Jeanne-Marie Kiely

Characterization of resident glomerular cells in the rat expressing Ia determinants and manifesting genetically restricted interactions with lymphocytes.

The existence of a subpopulation of rat glomerular cells bearing Ia determinants has been demonstrated with the aid of techniques for the enzymatic isolation and culture of glomerular cells. The Ia-positive cell is normally resident in the uninflamed glomerulus. It resembles a mononuclear phagocyte and consists of a functionally heterogeneous cell population with the capacity of Fc receptor display and phagocytosis, both in vivo and in vitro. A new technique for labeling these cells in situ in intact glomeruli has indicated that Ia-positive cells make up approximately 2% of the total glomerular cell population. The isolated glomerular cells can take up antigen and stimulate immune lymphocytes in an I-region-restricted interaction. They are strongly stimulatory in an allogeneic primary mixed lymphocyte culture. Characterization of this cell type suggests potential new insights into the pathogenesis of renal allograft rejection and immunologically mediated glomerulonephritis.

E-Selectin-Dependent Signaling Via the Mitogen-Activated Protein Kinase Pathway in Vascular Endothelial Cells

E-selectin, a cytokine-inducible adhesion molecule, supports rolling and stable arrest of leukocytes on activated vascular endothelium. Previous studies have suggested that this transmembrane protein can also transduce signals into the endothelial cell. We now demonstrate activation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured HUVEC in response to E-selectin-dependent leukocyte adhesion and Ab-mediated cross-linking of cell surface E-selectin. Adhesion of increasing numbers of HL60 cells to IL-1β-activated HUVEC stimulated robust increases in MAPK activity that were abrogated by an E-selectin blocking Ab. Cross-linking of cell surface E-selectin with Abs, as a mimic of multivalent ligand engagement, strongly stimulated MAPK/extracellular signal-related kinase (ERK) kinase (MEK)-dependent MAPK activation and concomitant up-regulation of mRNA for c-fos, an immediate early response gene, whereas Ab cross-linking of HLA class I molecules (present at comparable density) failed to do so. Coimmunoprecipitation documented Ras, Raf-1 and, phospho-MEK complex formation. Unactivated HUVEC transduced with a full-length adenoviral E-selectin construct also exhibited cross-link-induced MAPK activation, macromolecular complex formation, and c-fos up-regulation, whereas HUVEC transduced with a cytoplasmic domain deletion mutant failed to respond. These observations indicate that E-selectin can transduce an activating stimulus via the MAPK cascade into the endothelial cell during leukocyte adhesion.

Interleukin‐8 induces changes in human neutrophil actin conformation and distribution: relationship to inhibition of adhesion to cytokine‐activated endothelium

Interleukin‐8 (IL‐8) induces diverse biological responses in neutrophils, including inhibition of adhesion to cytokine‐activated endothelium, which we have termed the leukocyte adhesion inhibition (LAI) effect. Pretreatment of neutrophils with cytochalasin B abolished the LAI effect of IL‐8, suggesting a microfilament‐dependent mechanism. Interleukin‐8 induced a rapid increase (≤ 15 s) in the polymerization of actin filaments in human neutrophils that was blocked by pretreatment with cytochalasin B. F‐actin depolymerization occurred gradually at a rate inversely proportional to IL‐8 concentration. This temporal pattern of actin polymerization‐depolymerization was similar to that induced by other chemotactic factors such as C5a and N‐formylmethionyl‐leucyl‐phenylalanine, which also exhibit a marked LAI effect, but the lipid mediators, leu‐kotriene B4 and platelet‐activating factor, lack any significant LAI effect. Scanning confocal microscopy demonstrated that neutrophil actin microfilaments undergo a dramatic rearrangement prior to detachment of the neutrophil from a surface. We suggest that the ability of IL‐8 and certain other leukocyte agonists to regulate the actin polymer network of neutrophils may play an important role in adhesive interactions with the vascular endothelium.

Activation-Dependent Isolation and Culture of Murine Pulmonary Microvascular Endothelium

Objective: Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium. To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study.

Lipid Raft Localization of Cell Surface E-Selectin Is Required for Ligation-Induced Activation of Phospholipase Cγ

E-selectin, an endothelial cell surface adhesion receptor for leukocytes, also acts as a signaling receptor. Upon multivalent ligation, E-selectin transduces outside-in signals into the endothelium leading to changes in intracellular Ca2+ concentration and activation of the mitogen-activated protein kinase signaling pathway. In addition, following leukocyte engagement, E-selectin associates via its cytoplasmic domain with components of the actin cytoskeleton and undergoes alterations in phosphorylation state that result in changes in gene expression. In this study, we show that E-selectin is localized in cholesterol-rich lipid rafts at the cell surface, and that upon ligation E-selectin clusters and redistributes in the plasma membrane colocalizing with a fraction of caveolin-1-containing rafts. In addition, we demonstrate that leukocyte adhesion via E-selectin results in association with and activation of phospholipase Cγ (PLCγ). Moreover, we show that disruption of lipid rafts with the cholesterol-depleting drug methyl-β-cyclodextrin disrupts the raft localization of E-selectin as well as the ligation-induced association of E-selectin with PLCγ, and subsequent tyrosine phosphorylation of PLCγ. In contrast, cholesterol depletion has no effect on E-selectin-dependent mitogen-activated protein kinase activation. Thus, these findings demonstrate that the presence of E-selectin in lipid rafts is necessary for its association with, and activation of, PLCγ, and suggest that this subcellular localization of E-selectin is related to its signaling function(s) during leukocyte-endothelial interactions.

Molecular events in transmembrane signaling via E-selectin. SHP2 association, adaptor protein complex formation and ERK1/2 activation.

E-selectin is a cytokine-inducible adhesion molecule that is expressed by activated endothelial cells at sites of inflammation. In addition to supporting rolling and stable arrest of leukocytes, there is increasing evidence that E-selectin functions in transmembrane signaling into endothelial cells during these adhesive interactions. We have previously shown that adhesion of HL-60 cells (which express ligands for E-selectin), or antibody-mediated cross-linking of E-selectin, results in formation of a Ras/Raf-1/phospho-MEK macrocomplex, extracellular signal-regulated protein kinase (ERK1/2) activation, and c-fosup-regulation. All of these downstream signaling events appear to require an intact cytoplasmic domain of E-selectin. Here we demonstrate that tyrosine 603 in the cytoplasmic domain of E-selectin is required for the E-selectin-dependent ERK1/2 activation. Tyrosine 603 plays an important role in mediating the association of E-selectin with SHP2, and the catalytic domain of SHP2 is, in turn, critical for E-selectin-dependent ERK1/2 activation. An adapter protein complex consisting of Shc·Grb2·Sos bridges between SHP2 and the Ras·Raf·phospho-MEK macrocomplex. These molecular events thus outline a mechanism by which cross-linking of E-selectin by engagement of ligands on adherent leukocytes can initiate a multifunctional signaling pathway in the activated endothelial cell at sites of inflammation.

Immunoselective Targeting of an Anti-Thrombin Agent to the Surface of Cytokine-Activated Vascular Endothelial Cells

An immunoconjugate was designed to target hirudin, a potent and specific inhibitor of thrombin, to the surface of activated endothelial cells. Hirudin was covalently cross-linked to the monoclonal antibody H18/7 that recognizes the extracellular domain of E-selectin (CD62E), an endothelium-leukocyte adhesion molecule that is expressed only on cytokine-activated endothelium. The hirudin-H18/7 immunoconjugate selectively bound to interleukin-1-activated but not to unactivated cultured human umbilical vein endothelial cells with a temporal profile similar to that of inducible cell-surface procoagulant activity. When bound to activated endothelial cells, the hirudin-H18/7 immunoconjugate significantly inhibited endogenous thrombin activity generated from coincubated human plasma and fibrin clot formation on the monolayer surface. Cellular responses that are mediated via the thrombin receptor, such as increases in cytoskeletal F-actin content, also were significantly downregulated, and monolayers were protected from thrombin-induced disruption by this treatment. The ability to selectively antagonize thrombin-dependent processes at the endothelium-blood interface may provide new insights into complex pathophysiological processes, such as thrombosis, inflammation, and atherogenesis. These studies also demonstrate the general feasibility of selective targeting of therapeutic agents to endothelial cells based on recognition of an activation-dependent surface phenotype.

The Regulatory Role of Macrophages in Infection with Intracellular Pathogens

We summarize here published studies from our laboratories that deal with the analysis of antigen presentation and the regulation of this function as it pertains to the macrophage. Our published studies contain a review of the literature plus a more extensive discussion of the various issues reviewed here. Many of our studies on antigen presentation have included as antigen the intracellular pathogenic bacteria Listeria monocytogenes in the mouse. By analyzing the cellular immune reaction to this pathogen we have been able to define antigen processing, as well as the regulation of the expression of the class II histocompatibility molecules (the Ia molecules) and interleukin-1 (IL-1). More recently, we have analyzed some of the elements of nonspecific immunity by studying listeriosis in mice with the severe combined immunodeficiency (SCID) mutation.