We have identified two compounds that inhibit the expression of endothelial-leukocyte adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. These compounds act by inhibiting tumor necrosis factor-␣-induced phosphorylation of IB-␣, resulting in decreased nuclear factor-B and decreased expression of adhesion molecules. The effects on both IB-␣ phosphorylation and surface expression of E-selectin were irreversible and occurred at an IC 50 of approximately 10 M. These agents selectively and irreversibly inhibited the tumor necrosis factor-␣-inducible phosphorylation of IB-␣ without affecting the constitutive IB-␣ phosphorylation. Although these compounds exhibited other activities, including stimulation of the stress-activated protein kinases, p38 and JNK-1, and activation of tyrosine phosphorylation of a 130 -140-kDa protein, these effects are probably distinct from the effects on adhesion molecule expression since they were reversible. One compound was evaluated in vivo and shown to be a potent anti-inflammatory drug in two animal models of inflammation. The compound reduced edema formation in a dose-dependent manner in the rat carrageenan paw edema assay and reduced paw swelling in a rat adjuvant arthritis model. These studies suggest that inhibitors of cytokine-inducible IB␣ phosphorylation exert anti-inflammatory activity in vivo.The adhesion of circulating leukocytes to vascular endothelium is critical to inflammatory responses (reviewed in Refs. 1-3). Interaction of the selectin family of adhesion proteins and lectin counter-receptors is the predominant mechanism mediating initial adhesion between leukocytes and the vessel wall. The expression of endothelial-leukocyte adhesion molecule-1 (E-selectin, CD62E), vascular cell adhesion molecule-1 (VCAM-1, 1 CD106), and intercellular adhesion molecule-1 (ICAM-1, CD54) on the surface of endothelial cells is elevated at sites of inflammation (2, 4). Induction of these molecules by tumor necrosis factor-␣ (TNF␣) and other inflammatory cytokines is regulated at the level of gene transcription and requires binding of the transcription factor nuclear factor-B (NF-B) to the regulatory regions within the promoters of each of these genes (5-12).The NF-B/Rel transcription factor family plays an important role in cytokine-induced gene activation (13-15). The Rel family includes p50 (NFKB1), p52 (NFKB2), p65 (RelA), RelB, v-Rel, and c-Rel. In endothelial cells, the p50⅐p65 heterodimer is the predominant species that binds to B consensus sequences in the VCAM-1, ICAM-1, and E-selectin genes and activates gene transcription. NF-B is located in the cytoplasm of cells in an inactive form in association with the inhibitor IB-␣. In response to TNF␣ stimulation, IB-␣ is phosphorylated on 2 serine residues (Ser-32 and Ser-36), ubiquitinated, and degraded by a proteosome-dependent pathway allowing active NF-B to translocate to the nucleus where it can activate gene expression (16 -23). Many NF-B-dependent genes including the adhesion m...

We studied the effects of intravenous infusion of recombinant human tumor necrosis factor type a (rTNF-a;12 pg/kg) on lung fluid balance in sheep prepared with chronic lung lymph fistulas. Recent studies have proposed TNF-a as a mediator of endotoxemia (5) since TNF-a administration mimics the clinical course of endotoxemia (6, 7). Passive immunization against TNF-a reduces the mortality associated with endotoxin challenge (8, 9). Pathologic changes following TNF-a infusion include pulmonary inflammation and hemorrhage, with microaggregates of leukocytes and margination of polymorphonuclear leukocytes (neutrophils; PMNs) in the pulmonary microcirculation (7). Endothelial cells exposed to TNF-a develop cytoskeletal changes (10), procoagulant activity (11,12), and increased adhesiveness to lymphocytes (13) and PMNs (14). TNF-a also causes endothelial cells to release interleukin 1 (15) and platelet-activating factor (16). PMNs stimulated with TNF-a display enhanced adherence (14, 17), migration (18), phagocytic and cytotoxic activities (19), respiratory burst activity (20), and degranulation (20). These effects of TNF-a on endothelial cells and on PMNs suggest that TNF-a is involved in mediating the increase in lung vascular permeability associated with endotoxemia. In the present study, we examined whether recombinant TNF-a (rTNF-a) affected pulmonary vascular permeability by assessing alterations in pulmonary lymph flow in unanesthetized sheep following rTNF-a challenge and by determining transendothelial permeability in rTNF-a-treated endothelial monolayers. We also examined the role of PMNs in the response. MATERIALS AND METHODSChronic lymph fistulas were implanted in male sheep (n = 11) weighing -25 kg (21). This procedure allows collection of blood-free pulmonary lymph after a 3-or 4-day recovery period. Hydroxyurea (HDY; Squibb) was used to deplete circulating neutrophils. HDY (5 g) was dissolved in 200 ml of O.9%o NaCl and the pH was corrected to 7.4; this solution was administered i.v. once daily for 4 or 5 days (200 mg/kg per day). Daily antibiotic treatment was continued in these animals (5 ml i.m.; Ambipen; Butler, Columbus, OH). Animals were studied within 2 days of the last HDY treatment.Awake sheep were studied while standing in a metabolic cage with free access to food and water. A 7.5-F thermodilution catheter (American Edwards Laboratories, Santa Ana, CA) was positioned in the pulmonary artery. Pulmonary artery (Pp) and pulmonary artery wedge (Ppw) pressures were determined with Statham P23Db transducers (Statham, Hato Rey, PR). Pulmonary blood flow (QL) was determined by a thermodilution technique (Cardiac Output Computer, model 9520, American Edwards Laboratories). Pulmonary vascular resistance (PVR) was calculated as (Pp -PPw)/QL. Pulmonary lymph flow (Qlym) (a measure of net pulmonary transvascular fluid filtration rate) was determined by measuring the volume of lymph catheter effluent collected over 15-or 30-min periods. Lymph fluid was collected in plastic tubes containing 100 mg of...

All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a directacting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages. (Blood.