Adhesion-dependent modulation of macrophage K+ channels.

Integrin-mediated adhesion of monocytes not only triggers cell rolling and diapedesis, it also activates ionic permeability changes resulting in monocyte activation, maturation and differentiation. Mononuclear phagocytes possess voltage-dependent inwardly rectifying K+ (Kir) currents and delayed outwardly, rectifying K+ (Kdr) currents that are modulated by tissue origin, adherence, presence of growth factors or cytokines and the functional or differentiation state of the cells. This chapter reviews the exploration of Kir and Kdr channels in mononuclear phagocytes over the last 30 years with an emphasis on culturing conditions, modulation by substrates and role in macrophage function. It has only been recent that successful attempts have been made to study these K+ currents in monocytes/macrophages as they may be engaged in the human body which may serve as the foundation for the development of novel therapeutic agents targeting macrophage Kir/Kdr channel activity to favorably influence risk factors for hypertension, atherosclerosis and diabetes.

Multinucleate host cells induced by Vittaforma corneae (Microsporidia).

The microsporidium Vittaforma corneae develops within the target cell cytoplasm. In the present study, green monkey kidney (E6) cells infected at 30 degrees C, 35 degrees C or 37 degrees C with V. corneae developed enlarged multinucleate structures of up to 200 microm in any horizontal dimension made up either of a single cell or of multiple fused cells. A number of epithelial cell types (SW-480, HT-29, Caco-2 and HCT-8) were infected with V. corneae but did not induce the same highly organized structures, suggesting that for the structure to develop, the host cell must be capable of continued mitosis, and not be differentiated or be detaching from the surface matrix. Live cell imaging of infected E6 cells revealed large, multinucleate infected cells characterized by a central focus from which radiated parasite stages and host cell mitochondria. Immunocytochemistry identifying gamma and alpha tubulin suggested that a single centrally-located microtubule organizing centre governed the distribution of parasite stages and host cell organelles, with mitochondria and parasites being eventually transported towards the periphery of the structure. Whole cell patch clamp analysis of infected cells indicated an average five-fold increase in total membrane capacitance, consistent with an enlarged single cell. Scanning electron microscopy revealed cell-like protrusions around the periphery of the structure with the intervening space being made up of parasites and cell debris. Clearly in the case of V. corneae-infected E6 cells the parasite-host cell relationship involves subverting the host cell cytoskeleton and cell volume control, providing the parasite with the same protected niche as does a xenoma.

Clustering of very late antigen-4 integrins modulates K(+) currents to alter Ca(2+)-mediated monocyte function.

Endothelial cell vascular cell adhesion molecule-1 (VCAM-1) activates adherent monocytes by clustering their very late antigen-4 (VLA-4) receptors, resulting in the modulation of the inwardly rectifying (I(ir)) and delayed rectifying (I(dr)) K(+) currents, hyperpolarization of the cells, and enhanced Ca(2+) influx (Colden-Stanfield M and Gallin EK. Am J Physiol Cell Physiol 275: C267-C277, 1998; Colden-Stanfield M and Scanlon M. Am J Physiol Cell Physiol 279: C488-C494, 2000). The present study was undertaken to test the hypothesis that monoclonal antibodies (MAbs) against VLA-4 (MAbVLA-4) mimic VCAM-1 to cluster VLA-4 integrins, which play a key role in signaling an increase in the secretion of the proinflammatory cytokine interleukin-8 (IL-8). Whole cell ionic currents and IL-8 secretion from THP-1 monocytes that were incubated on polystyrene, VCAM-1-immobilized MAbVLA-4 or an isotype-matched MAb against CD45 (MAbCD45) were measured. Clustering of VLA-4 integrins with a cross-linked MAbVLA-4, but not a monovalent MAbVLA-4, modulated the K(+) currents in an identical manner to incubation of cells on VCAM-1. Similarly, cross-linked MAbVLA-4 or VCAM-1 augmented Ca(2+)-mediated IL-8 secretion from THP-1 monocytes and was completely abolished by exposure to CsCl, an I(ir) blocker. Thus VLA-4 integrin clustering by cross-linked MAbVLA-4 mimics VCAM-1/VLA-4 interactions sufficiently to be associated with events leading to monocyte differentiation, enhanced Ca(2+)-mediated macrophage function, and possibly atherosclerotic plaque formation.

Antiphospholipid antibodies induced in mice by immunization with a cytomegalovirus-derived peptide cause thrombosis and activation of endothelial cells in vivo.

OBJECTIVE: To characterize the binding and functional properties of antiphospholipid antibodies (aPL) induced by immunization with a viral peptide and to determine whether aPL are pathogenic in vivo. METHODS: Ten murine monoclonal aPL were generated from spleen cells of PL/J mice immunized with TIFI, a phospholipid-binding peptide spanning Thr(101)-Thr(120) of ULB0-HCMVA from human cytomegalovirus (CMV), which shares structural similarity with the phospholipid-binding site of beta(2)-glycoprotein I (beta(2)GPI). RESULTS: The antibodies generated had aPL activity that was inhibited by cardiolipin liposomes, and this inhibition was enhanced in the presence of beta(2)GPI. Some of the antibodies exhibited binding to cultured endothelial cells in vitro, and some had lupus anticoagulant activity. Injection with 2 of the monoclonal aPL in mice resulted in a significant increase in the number of leukocytes adhering to endothelial cells and enhanced thrombus formation in vivo. CONCLUSION: These results indicate that aPL induced by immunization with a phospholipid-binding CMV peptide are pathogenic in vivo. The results also suggest a mechanism (molecular mimicry) by which pathogenic aPL may be generated in patients with antiphospholipid syndrome.