Heme, a ubiquitous iron-containing compound, is present in large amounts in many cells and is inherently dangerous, particularly when it escapes from intracellular sites. The release of heme from damaged cells and tissues is supposed to be higher in diseases such as malaria and hemolytic anemia or in trauma and hemorrhage. We investigated here the role of free ferriprotoporphyrin IX (hemin) as a proinflammatory molecule, with particular attention to its ability to activate neutrophil responses. Injecting hemin into the rat pleural cavity resulted in a dosedependent migration of neutrophils, indicating that hemin is able to promote the recruitment of these cells in vivo. In vitro, hemin induced human neutrophil chemotaxis and cytoskeleton reorganization, as revealed by the increase of neutrophil actin polymerization. Exposure of human neutrophils to 3 M hemin activated the expression of the chemokine interleukin-8, as demonstrated by quantitative reverse-transcription polymerase chain reaction, indicating a putative molecular mechanism by which hemin induces chemotaxis in vivo. Brief incubation of human neutrophils with micromolar concentrations of hemin (1-20 M) triggered the oxidative burst, and the production of reactive oxygen species was directly proportional to the concentration of hemin added to the cells. Finally, we observed that human neutrophil protein kinase C was activated by hemin in vitro, with a K 1/2 of 5 M. Taken IntroductionHeme released from hemeproteins has been shown to promote the formation of oxygen radicals, playing a role as a catalyst in the oxidation of lipids, proteins, and DNA. [1][2][3] In addition, free heme can promptly bind to and oxidize low-density lipoprotein, acting as a biologically relevant lipoprotein oxidant. 4 Hemoglobin (probably because of the release of free heme and heme iron) may contribute to the acute renal failure often seen after episodes of intravascular hemolysis. 5,6 In fact, it has been proposed that heme could be considered one causative agent in organ failure after ischemia-reperfusion because heme-oxygenase is induced in heart and kidney. 7 In normal conditions, diverse species produce avid hemebinding plasma proteins, such as hemopexin, that efficiently remove most of the heme produced intravascularly, 8 thus preventing nonspecific cellular heme uptake and heme-catalyzed oxidation reactions. However, pathologic situations of increased hemolysis can lead to very high levels of free heme, as in malaria, 9 sickle cell disease, 10 HELLP (hemolysis, elevated liver levels, and low platelet count) syndrome, 11 or regions with turbulent blood flow. 12 Very little work has been done to assess the consequences of the interaction of free heme with intact cells. It has been demonstrated that free heme is promptly incorporated into endothelial cells in vitro and that this association potentiates the oxidative damage induced by chemical agents. 13 It is interesting to note that patients suffering from sickle cell disease often exhibit a low-grade chronic inflammation...

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.

The deregulation of inflammatory response during sepsis seems to reflect the overproduction of mediators, which suppress leukocyte functions. We investigated the intracellular mechanisms underlying the inability of neutrophils from severe septic patients to migrate toward chemoattractants. Patients IntroductionSepsis is a complex clinical syndrome resulting from a damaging host response to infection. 1 In the United States, more than 700 000 patients per year develop sepsis, with mortality rates reported to vary between 30% and 70%, despite the best available supportive care. 2 Polymorphonuclear neutrophils (PMNs) play the first line in the host defense against microorganisms, being recruited to the inflammatory sites by chemoattractants such as leukotriene B 4 (LTB 4 ) and chemokines. 3,4 Once emigrated, these leukocytes are able to phagocytose and to generate large amounts of reactive oxygen and nitrogen species, such as hydrogen peroxide and nitric oxide, which are crucial products for the microbicidal activity of these cells. 5,6 As neutrophils appear to play a crucial role in the control of the infectious process, one can hypothesize that a deficient migratory ability of neutrophils may aggravate infections. Indeed, impairment of neutrophil migration has been reported in leukemia, 7 diabetes, 8 and AIDS, 9 diseases associated with high susceptibility to infection. Furthermore, previous studies from our group showed that failure of neutrophil migration is observed in severe sepsis induced by cecal ligation and puncture and Staphylococcus aureus administration. 10,11 In these lethal models, failure of neutrophil migration to the site of infection was accompanied by increased numbers of bacteria in the peritoneal fluid and blood. Conversely, in sublethal infection in which massive neutrophil migration was observed, bacterial infection was restricted to the peritoneal cavity, and the animals exhibited a low mortality rate. 10,11 More recently, we have also reported that blood neutrophils obtained from patients with sepsis failed to respond in vitro to the chemotactic stimuli FMLP and LTB 4 . This unresponsiveness was directly associated to a poor prognosis. 12 Evidence from literature suggests that the high levels of circulating cytokines/chemokines observed in severe sepsis may mediate the impairment of neutrophil migration, in addition to being involved in the deleterious physiopathologic findings of the disease, such as coagulation disorders, cardiovascular collapse, and organ failure. 13 The intravenous administration of tumor necrosis factor-␣ (TNF-␣) or interleukin-8 (IL-8) inhibited neutrophil migration to mouse peritoneal cavity and anti-TNF-␣ antibody partially prevented the inhibition of neutrophil migration in endotoxemia model. 14 However, the molecular mechanisms involved in the reduced ability of neutrophils to migrate during sepsis were not completely clarified.Independent of their chemical nature, most chemoattractants exert their action via binding to specific G protein-coupled receptors (GP...