Instructive influences of phagocytic clearance of dying cells on neutrophil extracellular trap generation.

Coordinated programmes of resolution are thought to initiate early after an inflammatory response begins, actively terminating leucocyte recruitment, allowing their demise via apoptosis and their clearance by phagocytosis. In this review we describe an event that could be implicated in the resolution of inflammation, i.e. the establishment of a refractory state in human neutrophils that had phagocytosed apoptotic cells. Adherent neutrophils challenged with apoptotic cells generate neutrophil extracellular traps (NETs), filaments of decondensed chromatin decorated with bioactive molecules that are involved in the capture of various microbes and in persistent sterile inflammation. In contrast, neutrophils that had previously phagocytosed apoptotic cells lose their capacity to up-regulate ß2 integrins and to respond to activating stimuli that induce NET generation, such as interleukin (IL)-8. A defective regulation of NET generation might contribute to the persistent inflammation and tissue injury in diseases in which the clearance of apoptotic cells is jeopardized, including systemic lupus erythematosus and anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis.

Intravascular immunity as a key to systemic vasculitis: a work in progress, gaining momentum.

Vascular inflammation contributes to the defence against invading microbes and to the repair of injured tissues. In most cases it resolves before becoming apparent. Vasculitis comprises heterogeneous clinical entities that are characterized by the persistence of vascular inflammation after it has served its homeostatic function. Most underlying mechanisms have so far remained elusive. Intravascular immunity refers to the surveillance of the vasculature by leucocytes that sense microbial or sterile threats to vessel integrity and initiate protective responses that entail most events that determine the clinical manifestations of vasculitis, such as end-organ ischaemia, neutrophil extracellular traps generation and thrombosis, leucocyte extravasation and degranulation. Understanding how the resolution of vascular inflammation goes awry in patients with systemic vasculitis will facilitate the identification of novel pharmacological targets and bring us a step closer in each patient to the selection of more effective and less toxic treatments.

Translational mini-review series on immunology of vascular disease: mechanisms of vascular inflammation and remodelling in systemic vasculitis.

Vessel walls are the primary inflammatory sites in systemic vasculitides. In most cases the initiating event is unknown, and a self-sustaining circuit attracts and activates inflammatory leucocytes in the wall of vessels of various size and anatomical characteristics. Recent studies have revealed homeostatic roles of vascular inflammation and have identified the action of humoral innate immunity, in particular injury-associated signals and acute phase proteins, on the activation of circulating leucocytes, platelets and endothelial cells. These advances have provided clues to the molecular mechanisms underlying the vicious circle that maintains and amplifies vessel and tissue injury.

A Lucilia cuprina excretory-secretory protein inhibits the early phase of lymphocyte activation and subsequent proliferation.

The development of a protective immune response in sheep towards the presence of the larval stage of Lucilia cuprina has not been reported in the field. Upon investigation of the effects of larval excretory/secretory material on ovine T lymphocyte proliferation, we isolated a 56 kDa protein capable of inhibiting lymphocyte proliferation by at least 70%, compared with that in the presence of mitogen alone. This protein inhibited proliferation induced through cross-linking of the T-cell receptor as well as proliferation induced pharmacologically through the stimulation of the protein kinase C (PKC) pathway. The protein, named blowfly larval immunosuppressive protein (BLIP), was shown to bind directly to lymphocytes. Further investigation revealed that the BLIP prevented a proportion of lymphocytes from entering the first division following stimulation, by affecting the early events in lymphocyte activation. Subsequently, the BLIP reduced CD25 expression on T lymphocytes, reduced IL-2 mRNA expression, in addition to IFN-gamma, IL-4, IL-10 and IL-13 mRNA expression. Conversely, TNF-alpha and TGF-beta gene expression was up-regulated in response to the BLIP. These effects suggest suboptimal activation of T lymphocytes in the presence of the BLIP, and we propose that the BLIP presents an effective immune evasion tactic for the larvae of L. cuprina.

Human polymorphonuclear leukocytes produce and express functional tissue factor upon stimulation.

BACKGROUND: Blood-borne tissue factor (TF) plays a crucial role in thrombogenesis. AIM: To study whether polymorphonuclear leukocytes (PMN) are a source of TF. METHODS AND RESULTS: Human PMN were carefully separated from other blood cells and stimulated for 3 min with purified P-selectin or the chemotactic peptide formyl-MetLeuPhe (fMLP): they expressed both TF procoagulant activity, as identified by specific TF MoAb and inactivated factor VIIa blockade; and TF:Ag (four to six times), as shown by flow-cytometry and immunocytochemistry. About 40% of permeabilized PMN, both resting and stimulated, contained TF:Ag, indicating that stimulation only modifies the location of TF:Ag within PMN. By real time-polymerase chain reaction (RT-PCR), a very low amount of TF mRNA was detectable in resting PMN, but a 3- to 5-fold increase was observed after 1-h stimulation with P-selectin or fMLP, respectively. CONCLUSIONS: These findings suggest that TF is not constitutively expressed in peripheral PMN, but can be up-regulated and produced upon stimulation and specific gene transcription, as for instance during contact with activated platelets or endothelium. The stored TF is rapidly expressed in vitro as a functional molecule on the surface of activated PMN. The availability of PMN TF supports the relevance of inflammatory cells and their interaction with platelets for fibrin deposition and thrombus formation.

Inhibition of tissue factor expression by hydroxyurea in polymorphonuclear leukocytes from patients with myeloproliferative disorders: a new effect for an old drug?

BACKGROUND: Polymorphonuclear leukocytes (PMN) from healthy subjects can produce and store tissue factor (TF), which is expressed on PMN surface upon in vitro stimulation with P-selectin. RESULTS: We report here that platelets and PMN from 12 patients with myeloproliferative disorders (MPD) (six with polycythemia vera, six with essential thrombocythemia) show up regulation of P-selectin and TF, respectively, in the absence of any in vitro challenge. The number of circulating mixed platelet-PMN aggregates was also increased. PMN TF expression as well as mixed platelet-PMN aggregates, but not platelet P-selectin, were significantly reduced in six MPD patients after treatment with hydroxyurea (HU). In vitro studies performed on PMN separated from healthy donors confirmed HU effects (0-1400 microm). HU prevented both P-selectin-induced TF expression and mixed cell aggregate formation. The inhibitory effect of HU was specific for P-selectin-induced PMN activation, as it did not affect formyl-methionyl-leucyl-phenylalanine-induced PMN TF expression. CONCLUSIONS: In MPD patients, platelet P-selectin-mediated TF expression on circulating PMN may play a role in thrombus formation and represents a novel target for the antithrombotic activity of HU.

Enhanced response to chemotactic activation of polymorphonuclear leukocytes from patients with heart valve replacement.

Artificial surfaces activate blood components. Since anticoagulant and antiplatelet therapy fail to abolish thromboembolic complications in patients with mechanical heart valve replacement (MHVR), other mechanisms might contribute to switch on a thrombotic event. We therefore investigated the reactivity to chemotactic activation of PMN from patients with MHVR. PMN responses were analyzed in 3 groups: 130 patients with MHVR and oral anticoagulant therapy, with or without aspirin, 57 patients on a comparable antithrombotic regimen, but without MHVR and 50 healthy subjects. In vitro studies showed that the release of cathepsin G and elastase from fMLP-stimulated PMN was significantly higher in the MHVR group, the leukocyte content of alpha 1-antitrypsin (an inhibitor of both enzymes) being similar in all three groups. CD11b expression after stimulation with fMLP was also significantly higher on PMN from MHVR patients than from control patients or healthy volunteers, while PMN CD11b basal expression was similar in all three groups. This increased PMN response in vitro in the absence of an obvious activation in vivo, may reflect a modified reactivity of circulating PMN passing through the artificial valves. Increased reactivity to local stimuli might allow PMN to participate in thrombus formation, despite conventional antithrombotic therapy.

Polymorphonuclear leukocyte-platelet interaction: role of P-selectin in thromboxane B2 and leukotriene C4 cooperative synthesis.

In PMN/platelet suspensions stimulated by fMLP giant mixed aggregates are formed and TxB2 and LTC4 are synthesized as the result of the cooperation in the arachidonic acid (AA) metabolism during cell/cell contact. PMN-derived cathepsin G induced the expression of P-selectin on platelet surface. GE12, an antibody against P-selectin, significantly reduced mixed cell aggregates. GE12 did not affect platelet aggregation induced by PMN-derived supernatants, indicating that the inhibitory effect of GE12 on mixed cell aggregation depends on inhibition of PMN/platelet adhesion. GE12 significantly reduced TxB2 and LTC4 production in PMN/platelet mixed cell suspensions stimulated by fMLP. As previously reported, synthesis of 3H-TxB2 in 3H-AA-labeled PMN/unlabeled platelets indicates that platelets utilize 3H-AA from PMN. 3H-LTC4 production in unlabeled PMN/3H-AA-labeled platelets indicates that bidirectional routes are utilized in this system for LTC4 synthesis. GE12 significantly reduced 3H-TxB2 and 3H-LTC4 synthesis. These results show that cathepsin G released by activated PMN induces the expression of P-selectin on platelet membrane: this adhesive glycoprotein modulates cell-cell contact and transcellular metabolism of AA.

Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G.

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy.

Hormonal regulation on bioactive aortic substance (BAS) production.

Rat's vessel wall releases a protein named BAS (Bioactive Aortic Substance) whose antiaggregating effect on platelets and inotropy vascular properties have been already described. In this work we have investigated the effects of pregnancy, gonadectomy and gonadectomy with hormonal treatment on the BAS production from rat aortic rings. BAS production in pregnancy and ovariectomized rats was markedly decreased compared to normal rats. Return to normal values was obtained after estradiol treatment in ovariectomized rats. Castration resulted in an increased of BAS production which was suppressed by testosterone treatment.

Purification and partial characterization of a bioactive substance from rat's vessel wall independent of prostacyclin production.

The bioactive substance from rat's vessel wall was purified by Sephadex G-75 gel filtration and by a combination of DEAE Cellulose ion exchange and Sephadex G-50 gel filtration chromatographies. Purifications of 12.5 fold and 70 fold over the initial material were achieved. PAGE of the purified material resulted in a single major band with a molecular weight estimated at 55kd-65kd. Trypsin (0.3 mg/ml) and chymotrypsin (30 mg/ml) abolished platelet antiaggregating activity. Neuraminidase (1.2 units) had no effect on platelet antiaggregating activity. This is the report of purification of aortic vessel wall antiaggregating activity independent of prostacyclin production.

Intraplatelet levels of vWF:Ag and fibrinogen in myeloproliferative disorders.

Several platelet function abnormalities have been described in the myeloproliferative syndromes. We have measured the intraplatelet vWF:Ag and fibrinogen (FI) in the platelet lysates by Laurell technique in 11 patients with polycythemia vera (PV), 10 with essential thrombocythemia (ET), 14 with chronic myelocytic leukaemia (CML) and 3 with myelofibrosis (MF) and these results were correlated with platelet function abnormalities. Decreased intraplatelet levels of vWF:Ag and FI were found in all the patients with ET and MF, in 8 out of 11 PV and 3 out of 14 CML. A statistical significant correlation was observed between the intraplatelet levels of vWF:Ag and FI in the control group and in CML and PV, but no correlation was found in ET and MF. No correlation was observed between the plasmatic and the intraplatelet levels of vWF:Ag and FI in any group. Evidences of platelet activation (spontaneous platelet aggregation or circulating platelet aggregates) were observed in 40% of the cases with ET and PV, and all these cases had low intraplatelet levels of both antigens. None of the cases with MF had evidences of platelet activation and 2 out of 14 patients with CML had platelet activation. The deficiency of the dense bodies was less frequent than the depletion of the alpha granules (5 out of 11 PV, 4 out of 10 ET, 6 out of 14 CML and 2 out of 3 MF). The low intraplatelet contents of vWF: Ag and FI, more frequently observed in ET and PV, may be the result of platelet activation and in vivo release, but megakaryocyte dysfunction is more likely in myelofibrosis.

The influence of sex and different segments of thoracic aorta on bioactive aortic substance (BAS) and prostacyclin (PGI2) synthesis.

BAS is a protein generated by aortic rings isolated from rats. Our previous results clearly established that BAS inhibits platelet aggregation and modifies vascular tone. We have now examined the effect of separated segments of thoracic aorta and the effect of sex on the release of the BAS and PGI2. We evaluated three different segments of thoracic aorta: A = aortic arch, B = the upper segment and C = the lowest segment of the thoracic aorta. We measured the release of BAS and PGI2 from them. The BAS production increased in the first segment (A) when compared with the other two (B and C), whilst PGI2 production was the same along the thoracic aorta. On the other hand female and male thoracic aorta produced the same levels of BAS and 6-keto PGF1 cm.

Activated platelets present high mobility group box 1 to neutrophils, inducing autophagy and promoting the extrusion of neutrophil extracellular traps.

BACKGROUND: Increasing evidence implicates both platelets and neutrophils in the formation, stabilization, and growth of peripheral and coronary thrombi. Neutrophil extracellular traps (NETs) play a key role. The early events in the deregulated cross-talk between platelets and neutrophils are poorly characterized. OBJECTIVES: To identify at the molecular level the mechanism through which platelets induce the generation of NETs in sterile conditions. PATIENTS/METHODS: The presence of NETs was determined in 26 thrombi from patients with acute myocardial infarction by immunohistochemistry and immunofluorescence and markers of NETs assessed in the plasma. In vitro NET generation was studied in static and in physiological flow conditions. RESULTS: Coronary thrombi mainly consist of activated platelets, neutrophils, and NETs in close proximity of platelets. Activated platelets commit neutrophils to NET generation. The event abates in the presence of competitive antagonists of the high mobility group box 1 (HMGB1) protein. Hmgb1(-/-) platelets fail to elicit NETs, whereas the HMGB1 alone commits neutrophils to NET generation. Integrity of the HMGB1 receptor, Receptor for Advanced Glycation End products (RAGE), is required for NET formation, as assessed using pharmacologic and genetic tools. Exposure to HMGB1 prevents depletion of mitochondrial potential, induces autophagosome formation, and prolongs neutrophil survival. These metabolic effects are caused by the activation of autophagy. Blockade of the autophagic flux reverts platelet HMGB1-elicited NET generation. CONCLUSIONS: Activated platelets present HMGB1 to neutrophils and commit them to autophagy and NET generation. This chain of events may be responsible for some types of thromboinflammatory lesions and indicates novel paths for molecular intervention.

Circulating platelet/polymorphonuclear leukocyte mixed-cell aggregates in patients with mechanical heart valve replacement.

There is convincing evidence that cell adhesion plays an important role in cardiovascular pathology and is frequently associated to "in vivo" cellular activation. This study involves patients with mechanical heart valve replacement (MHVR patients) who have increased platelet polymorphonuclear leukocyte (PMN) reactivity. Dual-color cytometry was used to determine the expression of adhesive molecules on cellular surfaces, platelet, and PMN-bound fibrinogen as well as the presence of circulating platelet/PMN mixed-cell aggregates (MCA) in 55 MHVR patients, 49 control patients under oral anticoagulant therapy, and 22 healthy volunteers. The results demonstrated that (a) PMN from MHVR patients showed an increased PMN-bound fibrinogen (mean +/- SEM: 1,420 +/- 169 anti-fibrinogen fluorescence intensity, P= 0.0012), when compared to controls (mean +/- SEM: 747 +/- 32 anti-fibrinogen fluorescence intensity) and healthy volunteers (mean +/- SEM: 692 +/- 25 anti-fibrinogen fluorescence intensity; (b) platelet activation in MHVR patients was evidenced by the higher expression of CD62P (mean +/- SEM: 128 +/- 19 anti-CD62P fluorescence intensity, P = 0.003) compared to controls (mean +/- SEM: 65 +/- 15 and 50 +/- 10 anti CD62P fluorescence intensity) and by increased levels of platelet-bound fibrinogen (mean +/- SEM: 625 +/- 20 anti-fibrinogen fluorescence intensity, P = 0.0043 versus 496 +/- 45 and 480 +/- 30 for control patients and for healthy volunteers, respectively); and (c) the proportion of MCA in MHVR patients (15 +/- 2%) was significantly higher (P = 0.009) compared to controls (7 + 1%) and healthy volunteers (6 +/- 2%). The results indicate that the presence of stable circulating MCA represents another marker of "in vivo" PMN activation in MHVR patients.