The role of the macrophage in apoptosis: hunter,gatherer, and regulator

Clearance of cellular corpses is a critical feature of apoptosis in vivo during development, tissue homeostasis, and resolution of inflammation. As the professional phagocytes of the body, macrophages play a key role in this process. By recognizing emerging signals using several different receptors, macrophages engulf apoptotic cells swiftly and efficiently. In addition, the binding of apoptotic cells profoundly down-regulates the ability of the macrophage to produce inflammatory mediators by inducing the release of antiinflammatory mediators. Finally, macrophages may actually induce cell death in specific cells during embryogenesis. Abnormalities of apoptotic cell clearance may contribute to the pathogenesis of chronic inflammatory diseases, including those of autoimmune etiology. It is also possible that certain malignant tumor cells co-opt the mechanisms for apoptotic cell clearance to avoid immune surveillance by subverting macrophage and dendritic cell responses.     

Reduced ANXA5 mRNA and protein expression in pregnancies complicated by preeclampsia

Introduction

The placental anticoagulant protein Annexin A5 (ANXA5) is a multifunctional protein that is highly expressed on the apical surfaces of syncytiotrophoblasts, and plays an important role in haemostatic regulations, maintaining blood fluidity of the placenta. The aim of this study was to investigate the expression of ANXA5 in pregnancies complicated by preeclampsia (PE).

Materials and Methods

Placental tissue samples were collected from 23 pregnancies with PE and 34 normal pregnancies. ANXA5 mRNA levels were measured by quantitative Real-Time PCR (qPCR), while ANXA5 protein expression was measured by Western Blot (WB) and immunohistochemistry.

Results

ANXA5 mRNA expression in PE samples was lower than 1% of its expression in normal samples (mean ± SD: 0.002 ± 0.004 vs. 0.55 ± 0.38, p < 0.001), while ANXA5 protein levels in PE samples were approximately at 65% of the average normal expression (mean ± SD: 0.53 ± 0.30 vs. 0.81 ± 0.25, p = 0.001). Immunohistochemical analysis also verified the above results, since PE placentas tended to have low labelling indexes (LIs), in contrast to controls which demonstrated high LIs (p = 0.020). Statistical analysis of the WB data revealed that ANXA5 protein expression was increased in PE smokers vs. PE non-smokers (mean ± SD: 0.64 ± 0.23 vs. 0.41 ± 0.33, p = 0.027).

Conclusions

These results suggest that ANXA5 downregulation could be part of the pathophysiology of PE and the possible impairment in coagulation processes, which are seen in pregnancies that demonstrate PE. Further studies may investigate whether ANXA5 could be used as a biomarker for the early detection of PE and for the prediction of its severity.     

Autoantibodies and coagulation in reproductive medicine

Management of recurrent pregnancy loss (RPL) is considered to be difficult, in part because of cunfusion between autoantibodies and coagulation disorders. Autoantibodies and coagulation are related; two groups of multicenter studies concerning autoantibodies and coagulation reported that factor XII deficiency, hypofibrinolysis, anti-phosphatidylethanolamine (aPE), anti-beta2-glycoprotein I, anti-annexin A5, and lupus anticoagulant (LA) were found to be frequent risk factors in RPL women. Therefore, discrimination of autoantibodies and coagulation is important in understanding RPL well. We propose three types of pathways regarding reproduction, which are different and independent: (1) Negatively charged-phospholipid related antibodies (anti-phosphatidylserine; aPS, anti-cardiolipin; aCL, lupus anticoagulant; LA, anti-annexin A5; aANX), (2) factor XII–aPE–fibrinolysis: suppression of fibrinolysis, (3) protein C–protein S–factor V: loss of inactivation against activated factor V. Women with RPL and infertility showed similar findings in terms of the above clinical tests. Available data, however, is not enough to conclude whether these are pathogenic to infertile women.     

The apoptosis cascade — morphological and immunohistochemical methods for its visualization

 Apoptosis is involved in morphogenesis of embryonic tissues as well as in homeostasis of adult organs and tissues. It is the main process by which organs maintain cell mass and at the same time eliminate excess and aged cells that have lost their functional importance. The typical morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events, some of which are inextricably linked to the process of differentiation. Studies that analyze all stages of this cascade, rather than the final morphological stages of apoptotic death, are essential in order that specific link(s) between differentiation and apoptosis are appreciated. This review outlines the main stages of the apoptosis cascade together with current methods for their morphological visualization. Starting with (a) receptors and ligands known to induce apoptosis, we continue with (b) early initiator stages of apoptosis, and (c) proteins regulating and potentially inhibiting further progression of the cascade, into (d) irreversible execution stages of the cascade, and finally (d) the morphological events of apoptotic death. For each stage we present those aspects of the biochemical background that are morphologically relevant, together with proven methods for their visualization. We offer technical advice at each stage based upon our experience of studying differentiation and apoptosis in human placental trophoblast. Accepted: 21 January 1999     

Effect of phosphate on phosphatidylserine-mediated calcium transport

Summary Experiments were performed to study the effect of inorganic phosphate (Pi) on phosphatidylserine-mediated Ca²⁺ transport utilizing two- and three-compartment lipid-aqueous phase model systems. When the three-compartment model was used, the rate of Ca²⁺ transport from an aqueous donor compartment to an aqueous receiver compartment, separated by a nonaqueous phospholipid phase, was determined. This experiment showed that the rate of Ca²⁺ transport was proportional to the phosphatidylserine concentration and the pH. Pi modulated the rate of Ca²⁺ transport; even when the Pi concentration of the donor aqueous phase was low, there was a marked enhancement of transport. To determine whether the Pi-mediated rise in the Ca²⁺ transport rate was due to an increase in the uptake of Ca²⁺ into the lipid phase, or to an increase in the ability of the lipid phase to release Ca²⁺, a two-compartment model was used. It was found that the ability of the phosphatidylserine phase to take up Ca²⁺ increased as the Pi concentration of the aqueous donor phase was raised. With the increase in Ca²⁺ uptake there was a concomitant elevation in the rate of Ca²⁺ transport into an aqueous receiver phase. However, Pi did not stimulate Ca²⁺ release from the phosphatide. Thus it was concluded that Pi enhanced the interaction between Ca²⁺ and phosphatidylserine, possibly by forming a Ca-phospholipid-Pi complex. Once this interaction had taken place, Ca²⁺ release into the aqueous receiver compartment was independent of the Pi concentration.     

Blunting by chronic phosphatidylserine administration of the stress-induced activation of the hypothalamo-pituitary-adrenal axis in healthy men

Summary The effect of chronic administration of phosphatidylserine derived from brain cortex on the neuroendocrine responses to physical stress has been examined in a placebo-controlled study in 9 healthy men.Phosphatidylserine 800 mg/d for 10 days significantly blunted the ACTH and cortisol responses to physical exercise (P=0.003 and P=0.03, respectively), without affecting the rise in plasma GH and PRL.Physical exercise significantly increased the plasma lactate concentration both after placebo and phosphatidylserine.The results suggest that chronic oral administration of phosphatidylserine may counteract stress-induced activation of the hypothalamo-pituitary-adrenal axis in man.     

Inhibition of eryptosis and intraerythrocytic growth of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> by flufenamic acid

Non-selective (NSC) cation channels participate in the Ca(2+) leak of human erythrocytes. Sustained activity of these channels triggers suicidal erythrocyte death (eryptosis), which is characterized by Ca(2+)-stimulated cell shrinkage and phosphatidylserine (PS) exposure. PS-exposing erythrocytes are rapidly cleared from circulating blood. PGE(2) activates the NSC channels, and erythrocyte PGE(2) formation is stimulated by a decrease in intra- or extracellular Cl(-) concentration. In addition, the intraerythrocytic malaria parasite Plasmodium falciparum activates the NSC channels, most probably to accomplish Na(+) and Ca(2+) entry into the erythrocyte cytosol required for parasite development. By Ca(2+) uptake the parasite maintains a low Ca(2+) concentration in the erythrocyte cytosol and thus delays the suicidal death of the host erythrocyte. Flufenamic acid has previously been shown to inhibit NSC channels. The present study thus explored the effect of flufenamic acid on erythrocyte Ca(2+) entry, on suicidal erythrocyte death and on intraerythrocytic growth of P. falciparum. Within 48 h, replacement of extracellular Cl(-) with gluconate or application of PGE(2) (50 microM) increased Fluo3 fluorescence reflecting cytosolic Ca(2+) activity, decreased forward scatter reflecting cell volume and increased annexin V binding reflecting PS exposure in FACS analysis. All those effects were significantly blunted in the presence of flufenamic acid (10 microM). Flufenamic acid (25 microM) further significantly delayed the intraerythrocytic growth of P. falciparum and the PS exposure of the infected erythrocytes. The present observations disclose a novel effect of flufenamic acid, which may allow the pharmacological manipulation of erythrocyte survival and the course of malaria.     

Hemin-induced suicidal erythrocyte death

Several diseases, such as malaria, sickle cell disease, and ischemia/reperfusion may cause excessive formation of hemin, which may in turn trigger hemolysis. A variety of drugs and diseases leading to hemolysis triggers suicidal erythrocyte death or eryptosis, i.e., cell membrane scrambling and cell shrinkage. Eryptosis is elicited by increased cytosolic Ca²⁺ activity and by ceramide. The present study explored whether hemin stimulates eryptosis. Cell membrane scrambling was estimated from annexin V-binding to phosphatidylserine exposed at the cell surface, cell shrinkage from forward scatter in fluorescence-activated cell sorter analysis, cytosolic Ca²⁺ activity from Fluo3 fluorescence and ceramide formation from fluorescence-labeled antibody binding. Exposure to hemin (1–10 μM) within 48 h significantly increased annexin V-binding, decreased forward scatter, increased cytosolic Ca²⁺ activity, and stimulated ceramide formation. In conclusion, hemin stimulates suicidal cell death, which may in turn contribute to the clearance of circulating erythrocytes and thus to anemia.     

Weak platelet agonists and U46619 induce apoptosis-like events in platelets,in the absence of phosphatidylserine exposure

Platelets express apoptotic markers during storage, while aging and after stimulation with strong agonists thrombin and collagen. It is unknown if the weak agonists ADP and epinephrine or U46619, a thromboxane analog, induce the expression of apoptotic markers in platelets. To answer this question, we measured phosphatidylserine exposure, gelsolin cleavage and decrease in membrane mitochondrial potential after stimulation with these agonists. No phosphatidylserine exposure was evident, however, gelsolin cleavage and a platelet population with a decreased membrane mitochondrial potential appeared, suggesting that in platelets selective agonists can induce apoptosis in the absence of phosphatidylserine exposure. Interestingly, costimulation by thrombin plus collagen together with each of the other agonists increased the phosphatidylserine exposure induced by strong agonists. These findings may be of importance in platelet activation and apoptosis under pathophysiological conditions where multiple effectors are involved.