Guy A. Zimmerman

The Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome (ARDS) is an important cause of acute respiratory failure that is often associated with multiple organ failure. Several clinical disorders can precipitate ARDS, including pneumonia, sepsis, aspiration of gastric contents, and major trauma. Physiologically, ARDS is characterized by increased permeability pulmonary edema, severe arterial hypoxemia, and impaired carbon dioxide excretion. Based on both experimental and clinical studies, progress has been made in understanding the mechanisms responsible for the pathogenesis and the resolution of lung injury, including the contribution of environmental and genetic factors. Improved survival has been achieved with the use of lung-protective ventilation. Future progress will depend on developing novel therapeutics that can facilitate and enhance lung repair.

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.

Hydrogen peroxide stimulates the synthesis of platelet-activating factor by endothelium and induces endothelial cell-dependent neutrophil adhesion.

Oxidant-induced damage to the intima of pulmonary and systemic vessels is thought to be an important mechanism of injury in a variety of syndromes of vascular damage. Hydrogen peroxide (H2O2) is an active oxygen metabolite that may induce intimal injury by cytolytic attack or by inducing biochemical and functional alterations in the endothelial cells (EC); however, mechanisms involved in noncytolytic perturbation of EC are largely unknown. We found that H2O2 stimulated the synthesis of platelet-activating factor (PAF) by primary cultures of bovine pulmonary artery endothelium (BPAEC) and by human umbilical vein endothelium (HUVEC). In each cell type the incorporation of [3H]acetate into [3H-acetyl]PAF was concentration- and time-dependent and was temporally dissociated from severe plasma membrane disruption and cytolytic cell injury; the newly synthesized PAF remained associated with the EC. H2O2 caused permeabilization of EC to 45Ca2+ and an increase in intracellular Ca2+, suggesting that a transmembrane Ca2+ flux is the signal that initiates PAF synthesis. H2O2 also induced the endothelial cell-dependent adhesion of neutrophils to HUVEC monolayers. This response was rapid, with an onset within minutes and a subsequent time course that paralleled the time course of PAF accumulation, and was dependent on extracellular Ca2+ but not on de novo protein synthesis. These studies demonstrate that H2O2 can induce two rapid activation responses of endothelium, PAF synthesis and EC-dependent neutrophil adhesion, events that may be important in physiologic and pathologic inflammation.

Cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate.

Cultured human endothelial cells synthesize prostacyclin (PGI2), a potent inhibitor of platelet function, when stimulated with histamine, bradykinin, or ATP. Paradoxically, we report that these agonists also induced the rapid and sustained synthesis of platelet-activating factor (PAF) by endothelial cells. In fact, the synthesis of this potent activator of platelets and neutrophils was induced by stimulation of the same receptor subtype that induced PGI2 synthesis: stimulation of a histamine H1 or a bradykinin B2 receptor induced both PAF and PGI2 synthesis. However, two physiologically important differences exist between the production of PAF and PGI2 by endothelial cells. The synthesis of PGI2 proceeded for only 7.5 min before the abrupt termination of synthesis, whereas the synthesis of PAF was clearly detectable even 45 min after stimulation. Although maximal accumulation of PAF occurred after 10-15 min of stimulation, the prolonged synthesis resulted in the presence of PAF for up to 1 h after stimulation. Secondly, whereas PGI2 was released from the cell monolayer, PAF remained cell-associated without significant release to the external medium. Endothelial cell-generated PAF, therefore, does not function as a hormone. The prolonged association of this potent activator of platelets and neutrophils with endothelial cells may mediate some of the inflammatory properties of histamine and bradykinin. It may also be a factor in the formation of a thrombogenic vascular surface, an event suggested to play a primary role in the pathogenesis of thrombosis and atherosclerosis.

Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro.

Highly purified human thrombin stimulates the adherence of polymorphonuclear leukocytes (PMNs) to vascular endothelial cells (EC). When Indium-labeled PMNs were incubated with primary monolayers of cultured human umbilical vein EC, the basal adherence was 10 +/- 1% of the PMNs at 5 min. Addition of thrombin (2 U/ml) increased the mean adherence to 42 +/- 15%. Enhanced neutrophil adherence in response to thrombin was confirmed by experiments with unlabeled leukocytes, examined by phase contrast and scanning electron microscopy. The action of thrombin was on the EC, since it did not directly stimulate PMN adhesiveness when measured by aggregation or by adherence to nylon fiber columns. Furthermore, enhanced neutrophil adherence occurred when endothelial monolayers were treated with thrombin and washed before adding 111Indium (111In)-labeled PMNs. Thrombin that had been inactivated with antithrombin III and heparin did not enhance neutrophil adherence. Prothrombin, Factor Xa, and fibrinogen were also ineffective. The stimulated adherence of PMNs was maximal 5 min after incubation of the EC with thrombin, and decreased thereafter. The response was dose-dependent, with half-maximal stimulation at 0.2-0.25 U thrombin/ml. The enhanced PMN adherence caused by thrombin may result in part from the production of platelet-activating factor (PAF) by the stimulated EC since thrombin-stimulated EC synthesize PAF with a time course and concentration dependence that are similar to the time and concentration relationships for thrombin-stimulated PMN adherence, PAF itself promoted neutrophil adherence to the EC monolayers, and pretreatment of PMNs with PAF decreased the adherence stimulated by thrombin and PAF, but not adherence stimulated by N-formylmethionyl-leucyl-phenylalanine and C5a fragments, which indicates specific desensitization of PAF-mediated adherence. These studies demonstrate the endothelial cell-dependent stimulation of PMN adherence by thrombin, a novel mechanism of enhanced leukocyte adherence that may be important in interactions between the coagulation and inflammatory systems.

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.

Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells.

Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.