Phagocytic clearance of apoptotic cells plays a pivotal role in the resolution of inflammation. Recent evidence has shown that such processes can be regulated by endogenous mediators, suggesting that specific mimetics may have therapeutic potential in chronic inflammation and autoimmune disorders. Here we review the mechanisms underlying recognition and engulfment of apoptotic cells and regulation of these processes by lipoxins and lipoxin receptor agonists.

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac(2-26)) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mphi). Peptide Ac(2-26)-stimulated phagocytosis is accompanied by rearrangement of the Mphi actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mphi were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mphi significantly enhanced the ability of naive Mphi to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mphi from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.

Lipoxins are lipoxygenase-derived lipid mediators with both anti-inflammatory and proresolution properties that have been demonstrated in vivo and in vitro. The bioactivity profile of lipoxins in vitro suggests that they have therapeutic potential in acute renal failure and glomerulonephritis; predictions that have been borne out to date in experimental models of renal disease. We review recent developments on the molecular basis of lipoxin bioactions mediated through receptor crosstalk and the accumulating evidence that lipoxins may have potential as novel anti-inflammatory agents.

OBJECTIVE:Tissue factor (TF) is the main activator of the coagulation cascade occurring in physiologic and pathologic conditions. Recent data suggest that human platelets might contain TF that is possibly derived from leukocytes. In this study, we investigated whether intraplatelet TF can be exposed on the membrane by platelet agonists. The modulation of this process by antiplatelet drugs has been evaluated as well.METHODS AND RESULTS:Flow cytometric analysis of unstimulated platelets showed a small amount of membrane-associated immunoreactive TF (irTF) in whole blood, platelet-rich plasma, and washed platelets isolated from healthy subjects. ADP, thrombin receptor-activating peptide, and epinephrine significantly increased functionally active, membrane-associated irTF. ADP induced irTF exposure in a concentration- and time-dependent fashion. Agonist-induced irTF expression was completely inhibited by iloprost but not by aspirin. Interestingly, glycoprotein IIb/IIIa antagonists did not inhibit but rather potentiated the stimulatory effect of ADP on platelet irTF expression. Real-time polymerase chain reaction experiments showed detectable amounts of TF mRNA in unstimulated platelets.CONCLUSIONS:These findings indicate that platelet agonists and antiplatelet drugs might modulate platelet-associated irTF expression. Regulated TF expression establishes the potential for a previously unrecognized role for platelets in sustaining thrombus formation and growth via coagulation-mediated mechanisms.

Clearance of apoptotic cells by phagocytic cells plays a significant role in the resolution of inflammation, protecting tissue from harmful exposure to the inflammatory and immunogenic contents of dying cells. Apoptosis induces cell surface changes that are important for recognition and engulfment of cells by phagocytes. These changes include alterations in surface sugars, externalization of phosphatidylserine and qualitative changes in the adhesion molecule ICAM-3. Several studies have contributed to clarify the role of the receptors on the surface of phagocytes that are involved in apoptotic cell clearance. The phagocytic removal of apoptotic cells does not elicit pro-inflammatory responses; in contrast, apoptotic cell engulfment appears to activate signals that suppress release of pro-inflammatory cytokines. Therefore, clearance of apoptotic leucocytes is implicated in the resolution of inflammation and mounting evidence suggests that defective clearance of apoptotic cells contributes to inflammatory and autoimmune diseases. Defining the ligands on apoptotic cells and the corresponding receptors on phagocytes with which they engage, is likely to lead to the development of novel anti-inflammatory pro-resolution drugs. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. We will also discuss what is currently known about diseases that may reflect impaired phagocytosis and the consequences on inflammation and immune responses.

Lipoxins (LXs) are endogenously produced eicosanoids that inhibit neutrophil trafficking and stimulate nonphlogistic phagocytosis of apoptotic neutrophils by monocyte-derived macrophages. in this study we assessed the effect of LXs on cell ultrastructure and actin reorganization in human leukocytes and investigated the signaling events that subserve LX bioactivity in this context. LXA(4) (10(-9) mol/L), the stable synthetic analogues 15-(R/S)-methyl-LXA(4) and 16-phenoxy-LXA(4) (10(-11) mol/L), but not the LX precursor 15-(S)-HETE, induced marked changes in ultrastructure and rearrangement of actin in monocytes and macrophages. in contrast, LXA(4) did not modify actin distribution in neutrophils under basal conditions and after stimulation with leukotriene B-4. Blockade of Rho kinases by the inhibitor Y-27632 prevented LYA(4)-triggered actin reorganization in macrophages. To investigate the role of the specific small GTPases in LX-induced actin rearrangement we used THP-1 cells differentiated to a macrophage-like phenotype. THP-1 cells stimulated with LXs, but not with 15-(S)-HETE, showed an increase in membrane-associated RhoA and Rac by immunoblotting. Additionally, a twofold increase in Rho activity was seen in response to LXA(4). LX-induced actin rearrangement and RhoA activation were inhibited by the cell permeable cAMP analogue 8-Br-cAMP, whereas Rp-cAMP, an inhibitor of protein kinase A, mimicked the effect of LXA(4). These data demonstrate that LXs stimulate RhoA- and Rac-dependent cytoskeleton reorganization, contributing to the potential role of LXs in the resolution of inflammation.

Lipoxins (LX) are eicosanoids generated via transcellular biosynthetic routes during inflammation, hypersensitivity reaction, and after angioplasty. LXs are modulators of leukocyte trafficking and vascular tone. Their influence on the coagulation cascade has not been determined. In this study, we evaluated the influence of LXs on the expression of tissue factor (TF), a key regulator of coagulation. TF activity was measured in lysates of monocytes, human umbilical vein endothelial cells, and ECV304 cells using a one-stage clotting assay. LXA(4) stimulated TF activity in each cell type. The influence of LXA(4) on TF activity by ECV304 cells was studied further to explore the mechanism of induction of TF expression. LXA(4)-induced TF activity was dose dependent, cycloheximide sensitive, and associated with increased TF mRNA levels. Induction of TF activity was specific for LXA(4) and was not observed with LXB(4), the other major lipoxin generated by mammalian cells. Furthermore, ECV304 cell TF expression was not influenced by 15(R/S)-methyl-LXA(4) or 16-phenoxy-LXA(4), synthetic analogs of LXA(4) that activate the myeloid LXA(4) receptor, and was not modulated by SKF-104353, which blocks LXA(4) bioactivities transduced through the putative shared LXA(4)/LTD(4) receptor. LXA(4)-stimulated TF expression was blunted by pertussis toxin and by GF-109203X, an inhibitor of protein kinase C, and was not associated with degradation of IkappaBalpha. Our results establish that LXA(4) induces TF activity via cell signaling pathways with different structural and receptor requirements from those described for inhibition of leukocyte-endothelial cell interactions. They suggest a role for LXA(4) as a modulator of TF-related vascular events during inflammation and thrombosis.

Hemorrhagic complications are common among hemodialysis (HD) patients. The mechanisms by which HD perturbs the coagulation cascade are still being defined. This study evaluated the influence of HD serum on cellular expression of tissue factor (TF), a procoagulant membrane-associated protein that is a pivotal regulator of blood coagulation. Serum was collected immediately before dialysis and 15, 30, and 180 min into HD using polysulfone membranes. Serum was then assessed for its ability to influence basal and cytokine-stimulated TF activity in human umbilical vein endothelial cells and ECV304 cells. Predialysis serum did not influence basal levels of TF activity. HD was associated with the appearance of a serum factor that suppressed basal TF activity (TF units/microg protein: predialysis serum 8.2 +/- 0.9; 180-min dialysis serum 4.9 +/- 0.6; P < 0.05) and TF activity induced by the cytokine tumor necrosis factor-alpha (TNFalpha) (TF units/microg protein: TNFalpha alone 15.9 +/- 0.7; TNFalpha + 180-min dialysis serum 5.9 +/- 0.9; P < 0.01). This response was not mimicked by heparin, suggesting production of an endogenous inhibitor of TF activity during HD. Dialysis was associated with a striking increase in circulating levels of tissue factor pathway inhibitor (TFPI), a physiologic inhibitor of the TF/VIIa complex. The lack of temporal correlation between TFPI levels and suppression of TF activity, however, suggested the presence of additional TFPI independent pathway(s) for modulation of TF activity. Dialysis-related suppression of TF expression may contribute to hemorrhagic complications in HD patients.

Ischemic acute renal failure (ARF) is a common clinical syndrome, associated with high morbidity and mortality, for which there is no specific therapy. Polymorphonuclear neutrophils (PMN) recruited during reperfusion have been implicated as mediators of renal parenchymal injury ischemic ARF. Leukocyte adhesion molecules appear to facilitate PMN recruitment in this setting. Complementary studies using monoclonal antibodies, antisense oligonucleotides and gene "knockout" indicate that blockade of CD11/CD18 integrins and intercellular adhesion molecule-1 (ICAM-1) attenuates ARF in some experimental models of renal ischemia. These exciting observations may herald the development of novel anti-adhesion strategies for use in human disease.

This study addressed two questions: 1) whether a relatively low dose of n-3 fatty acid ethyl esters (n-3 FAs) administered to healthy volunteers for a prolonged period of time would exert beneficial effects on plasma lipids, platelet function, and thromboxane biosynthesis; and 2) whether a short-term loading treatment (6 wk) with 6 g n-3 FAs/d followed by 12 wk with 3 g/d results in more pronounced effects. After 6 wk treatment a reduction of plasma triglyceride concentration and an accumulation of EPA and DHA in plasma were observed. A longer period of treatment with n-3 FAs was necessary to affect platelet aggregation and thromboxane A2 biosynthesis. At 12 and 18 wk, platelet aggregation, thromboxane A2 formation, and the excretion of thromboxane metabolites in urine were reduced, particularly in subjects who received 6 g n-3 FAs/d during the initial 6 wk. After treatment ended, triglyceride and thromboxane A2 biosynthesis returned to baseline values within 4 wk, whereas platelet aggregation remained impaired for ≥ 14 wk. The longlasting impairment in platelet aggregation was accompanied by the retention of n-3 FAs in platelet phospholipids.

n-3 Fatty acids are known to influence several functions of monocytes, including adhesion, cytokine synthesis, and superoxide generation. Monocytes express tissue factor, a membrane-bound glycoprotein, that acts as a catalyst in the coagulation cascade. In this study we evaluated the effects of administration of n-3 fatty acid ethyl esters to healthy volunteers and to hypertriglyceridemic patients on tissue factor activity (TF activity) in adherent monocytes. n-3 Fatty acids containing 75% eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (ratio of EPA to DHA, 1.34) were administered (3 g/d) to normal volunteers for 18 weeks. In addition, the effects of this treatment were evaluated in 30 hypertriglyceridemic patients for 24 weeks by using a double-blind, placebo-controlled study. TF activity in adherent monocytes was evaluated with a one-stage clotting assay. Plasma and monocyte fatty acid compositions were determined by gas-liquid chromatography. In healthy volunteers, n-3 fatty acids significantly reduced TF activity in adherent monocytes either in the unstimulated condition or after exposure to endotoxin. The inhibitory effect was observed after 12 weeks of treatment and was more pronounced after 18 weeks (> 70%, P < .001 versus baseline). Concomitantly, levels of EPA and DHA increased in plasma and monocyte lipids. Interestingly, after stopping treatment, monocyte TF activity remained inhibited for at least 14 weeks. Treatment with n-3 fatty acids for 24 weeks also resulted in a significant reduction of TF activity in adherent monocytes from hypertriglyceridemic patients (-31% and -40% in unstimulated and endotoxin-stimulated cells; P < .05 versus baseline).(ABSTRACT TRUNCATED AT 250 WORDS)

In recent years the interactions between lipoxygenase enzymes present in platelets and leukocytes, resulting in the transcellular biosynthesis of eicosanoids, have been discovered and their relevance in thrombotic and inflammatory disorders recognized. However, attention has focused on the synthesis of novel products, not normally formed by the single-cell population. Less information is available on the changes in hydroxyacid formation during platelet-neutrophil interactions. In this study, we evaluated the quantitative changes of the levels of 12- and 5-hydroxyeicosatetraenoic acids, leukotriene B4, and thromboxane B2 occurring during the incubation of platelet-neutrophil mixed suspensions stimulated with the calcium ionophore A23187. Cell-to-cell interaction resulted in quantitative changes of the level of hydroxyacids; in mixed platelet-neutrophil suspensions, the levels of 12-hydroxyeicosatetraenoic acid were significantly increased with respect to those measured in platelets incubated alone. The amount of 5-hydroxyeicosatetraenoic acid formed by neutrophils was significantly decreased by the presence of platelets in the incubation medium. In addition, a slight but significant reduction in leukotriene B4 synthesis was observed. Concomitantly with these changes, the formation of thromboxane B2 by platelets was modified, indicating that--besides the lipoxygenase pathway--the arachidonic acid metabolism by cyclooxygenase is affected. Our study demonstrates that in mixed platelet-neutrophil suspensions profound quantitative modifications in hydroxyacid and thromboxane synthesis occur, indicating that the overall balance of arachidonic acid products may be altered in pathologic conditions in which early events of multicellular origin have been recognized.