Thomas M. McIntyre

Endothelial cell interactions with granulocytes: tethering and signaling molecules

The adhesion of granulocytes to endothelial cells requires regulated expression of molecules on both the endothelial cell and the granulocyte. These pro-adhesive molecules have diverse structures and mechanisms of expression, and act either to tether the two cells together or as signals that induce activation-dependent adhesion events. Combinations of tethering and signaling molecules regulate endothelial-cell-granulocyte interactions at the endothelial surface.

Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARγ agonist

Lysophosphatidic acid (LPA) is a pluripotent lipid mediator acting through plasma membrane-associated LPAx receptors that transduce many, but not all, of its effects. We identify peroxisome proliferator-activated receptor γ (PPARγ) as an intracellular receptor for LPA. The transcription factor PPARγ is activated by several lipid ligands, but agonists derived from physiologic signaling pathways are unknown. We show that LPA, but not its precursor phosphatidic acid, displaces the drug rosiglitazone from the ligand-binding pocket of PPARγ. LPA and novel LPA analogs we made stimulated expression of a PPAR-responsive element reporter and the endogenous PPARγ-controlled gene CD36, and induced monocyte lipid accumulation from oxidized low-density lipoprotein via the CD36 scavenger receptor. The synthetic LPA analogs were effective PPARγ agonists, but were poor ones for LPA1, LPA2, or LPA3 receptor transfected cells. Transfection studies in yeast, which lack nuclear hormone and LPAx receptors, show that LPA directly activates PPARγ. A major growth factor of serum is LPA generated by thrombin-activated platelets, and media from activated platelets stimulated PPARγ function in transfected RAW264.7 macrophages. This function was suppressed by ectopic LPA-acyltransferase expression. LPA is a physiologic PPARγ ligand, placing PPARγ in a signaling pathway, and PPARγ is the first intracellular receptor identified for LPA. Moreover, LPA produced by stimulated plasma platelets activates PPARγ in nucleated cells.

Activated platelets mediate inflammatory signaling by regulated interleukin 1β synthesis

Platelets release preformed mediators and generate eicosanoids that regulate acute hemostasis and inflammation, but these anucleate cytoplasts are not thought to synthesize proteins or cytokines, or to influence inflammatory responses over time. Interrogation of an arrayed cDNA library demonstrated that quiescent platelets contain many messenger RNAs, one of which codes for interleukin 1β precursor (pro–IL-1β). Unexpectedly, the mRNA for IL-1β and many other transcripts are constitutively present in polysomes, providing a mechanism for rapid synthesis. Platelet activation induces rapid and sustained synthesis of pro–IL-1β protein, a response that is abolished by translational inhibitors. A portion of the IL-1β is shed in its mature form in membrane microvesicles, and induces adhesiveness of human endothelial cells for neutrophils. Signal-dependent synthesis of an active cytokine over several hours indicates that platelets may have previously unrecognized roles in inflammation and vascular injury. Inhibition of β3 integrin engagement markedly attenuated the synthesis of IL-1β, identifying a new link between the coagulation and inflammatory cascades, and suggesting that antithrombotic therapies may also have novel antiinflammatory effects.

Escaping the Nuclear Confines: Signal-Dependent Pre-mRNA Splicing in Anucleate Platelets

Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.

Monocyte tethering by P-selectin regulates monocyte chemotactic protein-1 and tumor necrosis factor-alpha secretion. Signal integration and NF-kappa B translocation.

Adhesion molecules that tether circulating leukocytes to endothelial cells may also transduce or modulate outside-in signals for cellular activation, providing an initial regulatory point in the inflammatory response. Adhesion of human monocytes to P-selectin, the most rapidly expressed endothelial tethering factor, increased the secretion of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-alpha (TNF-alpha) by the leukocytes when they were stimulated with platelet-activating factor. Increased cytokine secretion was specifically inhibited by G1, an anti-P-selectin mAb that prevents P-selectin from binding to its ligand (P-selectin glycoprotein ligand-1) on myeloid cells. Moreover, tethering by P-selectin specifically enhanced nuclear translocation of nuclear factor-kappa B (NF-kappa B), a transcription factor required for expression of MCP-1, TNF-alpha, and other immediate-early genes. These results demonstrate that P-selectin, through its ligands on monocytes, may locally regulate cytokine secretion in inflamed tissues.

The Leukocyte Integrins

Integrins on Leukocytes Leukocytes are marrow-derived cells of diverse form and function that circulate in the blood in a quiescent state of low adhesiveness before migrating into tissues to defend against invading microbes, participate in immune functions and wound repair, or become fixed extracellular residents. Some, such as T-lymphocytes, recirculate and traverse blood, organ, and lymphatic compartments during long cycles of immune surveillance. Others, notably polymorphonuclear leukocytes (PMNs, neutrophils), are rapid response cells specialized for acute spatially targeted defensive actions that can be mounted in minutes. Leukocytes are also effectors of pathologic inflammation when their accumulation and actions are disregulated. Integrins on their surfaces, together with other plasma membrane adhesion molecules, are required for interactions of leukocytes with endothelial cells and other cell types and with matrix structures (1). The functional state, density, and topography of integrins on leukocytes are regulated by lipid, cytokine, and chemokine signaling molecules and by “cross-talk” from other surface adhesion molecules (1–6). Each class of leukocytes displays a particular pattern of integrins that can change in a signaland time-dependent fashion. For example, resting human T lymphocytes (T cells) express b1, b2, and b7 integrins, but this varies with the subclass and is altered by immune stimulation (5). Freshly isolated human monocytes express b1 and b2 integrins, but their culture and/or differentiation into macrophages changes the pattern and induces avb3 (5, 7). Human PMNs, once thought to express only b2 integrins, display b1 and b3 heterodimers and use them in motility and migration (8– 12). A common feature, however, is that each leukocyte subtype expresses one or more members of the b2 integrin family. Further, the b2 heterodimers are restricted to cells of the leukocyte lineage. The remainder of this minireview will focus on the structure and function of the b2 or “leukocyte” integrins, which were among the first adhesion molecules to be studied at the molecular level. The most recently identified member of the subfamily, aDb2, 3 is still being characterized (13–16).

Peroxisome Proliferators Enhance Cyclooxygenase-2 Expression in Epithelial Cells

The formation of prostaglandins requires the catalytic activity of cyclooxygenase (COX) which converts arachidonic acid to the prostaglandin endoperoxide PGH2, from which all other prostaglandins are formed. COX-2 is the highly inducible isozyme of COX which is responsible for much of the prostaglandin production in inflammation and is a key factor in colon carcinogenesis. Because COX-2 activity can be rate-limiting in prostaglandin formation, COX-2 expression must be regulated tightly. Numerous factors, including mitogens, tumor promoters, and cytokines have been found to stimulate the transcription of COX-2. We show that fatty acids, prostaglandins, and non-steroidal anti-inflammatory drugs, compounds that are substrates, products, and inhibitors, respectively, of COX enzymatic activity, also increase its expression. These compounds are members of a heterogeneous group of compounds known as peroxisome proliferators, and the prototypical peroxisome proliferator, WY-14,643, also enhanced COX-2 expression. We demonstrate that these compounds increase COX-2 transcription, and we identify a region of the COX-2 promoter containing a peroxisome proliferator response element that is responsible for the enhancement of COX-2 expression seen with these compounds.

Inflammatory roles of P-selectin.

Polymorphonuclear leukocytes (PMNs) bind rapidly and reversibly to endothelial cells induced to express P-selectin, a glycoprotein that mediates adhesive intercellular interactions. In addition, PMNs adherent to endothelium expressing P-selectin demonstrate an intracellular Ca2+ transient, functionally up-regulate beta-2-integrins (CD11/CD18 glycoproteins), become polarized in shape, and are primed for enhanced degranulation when subsequently stimulated with chemotactic factors. However, P-selectin induces none of these responses directly when used alone, when incorporated into model membranes, or when expressed by transfected cells. The absence of direct activation of the PMNs is not due to competing antiinflammatory effects of P-selectin; instead, purified P-selectin and P-selectin in membranes support agonist-stimulated PMN responses. Furthermore, tethering of PMNs to endothelial surfaces by P-selectin is required for priming to occur efficiently, as shown by experiments with blocking monoclonal antibodies. The priming event is directly mediated by the signaling molecule, platelet-activating factor (PAF), and is inhibited by blocking the PAF receptor on PMNs. Thus, P-selectin and PAF are components of an adhesion and activation cascade, but have distinct roles: P-selectin tethers and captures the PMN, whereas PAF mediates juxtacrine activation. In vivo, selectins may facilitate interaction of target cells with membrane-bound molecules that send intercellular signals, in addition to mediating rolling of leukocytes and other adhesive functions.

The platelet-activating factor signaling system and its regulators in syndromes of inflammation and thrombosis.

ObjectivesTo review the platelet-activating factor (PAF) signaling system, its regulation, and its dysregulation in acute inflammation and thrombosis and in syndromes that involve these cascades, including sepsis. Data SourcesA summary of published literature from MEDLINE search files and published reviews. Data Extraction, Synthesis, and SummaryPAF, a phospholipid signaling molecule, transmits outside-in signals to intracellular transduction systems and effector mechanisms in a variety of cell types, including key cells of the innate immune and hemostatic systems: neutrophils, monocytes, and platelets. Thus, the PAF signaling system is a point of convergence at which injurious stimuli can trigger and amplify both acute inflammatory and thrombotic cascades. The biological activities of PAF are regulated by several precise mechanisms that, together, constrain and control its action in physiologic inflammation. Unregulated synthesis of PAF or defects in the mechanisms that limit its biological activities have the potential to cause pathologic inflammation and thrombosis. In addition, nonenzymatic generation of oxidized phospholipids that are recognized by the PAF receptor can trigger inflammatory and thrombotic events. There is evidence that the PAF signaling system is dysregulated in sepsis, shock, and traumatic injury and that interruption or termination of its effector responses leads to beneficial outcomes. Plasma PAF acetylhydrolase, an enzyme that hydrolyzes PAF and structurally related oxidized phospholipids, yielding products that are no longer recognized by the PAF receptor, may be a particularly important signal terminator. ConclusionThe PAF signaling system can trigger inflammatory and thrombotic cascades, amplify these cascades when acting with other mediators, and mediate molecular and cellular interactions (cross talk) between inflammation and thrombosis. Evidence from in vitro experiments, studies of experimental animals, and clinical observations in humans indicates that the PAF signaling system is important in sepsis and other syndromes of inflammatory injury and that therapeutic strategies to interrupt or terminate signaling via the PAF signaling system may be useful in these conditions.

Hepatic Lipid Partitioning and Liver Damage in Nonalcoholic Fatty Liver Disease ROLE OF STEAROYL-CoA DESATURASE

Hepatic lipid overloading mainly in the form of triglycerides is considered a prerequisite for the development of nonalcoholic fatty liver disease (NAFLD). However, triglyceride accumulation in the liver in response to lipid overflow may represent a protective mechanism against lipotoxicity. Our aims were to assess the fundamental cellular mechanisms that link lipid compartmentation in hepatocytes to liver damage and disease progression in NAFLD by using both in vivo dietary models of NAFLD and in vitro cell models of lipid overloading. Exposure of murine or human hepatocytes to monounsaturated fatty acids (MUFAs) resulted in lipid accumulation without changes in cell viability. In contrast, cell incubation with saturated fatty acids (SFAs) significantly decreased cell viability and increased caspase activation and apoptosis, with only minor lipid droplet accumulation. Genetic or pharmacological inhibition of stearoyl-CoA desaturase-1 (SCD1), the enzyme that converts SFA to MUFA, sensitized cells to SFA-induced apoptosis. Hepatic SCD1 expression increased in experimental steatosis resulting from high fat diet and decreased in a methionine-choline-deficient (MCD) dietary model of steatohepatitis resulting in the latter situation in significantly increased hepatic SFA levels. SCD1–/– mice on the MCD diet had decreased steatosis and markedly increased hepatocellular apoptosis, liver injury, and fibrosis compared with the SCD1+/+, whereas MUFA feeding prevents the MCD-induced injury. In conclusion, this study suggests hepatic SCD1 plays a key role in prevention of steatohepatitis by partitioning excess lipid into MUFA that can be safely stored. This concept has important implications for the development of novel treatment strategies for patients with this condition.