Hydroxychloroquine inhibits proinflammatory signalling pathways by targeting endosomal NADPH oxidase.

OBJECTIVES: Hydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL). METHODS: For in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1ß (IL-1ß) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis. RESULTS: HCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1ß and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation. CONCLUSIONS: We describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS.

Endosomal Redox Signaling in the Antiphospholipid Syndrome.

PURPOSE OF REVIEW: It is well established that the antiphospholipid syndrome (APS) is caused by antiphospholipid antibodies (aPL). While several underlying mechanisms have been described in the past, many open questions remain. Here, we will review data on endosomal signaling and, in particular, redox signaling in APS. RECENT FINDINGS: Endosomal redox signaling has been implicated in several cellular processes including signaling of proinflammatory cytokines. We have shown that certain aPL can activate endosomal NADPH-oxidase (NOX) in several cell types followed by induction of proinflammatory and procoagulant cellular responses in vitro. Involvement of endosomes in aPL signaling has also been reported by others. In wild-type mice but not in NOX-deficient mice, aPL accelerate venous thrombus formation underscoring the relevance of endosomal NOX. Furthermore, hydroxychloroquine (HCQ) inhibits activation of endosomal NOX and prevents thrombus formation in aPL-treated mice. Endosomal redox signaling is an important novel mechanism involved in APS pathogenesis. This makes endosomes a potential target for future treatment approaches of APS.

Distribution of antiphospholipid antibodies in a large population-based German cohort.

BACKGROUND: Antiphospholipid syndrome (APS) is the most common acquired thrombophilia. Diagnosis is based on clinical criteria and the presence of antiphospholipid antibodies (aPLs) above the 99th percentile of a reference group. Data on the distribution of aPL in the population are limited. The distribution of aPL including diagnostic cutoffs should be determined in a population-based cohort. METHODS: The Gutenberg Health Study (GHS) is a population-based cohort aged 35-74 years. We determined the presence of antibodies against cardiolipin (aCL, IgG, and IgM), ß2-glycoprotein I (anti-ß2GPI, IgG, and IgM), and domain 1 of ß2-glycoprotein I (anti-domain 1, IgG) in a sample of 4979 participants. RESULTS: aPL titers were similar in the whole sample and in an apparently healthy subgroup of 1049 individuals. There was a strong age-dependent increase of both aCL and anti-ß2GPI IgM, while aPL IgG titers were stable or tended to decrease with age. A relevant decrease was observed for aCL IgG in women and anti-domain 1 IgG in both sexes. There was no association of aPL titers with a history of venous thromboembolism (VTE). CONCLUSIONS: Our data show that for IgM aPL, age-dependent reference ranges should be used. In fact, the controversy regarding the clinical utility of IgM aPL might be related to the use of inappropriate reference ranges among other causes. In our population, aPLs were not associated with a history of VTE.

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation.

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased - and, under conditions of decreased platelet adhesion, PDI inhibition reduced - fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet-secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.

Immune cell pathology in rabbit hemorrhagic disease.

AIM: The aim of this research was to study the effect of rabbit hemorrhagic disease virus (RHDV) on the host immune response by examining the cellular composition/pathology of lymphoid organs and serum levels of tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ). MATERIALS AND METHODS: Nine adult rabbits were inoculated with 1 ml of 10% infected liver homogenate, and three rabbits served as controls. The rabbit hemorrhagic disease (RHD)-induced animals were studied on 3 consecutive days post-infection. Diagnosis of RHD was made through routine hemagglutination tests and the polymerase chain reaction. Blood smears and tissue samples from bone marrow (BM), spleen, lymph nodes, and liver were analyzed for cell composition and cytopathology. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assay. RESULTS: RHD showed a decreased absolute cell count of blood as well as lymph nodes, spleen, and BM cell populations with marked left shift. This was seen as a progressive rise in immature and blast cells. Quantitative cellular changes were accompanied by an increase in specific inflammatory cytokines. Immunocytopathological alterations were evidenced by: Vacuolized, hyperactivated tissue macrophages, finding of Döhle bodies in neutrophils, and activated lymphocytes with increased nuclear-cytoplasmic ratio. Cytoplasmic eosinophilic viral inclusions found in tissue (liver, spleen, and BM) macrophages were shown for the 1st time in RHD. Megakaryocytic emperipolesis was a common feature of RHD. CONCLUSION: These studies suggest that RHDV induces pathology in leukocytes due to hyperactivation with left shift (toward immature stages of the different cell lineages). Macrophages are increased in number and show an expressed cytopathic effect often accompanied by viral eosinophilic cytoplasmic inclusions. They also developed a secretory activation (increased levels of pro-inflammatory cytokines).

Protein disulfide isomerase as a trigger for tissue factor-dependent fibrin generation.

Recent evidence suggests that protein disulfide isomerase (PDI) represents an injury response signal that can activate tissue factor (TF), a major initiator protein of blood coagulation. PDI was found to be specifically exposed at the site of vessel injury, originating both from disrupted vessel-wall cells and adhering platelets. The exposed PDI promotes TF-dependent fibrin deposition in different mouse models of thrombosis. In particular, PDI can mediate stimulation of circulating (intravascular) TF present on microparticles. It has been proposed that PDI activates TF by changing the disulfide status of the membrane-proximal Cys186-Cys209 pair of TF. Indeed, PDI was shown to cleave mixed disulfide bonds of TF with glutathione. This might enable the formation of an intrachain disulfide bond which is associated with an increased procoagulant efficiency of TF. The PDI-induced activation of TF could represent the primary step of the entire coagulation process.

Pathophysiological insights into the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is characterized by venous and/or arterial thrombosis and severe pregnancy morbidity in presence of antiphospholipid antibodies (aPL). While there is compelling evidence that aPL cause the clinical manifestations of APS, the underlying mechanisms are still a matter of scientific debate. This is mainly related to the broad heterogeneity of aPL. There are three major types of aPL: The first one binds to (anionic) phospholipids, e.g. cardiolipin, in absence of other factors (cofactor independent aPL). The second type binds to phospholipids only in presence of protein cofactors, e.g. ß2-glycoprotein I (ß2GPI) (cofactor dependent aPL). The third type binds to cofactor proteins directly without need for phospholipids. It is widely believed that cofactor independent aPL (type 1) are associated with infections and, more importantly, non-pathogenic, while pathogenic aPL belong to the second and in particular to the third type. This view, in particular with regard to type 1 aPL, has not been undisputed and novel research data have shown that it is in fact untenable. We summarize the available data on the pathogenetic role of aPL and the implications for diagnosis of APS and future research.

Mice deficient in the anti-haemophilic coagulation factor VIII show increased von Willebrand factor plasma levels.

Von Willebrand factor (VWF) is the carrier protein of the anti-haemophilic Factor VIII (FVIII) in plasma. It has been reported that the infusion of FVIII concentrate in haemophilia A patients results in lowered VWF plasma levels. However, the impact of F8-deficiency on VWF plasma levels in F8-/y mice is unresolved. In order to avoid confounding variables, we back-crossed F8-deficient mice onto a pure C57BL/6J background and analysed VWF plasma concentrations relative to C57BL/6J WT (F8+/y) littermate controls. F8-/y mice showed strongly elevated VWF plasma concentrations and signs of hepatic inflammation, as indicated by increased TNF-α, CD45, and TLR4 transcripts and by elevated macrophage counts in the liver. Furthermore, immunohistochemistry showed that expression of VWF antigen was significantly enhanced in the hepatic endothelium of F8-/y mice, most likely resulting from increased macrophage recruitment. There were no signs of liver damage, as judged by glutamate-pyruvate-transaminase (GPT) and glutamate-oxalacetate-transaminase (GOT) in the plasma and no signs of systemic inflammation, as white blood cell subsets were unchanged. As expected, impaired haemostasis was reflected by joint bleeding, prolonged in vitro clotting time and decreased platelet-dependent thrombin generation. Our results point towards a novel role of FVIII, synthesized by the liver endothelium, in the control of hepatic low-grade inflammation and VWF plasma levels.

Antiphospholipid antibodies in a large population-based cohort: genome-wide associations and effects on monocyte gene expression.

The antiphospholipid syndrome (APS) is characterised by venous and/or arterial thrombosis and pregnancy morbidity in women combined with the persistent presence of antiphospholipid antibodies (aPL). We aimed to identify genetic factors associated with the presence of aPL in a population based cohort. Furthermore, we wanted to clarify if the presence of aPL affects gene expression in circulating monocytes. Titres of IgG and IgM against cardiolipin, ß2glycoprotein 1 (anti-ß2GPI), and IgG against domain 1 of ß2GPI (anti-domain 1) were determined in approx. 5,000 individuals from the Gutenberg Health Study (GHS) a population based cohort of German descent. Genotyping was conducted on Affymetrix Genome-Wide Human SNP 6.0 arrays. Monocyte gene expression was determined in a subgroup of 1,279 individuals by using the Illumina HT-12 v3 BeadChip. Gene expression data were confirmed in vitro and ex vivo by qRT-PCR. Genome wide analysis revealed significant associations of anti-ß2GPI IgG and APOH on chromosome 17, which had been previously identified by candidate gene approaches, and of anti-domain1 and MACROD2 on chromosome 20 which has been listed in a previous GWAS as a suggestive locus associated with the occurrence of anti-ß2GPI antibodies. Expression analysis confirmed increased expression of TNFα in monocytes and identified and confirmed neuron navigator 3 (NAV3) as a novel gene induced by aPL. In conclusion, MACROD2 represents a novel genetic locus associated with aPL. Furthermore, we show that aPL induce the expression of NAV3 in monocytes and endothelial cells. This will stimulate further research into the role of these genes in the APS.

Targeting platelet migration in the postischemic liver by blocking protease-activated receptor 4.

BACKGROUND: Platelets play a critical role during hepatic ischemia/reperfusion (I/R). Antiplatelet strategies during liver transplantation are, however, limited because of bleeding complications. Thrombin is activated during reperfusion and regulates platelet and endothelial cell function via protease-activated receptor 4 (PAR-4). Interventions at the level of PAR-4, the main platelet receptor for thrombin, are assumed to attenuate the proinflammatory effects of thrombin without affecting blood coagulation. The aim of our study was to analyze the impact of PAR-4 blockade on platelet recruitment and microvascular injury during hepatic I/R. METHODS: C57BL/6 mice undergoing hepatic I/R (90 min/60 min and 240 min) were treated either with a selective PAR-4 antagonist TcY-NH2 or vehicle. Sham-operated animals served as controls. Recruitment of freshly isolated and fluorescence-labeled platelets and CD4 T cells was analyzed using intravital video fluorescence microscopy. Parameters of tissue injury, regeneration, and blood coagulation were assessed in tissue/blood samples. RESULTS: Results show that treatment with TcY-NH2 attenuated I/R-induced platelet and CD4 T-cell recruitment, improved sinusoidal perfusion failure, and reduced apoptotic and necrotic injury. The protective effect of PAR-4 blockade did not suppress hemostasis or liver regeneration. CONCLUSION: Our in vivo data suggest PAR-4 as a potential target for future therapeutic strategies against platelet-mediated liver injury on transplantation.

Nature and dynamics of nucleosome release from neoplastic and non-neoplastic cells.

BACKGROUND: Circulating nucleosomes are elevated in the blood of patients with malignant and non-malignant diseases. Here, we investigated the nature and the dynamics of their release in functional cell studies. MATERIALS AND METHODS: Leukemia blasts were exposed to the intrinsic inducers of apoptotic cell death, cytosine arabinoside (AraC; 10 µg/ml) and etoposide (50 µg/ml), and cell death markers lactate dehydrogenase (LDH) and the nucleosomes were measured in the supernatants at 0, 24, 48, 72, and 96 hours after drug application. In addition, HepG2 cells were exposed to extrinsic apoptosis-inducing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL; 0.5 and 1.0 ng/ml) and the nucleosomes were measured in the supernatants after 0, 24, 48, and 72 hours. Finally, neutrophils preactivated by phorbol myristate acetate (PMA) were co-incubated with platelet-rich plasma (PRP) in the presence of collagen (type I; 8 µg/ml) for 15 or 30 minutes at 37°C, and the nucleosome release into the supernatant was quantified. RESULTS: During treatment with AraC, cell viability constantly decreased. LDH and nucleosome levels increased at 24 h and peaked at 48 h after exposure to AraC and etoposide. While LDH declined after 96 h, the nucleosomes' levels were still elevated. Similarly, nucleosomes increased dose-dependently 24 h after exposure to TRAIL and reached a peak at 48 h. After 72 h, the nucleosomes' levels decreased again. While there was only a minor release of nucleosomes from PMA-stimulated neutrophils, co-incubation with PRP resulted in a strongly increased nucleosome release after 30 minutes. CONCLUSION: Nucleosomes are released from cells stimulated intrinsically or extrinsically to undergo apoptotic cell death in a time- and dose-dependent manner. Further mechanisms of release may be their active secretion from stimulated neutrophils when co-incubated with PRP, as may be observed during bacterial inflammation and thrombosis.

Monocytes, neutrophils, and platelets cooperate to initiate and propagate venous thrombosis in mice in vivo.

Deep vein thrombosis (DVT) is a major cause of cardiovascular death. The sequence of events that promote DVT remains obscure, largely as a result of the lack of an appropriate rodent model. We describe a novel mouse model of DVT which reproduces a frequent trigger and resembles the time course, histological features, and clinical presentation of DVT in humans. We demonstrate by intravital two-photon and epifluorescence microscopy that blood monocytes and neutrophils crawling along and adhering to the venous endothelium provide the initiating stimulus for DVT development. Using conditional mutants and bone marrow chimeras, we show that intravascular activation of the extrinsic pathway of coagulation via tissue factor (TF) derived from myeloid leukocytes causes the extensive intraluminal fibrin formation characteristic of DVT. We demonstrate that thrombus-resident neutrophils are indispensable for subsequent DVT propagation by binding factor XII (FXII) and by supporting its activation through the release of neutrophil extracellular traps (NETs). Correspondingly, neutropenia, genetic ablation of FXII, or disintegration of NETs each confers protection against DVT amplification. Platelets associate with innate immune cells via glycoprotein Ibα and contribute to DVT progression by promoting leukocyte recruitment and stimulating neutrophil-dependent coagulation. Hence, we identified a cross talk between monocytes, neutrophils, and platelets responsible for the initiation and amplification of DVT and for inducing its unique clinical features.

Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases.

Blood neutrophils provide the first line of defense against pathogens but have also been implicated in thrombotic processes. This dual function of neutrophils could reflect an evolutionarily conserved association between blood coagulation and antimicrobial defense, although the molecular determinants and in vivo significance of this association remain unclear. Here we show that major microbicidal effectors of neutrophils, the serine proteases neutrophil elastase and cathepsin G, together with externalized nucleosomes, promote coagulation and intravascular thrombus growth in vivo. The serine proteases and extracellular nucleosomes enhance tissue factor- and factor XII-dependent coagulation in a process involving local proteolysis of the coagulation suppressor tissue factor pathway inhibitor. During systemic infection, activation of coagulation fosters compartmentalization of bacteria in liver microvessels and reduces bacterial invasion into tissue. In the absence of a pathogen challenge, neutrophil-derived serine proteases and nucleosomes can contribute to large-vessel thrombosis, the main trigger of myocardial infarction and stroke. The ability of coagulation to suppress pathogen dissemination indicates that microvessel thrombosis represents a physiological tool of host defense.

Eosinophils are a major intravascular location for tissue factor storage and exposure.

Blood cell progenitors were scanned for the presence of the coagulation starter protein tissue factor (TF) by immunoelectron microscopy. Thereby, substantial TF expression was observed in the precursor cells of eosinophils. TF levels were lower in basophil precursors and barely detectable in neutrophil progenitors. In peripheral blood immediately processed to avoid activation of the TF gene, mature eosinophils were found to considerably express TF, unique among the granulocyte and monocyte fractions. TF was preferentially located in the specific granules in resting eosinophils. Platelet-activating factor (PAF), and more pronounced, granulocyte-macrophage colony-stimulating factor (GM-CSF) plus PAF, caused translocation of preformed TF to the eosinophil cell membrane. GM-CSF/PAF also increased the TF transcript levels. The activated eosinophils exhibited procoagulant activity that was abrogated by TF inhibition. Targeting the extracellular domain of TF with specific antibodies markedly suppressed the initial phase of the eosinophil passage across the IL-4-activated endothelium. Eosinophil rolling and firm adhesion remained unaffected. This suggests that TF specifically facilitates the early transendothelial migration of the eosinophils. In summary, eosinophils maintain a high TF expression during maturation, providing a main source of preformed TF in blood, which might be relevant for the thrombogenesis promoted by hypereosinophilic conditions.