Christina Barja-Fidalgo

Nickel induces apoptosis in human neutrophils

Nickel is an ubiquitous transition metal that is industrially applied in many forms, which inevitably leads to a high degree of occupational and environmental exposure. Over-exposure to nickel can produce a variety of adverse effects on human health, including allergy and lung and nasal cancers. In the present study, it is demonstrated, for the first time, that nickel [(Ni(II)] (as a nickel nitrate salt) at concentrations that may be attained in vivo, induces neutrophils’ apoptosis by the intrinsic pathway. The use of diphenyleneiodonium, a NADPH oxidase inhibitor, delayed Ni(II)-induced apoptosis, suggesting that NADPH oxidase-derived reactive oxygen species and subsequent signaling could contribute to this event. This is an important finding since increased apoptosis mediated by nickel may disrupt the physiological activities of neutrophils, with potential impact in its immunological and antimicrobial role.

Heme oxygenase/carbon monoxide-biliverdin pathway down regulates neutrophil rolling, adhesion and migration in acute inflammation

Heme oxygenase (HO) activity is known to down‐regulate inflammatory events. Here, we address the role of HO and its metabolites, carbon monoxide (CO) and biliverdin (BVD), in leukocyte rolling, adhesion and neutrophil migration during inflammatory processes.

Heme Inhibits Human Neutrophil Apoptosis: Involvement of Phosphoinositide 3-Kinase, MAPK, and NF-κB

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1–50 μM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-κB translocation reversed heme antiapoptotic effect. NF-κB (p65 subunit) nuclear translocation and IκB degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.

Novel lipid mediator aspirin-triggered lipoxin A4 induces heme oxygenase-1 in endothelial cells

Lipoxins (LX) and aspirin-triggered LX (ATL) are eicosanoids generated during inflammation via transcellular biosynthetic routes that elicit distinct anti-inflammatory and proresolution bioactions, including inhibition of leukocyte-mediated injury, stimulation of macrophage clearance of apoptotic neutrophils, repression of proinflammatory cytokine production, and inhibition of cell proliferation and migration. Recently, it was reported that aspirin induces heme oxygenase-1 (HO-1) expression on endothelial cells (EC) in a COX-independent manner, what confers protection against prooxidant insults. However, the underlying mechanisms remain unclear. In this study, we investigated whether an aspirin-triggered lipoxin A(4) stable analog, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A(4) (ATL-1) was able to induce endothelial HO-1. Western blot analysis showed that ATL-1 increased HO-1 protein expression associated with increased mRNA levels on EC in a time- and concentration-dependent fashion. This phenomenon appears to be mediated by the activation of the G protein-coupled LXA(4) receptor because pertussis toxin and Boc-2, a receptor antagonist, significantly inhibited ATL-1-induced HO-1 expression. We demonstrate that treatment of EC with ATL-1 inhibited VCAM and E-selectin expression induced by TNF-alpha or IL-1beta. This inhibitory effect of the analog is modulated by HO-1 because it was blocked by SnPPIX, a competitive inhibitor that blocks HO-1 activity. Our results establish that ATL-1 induces HO-1 in human EC, revealing an undescribed mechanism for the anti-inflammatory activity of these lipid mediators.

Circulating cell membrane microparticles transfer heme to endothelial cells and trigger vasoocclusions in sickle cell disease

Intravascular hemolysis describes the relocalization of heme and hemoglobin (Hb) from erythrocytes to plasma. We investigated the concept that erythrocyte membrane microparticles (MPs) concentrate cell-free heme in human hemolytic diseases, and that heme-laden MPs have a physiopathological impact. Up to one-third of cell-free heme in plasma from 47 patients with sickle cell disease (SCD) was sequestered in circulating MPs. Erythrocyte vesiculation in vitro produced MPs loaded with heme. In silico analysis predicted that externalized phosphatidylserine (PS) in MPs may associate with and help retain heme at the cell surface. Immunohistology identified Hb-laden MPs adherent to capillary endothelium in kidney biopsies from hyperalbuminuric SCD patients. In addition, heme-laden erythrocyte MPs adhered and transferred heme to cultured endothelial cells, inducing oxidative stress and apoptosis. In transgenic SAD mice, infusion of heme-laden MPs triggered rapid vasoocclusions in kidneys and compromised microvascular dilation ex vivo. These vascular effects were largely blocked by heme-scavenging hemopexin and by the PS antagonist annexin-a5, in vitro and in vivo. Adversely remodeled MPs carrying heme may thus be a source of oxidant stress for the endothelium, linking hemolysis to vascular injury. This pathway might provide new targets for the therapeutic preservation of vascular function in SCD.

Jararhagin, a snake venom metalloproteinase, induces a specialized form of apoptosis (anoikis) selective to endothelial cells

Jararhagin is a snake venom metalloproteinase (SVMP) from Bothrops jararaca involved in several hemostatic and inflammatory disorders that occur in human envenomings. In this study, we evaluated the effect of jararhagin on endothelial cells (tEnd). The exposure of tEnd to jararhagin (20 and 40μg/ml) resulted in apoptosis with activation of pro-caspase-3 and alterations in the ratio between Bax/Bcl-xL. We observed that apoptosis was followed by decrease of cell viability and the loss of cell adhesion. Jararhagin induced changes in cell shape with a decrease in cell spreading, rounding up and detachment. This was accompanied by a rearrangement of actin network and a decrease in FAK association to actin and in tyrosine phosphorylated proteins. Morphological alterations and apoptosis were abolished when jararhagin catalytic activity was inhibited, indicating the importance of catalysis. Treatment of murine peritoneal adherent cells or fibroblasts with jararhagin did not result in apoptosis. The data indicate that the pro-apoptotic effect of jararhagin is selective to endothelial cells, interfering with the adhesion mechanisms and inducing anoikis. The present model might be useful for the study of the relationships between the architectural changes in the cytoskeleton and the complex phenomenon named anoikis.

Aspirin-triggered lipoxin A4 blocks reactive oxygen species generation in endothelial cells: A novel antioxidative mechanism

Lipoxins and their aspirin-triggered carbon-15 epimers have emerged as mediators of key events in endogenous anti-inflammation and resolution. However, the implication of these novel lipid mediators on cardiovascular diseases such as hypertension, atherosclerosis, and heart failure has not been investigated. One of the major features shared by these pathological conditions is the increased production of reactive oxygen species (ROS) generated by vascular NAD(P)H oxidase activation. In this study, we have examined whether an aspirin-triggered lipoxin A (4) analog (ATL-1) modulates ROS generation in endothelial cells (EC). Pre-treatment of EC with ATL-1 (1 - 100 nM) completely blocked ROS production triggered by different agents, as assessed by dihydrorhodamine 123 and hydroethidine. Furthermore, ATL-1 inhibited the phosphorylation and translocation of the cytosplamic NAD(P)H oxidase subunit p47 (phox) to the cell membrane as well as NAD(P)H oxidase activity. Western blot and immunofluorescence microscopy analyses showed that ATL-1 (100 nM) impaired the redox-sensitive activation of the transcriptional factor NF- kappaB, a critical step in several events associated to vascular pathologies. These results demonstrate that ATL-1 suppresses NAD(P)H oxidase-mediated ROS generation in EC, strongly indicating that lipoxins may play a protective role against the development and progression of cardiovascular diseases.

Induction of NOS in rat blood PMN in vivo and in vitro: modulation by tyrosine kinase and involvement in bactericidal activity.

Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NOx) by blood polymorphonuclear neutrophils (PMN) in vitro. Both dexamethasone and L‐NMMA, added in vitro to neutrophil cultures, inhibited the production of NO. On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5‐lipoxygenase or with a platelet‐activating factor (PAF) antagonist. The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B4, and interleukin‐8) and different cytokines [interleukin‐1, tumor necrosis factor α, and interferon‐γ (IFN‐γ)] showed that only IFN‐γ was able to induce the production of high amounts of NO. This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN. The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN‐γ, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase. We also showed that NO produced by IFN‐γ activated rat blood PMN involved in the killing of Pseudomonas aeruginosa. J. Leukoc. Biol. 65: 508–514; 1999.

Host Cell Lipid Bodies Triggered by Trypanosoma cruzi Infection and Enhanced by the Uptake of Apoptotic Cells Are Associated With Prostaglandin E2 Generation and Increased Parasite Growth

Lipid bodies (lipid droplets) are lipid-rich organelles with functions in cell metabolism and signaling. Here, we investigate the mechanisms of Trypanosoma cruzi-induced lipid body formation and their contributions to host-parasite interplay. We demonstrate that T. cruzi-induced lipid body formation in macrophages occurs in a Toll-like receptor 2-dependent mechanism and is potentiated by apoptotic cell uptake. Lipid body biogenesis and prostaglandin E₂ (PGE₂) production triggered by apoptotic cell uptake was largely dependent of α(v)β₃ and transforming growth factor-β signaling. T. cruzi-induced lipid bodies act as sites of increased PGE synthesis. Inhibition of lipid body biogenesis by the fatty acid synthase inhibitor C75 reversed the effects of apoptotic cells on lipid body formation, eicosanoid synthesis, and parasite replication. Our findings indicate that lipid bodies are highly regulated organelles during T. cruzi infection with roles in lipid mediator generation by macrophages and are potentially involved in T. cruzi-triggered escape mechanisms.

ATL-1, an analogue of aspirin-triggered lipoxin A4, is a potent inhibitor of several steps in angiogenesis induced by vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is the most important proangiogenic protein. We have demonstrated that ATL‐1, a synthetic analogue of aspirin‐triggered lipoxin A4, inhibits VEGF‐induced endothelial cell (EC) migration. In the present study, we investigated the effects of ATL‐1 in several other actions stimulated by VEGF.