,4,5). In the lung, however, leukocyte extravasation takes place via capillaries. This may be due to the specialized architecture of the lung vasculature (e.g., large numbers of branch points), or because of the differing expression of surface adhesion molecules that are required for leukocyte recruitment (1,6). In addition, local concentrations of cytokines, chemokines or other chemoattractant factors will play a role in the site and degree of leukocyte infiltration (7,8) through acute local activation of endothelial cells.