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PHAGOCYTES
From the Departamento de Farmacologia, Instituto de
Biologia, Universidade do Estado do Rio de Janeiro; and Departamento de
Bioquimica Médica, Instituto de Ciências Biomédicas,
Centro de Ciências da Saúde, Universidade Federal do Rio de
Janeiro, Brazil.
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 dose-dependent 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 µM 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 µM) 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 K1/2 of 5 µM. Taken together,
these results suggest a role for hemin as a proinflammatory agent able
to induce polymorphonuclear neutrophil activation in situations of
clinical relevance, such as hemolysis or hemoglobinemia.
(Blood. 2002;99:4160-4165) Heme 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.1-3
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 heme-binding 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. This state correlates with the enhanced expression of
adhesion molecules on activated endothelial cells, leukocytes, and
reticulocytes,14-16 but the specific agent that triggers
this process remains unknown.
Leukocyte migration into tissues is the hallmark of all types of
inflammatory responses. Polymorphonuclear neutrophils (PMNs) are
blood-borne inflammatory cells with oxidative and proteolytic potential
that are usually the first cells involved in pathogen recognition,
playing an essential role in the host defense against invading
microorganisms. The inflammatory process can be amplified by the
neutrophils themselves through the production of arachidonic acid-derived bioactive lipids such as leucotriene B4 (LTB4), cytokines (ie, interferon- In the present paper, we provide evidence that heme can modulate
several neutrophil-related responses either by a PKC-dependent pathway
(such as migration, actin cytoskeleton reorganization, and ROS
production) or by a PKC-independent mechanism (IL-8 expression). The
data reported here indicate that heme can act as a physiologically relevant proinflammatory signaling molecule that may be associated with
the development of inflammation in hemolytic diseases.
Animals
Hemin
Acute pleurisy in rats Acute pleurisy was induced as described earlier.22 Briefly, hemin was injected into the thoracic cavity of rats (30-300 nmol/cavity) in a volume of 100 µL. The animals were killed 4 hours after injection under ether anesthesia, and their thoracic cavities were opened and rinsed with 3 mL PBS containing heparin (10 IU/mL). Total leukocytes in the pleural fluid were determined on Neubauer chambers after dilution in Turk solution (Laborclin, São Paulo, Brazil). Differential counting of leukocytes was carried out on May-Grünwald-Giemsa-stained slides.Human neutrophil isolation Neutrophils were isolated from ethylenediaminetetraacetic acid (EDTA) (0.5%)-treated peripheral venous blood of healthy human volunteers using a 4-step discontinuous Percoll (Sigma) gradient.23 Erythrocytes were removed by hypotonic lysis, and PMNs were resuspended in RPMI-1640 medium (Sigma) or Dulbecco modified Eagle medium (DMEM). Neutrophil purity and viability were always higher than 99% and 96%, respectively. The trypan blue assay was used to evaluate hemin toxicity, and no cellular lysis was observed in the presence of up to 50 µM hemin for 2 hours (data not shown).Neutrophil chemotaxis Chemotaxis was assayed in a 48-well Boyden chamber (Neuprobe Microchemotaxis System) using 5 µM polyvinylpyrrolidone-free polycarbonate filters, as described earlier (Cabin John, MD).24 Inducers of neutrophil chemotaxis were added to the bottom wells as described in the figure legends. Hemin was added in the presence or in the absence of 1% human serum albumin. Neutrophils suspended in RPMI-1640 medium (106 cells/mL; 50 µL) were added to the top wells and incubated for 60 minutes at 37°C under a 5% CO2 atmosphere. Following incubation, the filters were removed from the chambers, fixed, and stained with a Diff-Quick stain kit (Baxter Travenol Labs, ON, Canada). Neutrophils that migrated to the bottom of the filters were counted using a 100× objective in at least 5 randomly chosen fields. Results were representative of 3 different experiments performed in triplicate for each sample. Neutrophil migration toward RPMI-1640 medium alone (random chemotaxis) was used as a negative control.Actin content of cytoskeleton Cytoskeleton fractions were derived from human neutrophils treated as indicated in the figure legends. Briefly, suspensions of 5 × 106 cells/mL were washed 3 times with ice-cold PBS and lysed in 50 mM morpholinoethanol sulfonide (MES) buffer, pH 6.4, 1 mM MgCl2, 10 mM EDTA, 1% Triton X-100 (Amersham Pharmacia Biotech), 1 µg/mL DNase, 0.5 µg/mL RNase, and the following protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 µM aprotinin, 1 µM leupeptin, and 1 µM soybean trypsin inhibitor (Sigma). After centrifugation at 13 000g for 10 minutes at 25°C, supernatants were collected and pellets were suspended in the same buffer under vigorous agitation. Protein content of both pellets and supernatants was determined by the Bradford method.25 Sodium dodecyl sulfate (SDS) sample buffer was added and fractions were boiled for 3 minutes; 30-mg protein samples were separated on 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).26 After that, proteins were transferred to a nitrocellulose sheet (Hybond-C Extra; Amersham Life Science), followed by incubation with Tween-TBS (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 0.1% Tween-20) containing 1% bovine serum albumin. Actin was probed by overnight incubation at 4°C with monoclonal anti- -actin antibody
(1:500; Santa Cruz Biotechnology, Santa Cruz, CA). After
extensive washing in Tween-TBS, nitrocellulose sheets were incubated
with anti-mouse IgG antibody conjugated to biotin (1:1000; Sigma) for 1 hour and then incubated with streptavidin-conjugated horseradish
peroxidase (1:1000; Caltag Laboratories, Burlingame, CA).
Immunoreactive proteins were visualized by 3,3'-diaminobenzidine (Sigma) staining.
Assays of superoxide generation Nitroblue tetrazolium reduction. To observe superoxide production, we incubated human PMNs (106 cells/mL) with 0.05% nitroblue tetrazolium reduction (NBT) (Sigma) in DMEM in the presence or absence of phorbol 14-myristate 13-acetate (PMA; Sigma) or hemin for 1 hour at 37°C. After that, cells were cytocentrifuged onto glass slides, stained with 1% safranin (Sigma),27 and observed under a light microscope. Cells showing formazan deposits were counted in at least 5 random fields. Results were representative of 3 independent experiments. Cytochrome c reduction.
The production of superoxide by human neutrophils was also measured by
the superoxide dismutase-inhibitable reduction of ferricytochrome c (Sigma), as previously described.28 Briefly,
PMNs (106 cells/mL) were preincubated for 5 minutes with or
without 100 µM diphenyleneiodonium (DPI; Sigma) in DMEM. The
cells were then stimulated with heme at different concentrations, as
described in the figure legend. Cytochrome c reduction was
quantified 30 minutes later as an increase in
A550
( Neutrophil PKC activity Human neutrophils (2 × 105 cells/microtube) were treated for 30 minutes with different concentrations of hemin, as indicated in the figure legends. After that, cells were quickly disrupted by sonication in 0.5 M sucrose, 5 mM ethyleneglycoltetraacetic acid (EGTA), 2 mM EDTA, 2 mM dithiothreitol, and 0.1% Triton X-100 and centrifuged at 40 000 rpm for 10 minutes to remove cell debris, and the supernatant was immediately used for enzyme activity determination. PKC activity was measured by [ 32P]-adenosine triphosphate (ATP) phosphorylation of
myelin basic protein (MBP4-14; Calbiochem) substrate
peptide in the presence or absence of 10 nM bis-indoylmaleimide
III (BIM; Calbiochem). Briefly, a 20-µL aliquot of the
previously centrifuged cell lysate was added to 80 µL reaction buffer
containing 12.5 mM HEPES
(4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid) (pH 8.0),
12.5 mM magnesium acetate, 0.4 mM calcium chloride, 0.1 mg/mL
MBP4-14 peptide, and 20 µg phosphatidylserine. Reactions were started by the addition of [32P]-ATP to a final
concentration of 10 µM (500-1000 cpm/pmol). After 5 minutes at
30°C, 25-µL aliquots of the reaction medium were transferred to a
phosphocellulose filter and washed 3 times for 15 minutes each with
ice-cold 20% trichloroacetic acid. Incorporated radioactivity was
determined by liquid scintillation. Each treatment was assayed in
duplicate. Background reactions to determine nonspecific phosphorylation received the same components except for
phosphatidylserine, which was replaced by 5 mM EGTA.
Reverse transcription-polymerase chain reaction analysis of IL-8 transcripts Neutrophils (5 × 106 cells) were incubated for 4 hours with different effectors, as indicated in the figure legend. Total RNA was then extracted with the QuickPrep RNA extraction kit (Amersham Pharmacia) following the manufacturer's instructions. Semiquantitative RT-PCR was performed basically according to the reverse transcription-polymerase chain reaction (RT-PCR) kit from Perkin-Elmer. RT-PCR for IL-8 expression was performed with primer IL-8for (5'-TGGCTCTCTTGGCAGCCTTC-3') and primer IL-8rev (5'-TCTCCACAACCCTCTGCACC-3'). Control reactions were performed with primers GAP1for (5'-GGTGAAGGTCGGAGTCAACGGA-3') and GAP2rev (5'-GAGGGATCTCGCTCCTGGAAGA-3'), which are specific for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Conditions for PCR were: 94°C, 1 minute; 50°C, 1 minute; and 72°C, 40 seconds. To achieve quantitative amplification of IL-8 transcripts, we used 18 cycles to keep the reaction within its exponential phase. All reactions were carried out in the presence of 1 µCi (0.037 MBq)[32P]-CTP per tube. The amplification
products were separated using 8% PAGE, dried, and exposed to Kodak
X-Omat film. Data are presented as the ratio of IL-8 to GAPDH product
obtained by densitometry from the autoradiograms (BioImager; BioRad).
Hemin induces neutrophil migration in vivo and in vitro The effect of hemin on neutrophil recruitment was investigated in vivo and in vitro using different experimental approaches. As illustrated in Figure 1, free hemin can promote neutrophil recruitment in vivo. The intrathoracic administration of hemin induced a dose-dependent neutrophil accumulation in rat pleural cavities, which was observed 4 hours after the injection. The stimulatory effect was already evident with 30 nmol/cavity and increased 7-fold at the highest dose administered (300 nmol/cavity). Four hours after hemin injection, only the pleural neutrophil population was increased, and no changes in the number of mononuclear cells were observed.
On the basis of these results, we investigated whether hemin could also
directly stimulate neutrophil migration in vitro. For these studies,
human PMNs were allowed to migrate toward hemin in a Boyden chamber.
Because albumin is a plasma constituent able to bind heme, we checked
whether the presence of 150 µM human serum albumin (HSA) would modify
neutrophil recruitment. The binding constant of the association of
hemin to HSA is 3.6 × 107
M
Hemin induces alterations in actin cytoskeleton dynamics Motile activities of neutrophils (chemotaxis and phagocytosis) are generated by dynamic alterations of actin filaments. Under resting conditions, human PMNs exhibit a high content of unpolymerized actin (G-actin), whereas the polymerized actin (F-actin), associated with the Triton-insoluble cytoskeleton fraction, is almost undetectable (Figure 2B; control). Incubation with PMA increased actin polymerization, decreasing G-actin content (Figure 2B; PMA). It is clear that hemin induced alterations in actin cytoskeleton dynamics similar to those generated by incubation with PMA (Figure 2B; hemin). However, in the presence of BIM (Figure 2B; hemin + BIM), hemin-induced actin polymerization was prevented, and cells showed a G/F-actin profile similar to that in the control group.Hemin induces superoxide production by human neutrophils One of the most characteristic aspects of phagocyte activation is the increase in the production of superoxide anion, the so-called oxidative burst. Stimulation of neutrophils by hemin resulted in increased superoxide formation, assessed through the intracellular precipitation of the insoluble formazan crystals induced by the reduction of NBT by superoxide (Figure 3A). Activation of superoxide production by hemin was already noted at 2 µM hemin, reaching maximal values at 20 µM, at which point 70% of the cells presented intracellular formazan crystals. Incubation of neutrophils with 30 nM PMA, used as routine positive control, resulted in 96% of cells producing formazan, compared with the control group incubated in medium alone, in which only 8% of the cells were activated. The ability of hemin to trigger the oxidative burst in neutrophils was further investigated using the method of Pick and Mizel,28 with minor modifications. When neutrophils were incubated with hemin, superoxide production increased up to 4.4-fold in a hemin dose-dependent fashion (Figure 3B), confirming that hemin can induce the oxidative burst in human PMNs. Preincubation of neutrophils with the NADPH oxidase inhibitor DPI abrogated superoxide production evoked by hemin, suggesting that the stimulatory effect promoted by hemin was actually on the NADPH oxidase system.
Hemin activates human neutrophil PKC Because migration and superoxide production by neutrophils are functions that can be modulated by a PKC pathway, and on the basis of our previously published data showing that hemin stimulated PKC activity in an insect cell,31 we investigated whether hemin could also activate PKC in neutrophils. To address this question, we incubated human neutrophils with hemin (0.1-20 µM) and evaluated PKC activity through phosphorylation of MBP peptide. As shown in Figure 4, PKC activity stimulation by hemin was dose-dependent, with a K1/2 of 5 µM, and was prevented by BIM, indicating that the PKC up-regulation observed with the insect model also occurs in human neutrophils.
Hemin activates IL-8 expression in human neutrophils Recruitment of neutrophils into inflammatory sites can be modulated by the presence of several chemokines. One of the most important chemoattractants for neutrophils is the cytokine IL-8, which also stimulates adhesion, respiratory burst, and degranulation.32 To investigate an additional parameter of neutrophil activation, we evaluated the mRNA levels of IL-8 in cells treated with hemin. Incubation of human neutrophils with 3 µM heme resulted in a 40-fold increase in IL-8 expression compared with the control group (Figure 5). When heme was added in the presence of albumin, induction of IL-8 expression was also observed, but to a lesser extent, suggesting that binding of heme to plasma components can modulate the biologic effects of this molecule. In contrast to the effects on recruitment and oxidative burst, induction of IL-8 expression promoted by hemin was not reversed in the presence of BIM, ruling out the participation of PKC in this phenomenon. As a positive control of IL-8 expression, neutrophils were incubated with lipopolysaccharide (LPS) and produced the expected increase in mRNA.
Hemorrhagic episodes and severe hemolysis are often associated with tissue injury that can trigger the inflammatory response characterized by intense leukocyte infiltration. We show here that heme can promote neutrophil migration in vivo, suggesting a direct proinflammatory effect of this blood component. The heme toxicity may arise in environments where there is pronounced hemolysis, for instance, localities exposed to high erythrocyte shear forces or turbulent blood flow. Very high heme plasma levels (>20 µM) have been reported in hemolytic diseases such as sickle cell anemia.33 The demonstration that hemopexin levels are decreased in patients with a variety of hematologic disorders suggests the importance of other plasma constituents able to bind heme during hemolytic episodes.34 Although albumin is capable of drawing hemin away from red cell membranes, the detection of heme-albumin complexes portends an extremely poor prognosis for patients with life-threatening hemorrhagic shock.35 In our experiments with rats, intrathoracic injection of hemin induced an acute inflammatory reaction, characterized by edema formation (data not shown) and intense accumulation of neutrophils in the pleural cavities. The stimulation of neutrophil migration in vivo and in vitro reported here suggests that free heme has the potential to serve as an endogenous chemoattractant. Association of hemin with human albumin did not reduce its propensity to promote neutrophil migration. This is consistent with the fact that binding of heme to albumin36 does not prevent heme uptake, and therefore, after dissociation, heme will eventually diffuse into circulating and endothelial cells. A primary event in the inflammatory response is the recruitment of neutrophils into sites of inflammation. In vivo or in vitro, leukocyte migration induced by different chemoattractants is promptly stimulated by phorbol esters, indicating that PKC stimulation can lead to neutrophil chemotaxis. In our experiments, the PKC inhibitors BIM and calphostin C efficiently blocked neutrophil migration induced by hemin in vitro, consistent with a link between migration and PKC. Another important event in the inflammatory response is the production of ROS by activated phagocytes. We show in the present study that hemin triggers the oxidative burst and promotes actin polymerization in human PMNs, indicating that hemin is a potent neutrophil activator such as formyl peptides, IL-8, platelet activation factor, and C5a.37 Incubation with hemin induced profound alterations in the actin network and an increase of F-actin content. This event seems to be related to the PKC activation promoted by hemin because the PKC inhibitor BIM counteracts this hemin-induced actin polymerization. Thus, it is conceivable to suggest that hemin triggers human neutrophils by means of PKC up-regulation. Incubation of human neutrophils with hemin promptly activated PKC in a concentration-dependent manner, and this effect was completely inhibited by a selective PKC inhibitor. This is consistent with our previous report, in which heme was shown to be a potent activator of PKC in an insect cell model.31 We are currently investigating the molecular basis of the PKC activation reported here. Because of its highly hydrophobic nature and considering that at neutral pH, the 2 lateral carboxyl groups of hemin are dissociated, we believe that a possible explanation for the PKC activator effect reported here might be interaction of hemin carboxyl groups with the phospholipid-binding regulatory domains of the enzyme. However, an indirect effect, such as oxidation of regulatory and/or binding domains, should not be discarded because it has been shown that PKC can be activated by superoxide anion, redox cycling quinones, and micromolar levels of periodate.38,39 Because hemin was able to activate PKC in PMNs, a possible direct
implication is the modulation of PKC-related gene expression. Expression of proinflammatory and immunoregulatory cytokines rapidly increases in the lung after hemorrhage,40 but the specific
agent responsible for the onset of these proinflammatory responses
remains unknown. It was recently shown that hemin can boost the
expression of We also investigated whether hemin could modulate the expression
of inflammatory mediators by human neutrophils. A key mediator for the
migration of neutrophils from the circulation is the Taken together, the data reported here are consistent with the assumption that hemin could act as a signaling molecule involved in the triggering of inflammatory processes often associated with hemolysis.
We express our gratitude to Dr Patrícia T. Bozza and Martha Sorenson for a critical reading of the manuscript and to Rosane M. M. Costa and S. R. Cássia for the excellent technical assistance.
Submitted May 14, 2001; accepted January 18, 2002.
Supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenadoria de Aperfeiçoamento do Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (Finep), Programas de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT), Howard Hughes Medical Institute (HHMI), John Simon Guggenheim Memorial Foundation, Fundação de Apoio a Pesquisa do Estado do Rio de Janeiro (FAPERJ), and Programa de Núcleos de Excelência (PRONEX).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Aurélio V. Graça-Souza, Departamento de Farmacologia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; e-mail: avsouza{at}bioqmed.ufrj.br.
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© 2002 by The American Society of Hematology.
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J. D. Dimitrov, L. T. Roumenina, V. R. Doltchinkova, N. M. Mihaylova, S. Lacroix-Desmazes, S. V. Kaveri, and T. L. Vassilev Antibodies Use Heme as a Cofactor to Extend Their Pathogen Elimination Activity and to Acquire New Effector Functions J. Biol. Chem., September 14, 2007; 282(37): 26696 - 26706. [Abstract] [Full Text] [PDF]
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B. N. Porto, L. S. Alves, P. L. Fernandez, T. P. Dutra, R. T. Figueiredo, A. V. Graca-Souza, and M. T. Bozza Heme Induces Neutrophil Migration and Reactive Oxygen Species Generation through Signaling Pathways Characteristic of Chemotactic Receptors J. Biol. Chem., August 17, 2007; 282(33): 24430 - 24436. [Abstract] [Full Text] [PDF]
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R. T. Figueiredo, P. L. Fernandez, D. S. Mourao-Sa, B. N. Porto, F. F. Dutra, L. S. Alves, M. F. Oliveira, P. L. Oliveira, A. V. Graca-Souza, and M. T. Bozza Characterization of Heme as Activator of Toll-like Receptor 4 J. Biol. Chem., July 13, 2007; 282(28): 20221 - 20229. [Abstract] [Full Text] [PDF]
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 C. Pierrot, E. Adam, D. Hot, S. Lafitte, M. Capron, J. D. George, and J. Khalife Contribution of T Cells and Neutrophils in Protection of Young Susceptible Rats from Fatal Experimental Malaria J. Immunol., February 1, 2007; 178(3): 1713 - 1722. [Abstract] [Full Text] [PDF]
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M. Walther, J. Woodruff, F. Edele, D. Jeffries, J. E. Tongren, E. King, L. Andrews, P. Bejon, S. C. Gilbert, J. B. De Souza, et al. Innate Immune Responses to Human Malaria: Heterogeneous Cytokine Responses to Blood-Stage Plasmodium falciparum Correlate with Parasitological and Clinical Outcomes J. Immunol., October 15, 2006; 177(8): 5736 - 5745. [Abstract] [Full Text] [PDF]
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 D. J. Schaer, C. A. Schaer, P. W. Buehler, R. A. Boykins, G. Schoedon, A. I. Alayash, and A. Schaffner CD163 is the macrophage scavenger receptor for native and chemically modified hemoglobins in the absence of haptoglobin Blood, January 1, 2006; 107(1): 373 - 380. [Abstract] [Full Text] [PDF]
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 R. Dey, A. Sarkar, N. Majumder, S. Bhattacharyya (Majumdar), K. Roychoudhury, S. Bhattacharyya, S. Roy, and S. Majumdar Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis Infect. Immun., December 1, 2005; 73(12): 8334 - 8344. [Abstract] [Full Text] [PDF]
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 V. Nascimento-Silva, M. A. Arruda, C. Barja-Fidalgo, C. G. Villela, and I. M. Fierro Novel lipid mediator aspirin-triggered lipoxin A4 induces heme oxygenase-1 in endothelial cells Am J Physiol Cell Physiol, September 1, 2005; 289(3): C557 - C563. [Abstract] [Full Text] [PDF]
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 F. A. Lara, U. Lins, G. H. Bechara, and P. L. Oliveira Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus J. Exp. Biol., August 15, 2005; 208(16): 3093 - 3101. [Abstract] [Full Text] [PDF]
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 R. P. Rother, L. Bell, P. Hillmen, and M. T. Gladwin The Clinical Sequelae of Intravascular Hemolysis and Extracellular Plasma Hemoglobin: A Novel Mechanism of Human Disease JAMA, April 6, 2005; 293(13): 1653 - 1662. [Abstract] [Full Text] [PDF]
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 M. Bitzan, B. B. Bickford, and G. H. Foster Verotoxin (Shiga Toxin) Sensitizes Renal Epithelial Cells to Increased Heme Toxicity: Possible Implications for the Hemolytic Uremic Syndrome J. Am. Soc. Nephrol., September 1, 2004; 15(9): 2334 - 2343. [Abstract] [Full Text] [PDF]
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M. A. Arruda, A. G. Rossi, M. S. de Freitas, C. Barja-Fidalgo, and A. V. Graca-Souza Heme Inhibits Human Neutrophil Apoptosis: Involvement of Phosphoinositide 3-Kinase, MAPK, and NF-{kappa}B J. Immunol., August 1, 2004; 173(3): 2023 - 2030. [Abstract] [Full Text] [PDF]
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 N. Osakabe, A. Yasuda, M. Natsume, and T. Yoshikawa Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine two-stage skin model Carcinogenesis, April 1, 2004; 25(4): 549 - 557. [Abstract] [Full Text] [PDF]
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X. Liu and Z. Spolarics Methemoglobin is a potent activator of endothelial cells by stimulating IL-6 and IL-8 production and E-selectin membrane expression Am J Physiol Cell Physiol, November 1, 2003; 285(5): C1036 - C1046. [Abstract] [Full Text] [PDF]
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F. A. D. T. G. Wagener, H. E. van Beurden, J. W. von den Hoff, G. J. Adema, and C. G. Figdor The heme-heme oxygenase system: a molecular switch in wound healing Blood, July 15, 2003; 102(2): 521 - 528. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
), hydrogen peroxide
(H2O2), hydroxyl radical (.OH), and
hypochlorous acid (HOCl).
550 = 21 × 103 M
1 and glycoprotein IV (CD 36) are expressed on circulating reticulocytes in sickle cell anemia.
Blood.
1993;82:3548-3555
B regulation and cytokine expression.
J Clin Invest.
1997;99:1516-1524