Departments of 1Cell Biology and Molecular Medicine and 2Surgery, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103
Submitted 25 April 2003 ; accepted in final form 25 June 2003
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ABSTRACT |
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human umbilical vein endothelial cells; cytokine; chemokine; adhesion molecule; hemolysis; hemoglobin; hemin; nuclear factor-B
It is well known that hemoglobin and hemin sensitize endothelial cells to oxidative damage. It has been shown by several independent studies that the presence of heme or hemin augments H2O2-mediated endothelial cell injury (1, 11, 22, 34). It has also been demonstrated that hemoglobin, methemoglobin, and hemin markedly upregulate hemoxygenase (HO)-1 (2, 18, 48). Heme metabolism through HO produces carbon monoxide and free iron, which have been shown to play an important role in the regulation of vascular tone and oxidant damage, respectively (1, 33). HO-1 has also been shown to function as a chaperone and is considered to be a heat shock protein with cytoprotective effects upon its induction (4, 8, 13). These previous investigations mainly focused on the sensitizing effects of hemoglobin, heme, and hemin on oxidative stress and associated cell injury; however, only a few studies have investigated the potential role of hemoglobin derivatives in the inflammatory response by endothelial cells.
Studies testing the effects of liposome-encapsulated hemoglobin, a potential blood substitute, have demonstrated that free circulating heme or hemin stimulates IL-6 expression in macrophage-rich tissues (49). Acute hemolysis increased blood cytokines in humans (12). Hemoglobin was also shown to increase IL-8 release by polymorphonuclear phagocytes (29) and to augment IL-1-induced TNF-
release by macrophages (43a). Hemin in vivo induced neutrophil migration, IL-8 release, and oxidative burst (16). Whereas heme was shown to potentiate TNF-
-induced ICAM-1 and E-selectin expression by endothelial cells (43), no study compared the effects of hemoglobin, methemoglobin, and hemin on a representative set of endothelial cell activation markers. Therefore, it remains unknown whether hemoglobin or methemoglobin results in the activation of pathophysiologically relevant genes representative of endothelial cell activation in addition to their well-known stimulatory effects on HO-1 expression and associated changes in oxidative stress. Because, during infections, methemoglobin appears in RBCs that may be released into the circulation after hemolysis or free hemoglobin may be converted to methemoglobin at injury sites, methemoglobin-induced endothelial cell activation is a potentially important event in the cascade of the inflammatory response (17, 45). Thus we hypothesized that free hemoglobin and methemoglobin result in the activation of endothelial cells to different degrees. To test this hypothesis, we compared the effects of hemoglobin, methemoglobin, and hemin on the production of a representative set of endothelial cell inflammatory markers, including the chemokine IL-8 and the proinflammatory cytokine IL-6. We also compared their effects on the expression and membrane content of the adhesion molecule E-selectin in endothelial cells. We found that whereas hemoglobin, methemoglobin, and hemin are efficient activators of HO-1 expression, only methemoglobin stimulates the production of the investigated set of endothelial cell inflammatory markers.
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MATERIALS AND METHODS |
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Cell isolation and culture. Human umbilical vein endothelial cells (HUVECs; Cambrex) and HUVECs isolated from umbilical cords were used in the experiments. HUVECs were isolated from umbilical cords as previously described (1). Briefly, the umbilical cord was immersed in PBS before isolation. The umbilical vein was filled with 0.2% collagenase in PBS containing penicillin, streptomycin, amphotericin B, and gentamicin and incubated for 30 min at 37°C in a humidified incubator. Endothelial cells were collected and plated onto 25-cm2 gelatin-coated flasks (Corning, NY) in medium 199 supplemented with 15% fetal bovine serum, glutamine-penicillin-streptomycin mixture, 1.67 U/ml heparin, 0.1 mg/ml bovine brain extract, and 50 µg/ml gentamicin. After reaching 90% confluence, HUVECs were detached by trypsin-EDTA treatment and subcultured in complete endothelial growth medium. Cell cultures were tested for von Willebrand factor-related antigen positivity as described previously (19). Briefly, HUVECs were subcultured into eight-well glass slides (40,000 cells/well). Cells were fixed with acetone-methanol (1:1, vol/vol). After two washes (PBS), 5% goat serum was added as a blocker (0.5 ml/well), and the cells were incubated for 10 min. After two additional washes (PBS), cells were incubated with anti-human von Willebrand factor for 1 h, washed twice, and then incubated with anti-mouse antibody conjugated with FITC for 30 min in the dark. Anti-human IgG was used in parallel as negative control. Cells were analyzed under a confocal fluorescent microscope. von Willebrand factor-related antigen-positive cell preparations were subcultured and used in the experiments.
Treatment protocols and assays. HUVECs were subcultured into 96-well plates 16-18 h before the experiments. Before the experiment, medium was replaced with fresh medium before the treatment regimens were initiated. Methemoglobin, hemoglobin, and hemin solutions were prepared before every experiment. All agents used in the experiments were dissolved in medium as vehicle, unless otherwise stated. After various treatments, supernatants were collected for analyses. IL-6 and IL-8 in cell medium were determined by ELISA according to the manufacturer's protocol. Standards were run on each ELISA plate and analyzed in parallel with the test samples.
E-selectin membrane content was determined on confluent cells. After removal of conditioned medium, cells were fixed by addition of 1.5% paraformaldehyde in PBS (pH 7.4) and incubated for 15 min. After two washes (PBS), cells were incubated with 2% bovine serum albumin in PBS for 30 min (blocking). Cells were incubated with mouse anti-human E-selectin antibody (0.2 µg/ml) in the presence of 1% bovine serum albumin in PBS for 60 min. After three washes, 1:500 diluted alkaline phosphatase-conjugated anti-mouse antibody was added, and the cells were incubated for 60 min. Color reaction was developed in the presence of p-nitrophenyl disodium phosphate (0.1 ml/well). After 30 min, the reaction was terminated by addition of 0.05 ml of 3 M NaOH. Optical density was measured in a microplate reader at 405 nm.
RNA determinations. The final cell pellets containing 106 endothelial cells were lysed in 2.5 ml of TriReagent. Total cellular RNA was isolated according to the manufacturer's protocol. The final RNA pellet was dissolved in 0.05-0.1 ml of Formazol (Molecular Research Center). Deoxyoligonucleotide primers used for HO-1 (5), GAPDH (5), E-selectin (28), IL-6 (41), and IL-8 (35) determinations were synthesized in the Molecular Biology Core Laboratory of the University of Medicine and Dentistry of New Jersey. GAPDH expression controls were carried out throughout all RT-PCR reactions. The sequences of the employed oligonucleotide primers were as follows: 5'-GCT CAA CAT CCA GCT CTT TGA GG-3' (forward) and 5'-GAC AAA GTT CAT GGC CCT GGG A-3' (reverse) for HO-1, 5'-GTC TTC ACC ACC ATG GAG AA-3' (forward) and 5'-ATC CAC AGT CTT CTG GGT GG-3' (reverse) for GAPDH, 5'-GAT GTG GGC ATG TGG AAT GAT G-3' (forward) and 5'-AGG TAC ACT GAA GGA TCT GG-3' (reverse) for E-selectin, 5'-TGA ACT CCT TCT CCA CAA GCG-3' (forward) and 5'-TCT GAA GAG GTG AGT GGC TGT C-3' (reverse) for IL-6, and 5'-GGA CAA GAG CCA GGA AGA AAC CAC C-3' (forward) and 5'-GCA ACC CTA CAA CAG ACC CAC AC-3' (reverse) for IL-8.
Serial dilutions of RNA samples were subjected to RT-PCR using the GeneAmp Thermostable rTth reverse transcriptase RNA PCR kit (Roche, Brunchburg, NJ). After RT, Mn2+ was chelated and MgCl2 was added for the amplification steps (2.0 mM) in the presence of dNTP mixtures (2 mM each). Amplification was carried out in the GeneAmp 2400 instrument (Perkin-Elmer) as follows: RT at 70°C for 15 min and PCR at 95°C for 1 min, 95°C for 10 s, and 60°C for 15 s for 35 cycles followed by 60°C for 7 min. The mixture was cooled to 4°C to terminate the reaction and stored in ice. Aliquots of the reaction samples were subjected to PAGE.
Electrophoretic mobility shift assays. After the treatments, the cells were washed and then detached by trypsinization. After they were neutralized and washed, 3 x 106 cells were suspended in 0.1 ml of whole cell extraction buffer and homogenized (10 mM Tris, pH 7.5, 1.5 mM MgCl2, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 20% glycerol, 2 mM DTT, 1 µg/ml each aprotinin, leupeptin, and pepstatin, 1 mM NaVO3, and 0.5 mM PMSF), incubated on ice for 30 min, and then centrifuged at 16,000 g for 10 min at 4°C. The supernatant was collected, the protein concentration was determined, and the supernatant was stored in aliquots until NF-B determinations. The double-stranded NF-
B consensus oligonucleotide probe (5'-AGT TGA GGG GAC TTT CCC AGG C-3') was end-labeled with 10 µCi of [
-32P]ATP at 222 TBq/mmol (Amersham, Arlington Heights, IL). Binding reactions containing 35 fmol of oligonucleotide and 10 µg of cellular protein were carried out for 20 min at room temperature. For competition reactions, a 100-fold excess of unlabeled oligonucleotide was added 5 min before addition of the labeled probe. For supershift analysis, 1 µg of each antibody was added to the reaction mixture, and the mixture was incubated for 30 min before addition of the radiolabeled probe. After the binding reactions, samples were subjected to nondenaturing 4% PAGE in low ionic strength buffer (80 mM Tris-borate and 2 mM EDTA). Gels were vacuum dried, and signals were quantified using the PhosphorImager SI analyzer and Imagequant version 4.1 program (Molecular Dynamics, Sunnyvale, CA).
Statistical analysis. Statistical calculations were performed using JMP software (SAS Institute, Cary, NC). Results were analyzed using ANOVA followed by t-test for pairwise comparisons or Tukey-Kramer's test for multiple comparisons. Statistically significant difference was concluded at P < 0.05.
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RESULTS |
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Methemoglobin acts through increasing gene transcription. To test whether methemoglobin results in elevated expression of these activation markers, we determined cellular mRNA levels using semiquantitative RT-PCR after these treatments. Under nonstimulated conditions (vehicle), the cellular levels of E-selectin, IL-6, and IL-8 mRNA were below the detectable levels (Fig. 2). Methemoglobin administration resulted in readily detectable, elevated cellular mRNA for E-selectin, IL-6, and IL-8. The effect of 12.5 µM methemoglobin on E-selectin and IL-8 mRNA expression was similar to that observed after TNF- (5 ng/ml) treatment. Furthermore, IL-6 mRNA levels were more efficiently stimulated by methemoglobin than by TNF-
, in agreement with the more pronounced increase in IL-6 protein release after treatment with methemoglobin than after TNF-
treatment. As expected, and in agreement with previous observations, hemin, hemoglobin, and methemoglobin markedly increased HO-1 mRNA expression, whereas they did not increase E-selectin, IL-6, or IL-8 mRNA levels in endothelial cells (Fig. 2).
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To further elucidate whether methemoglobin acts through transcriptional stimulation, we tested the effect of the transcriptional inhibitor actinomycin D on the methemoglobin-induced responses. Methemoglobin-induced responses were inhibitable by actinomycin D (Fig. 3). The inhibitory effects of actinomycin D on the TNF-- and methemoglobin-induced E-selectin expression and IL-6 and IL-8 production were similar (Fig. 3).
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Mechanistic aspects of methemoglobin effects. In subsequent experiments, we tested whether methemoglobin uptake and heme metabolism through HO-1 are required for its stimulatory actions on endothelial cells. Haptoglobin, a natural methemoglobin-binding protein, decreased methemoglobin-induced E-selectin membrane content and IL-6 and IL-8 production (Fig. 4A).
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The HO inhibitor zinc protoporphyrin IX caused no inhibition of methemoglobin effects (Fig. 4B); rather, it augmented the methemoglobin-induced responses. In contrast, zinc protoporphyrin IX treatment did not augment the TNF-- or IL-1
-induced E-selectin membrane expression and IL-6 and IL-8 production (data not shown). We also tested the effect of the iron chelator desferroxamine on the methemoglobin-induced responses. Desferroxamine had no effects on the methemoglobin-induced increase in E-selectin membrane content or IL-6 and IL-8 release (data not shown).
To assess whether internalization of methemoglobin is required for the described responses, we tested the effects of cytochalasin D, an endocytosis inhibitor, on the methemoglobin- or TNF--induced responses. Cytochalasin D markedly inhibited the methemoglobin-induced increase in E-selectin expression and IL-6 and IL-8 production (Fig. 5). The effect of cytochalasin D was less pronounced on the TNF-
- than on the methemoglobin-induced E-selectin response. Furthermore, cytochalasin D did not inhibit TNF-
-induced IL-8 production (Fig. 5). The inhibitory effect of cytochalasin D on the IL-1
-induced response was similar to that observed after TNF-
treatment (not shown).
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Because activation of the transcription factor NF-B plays an important role in the regulation of E-selectin production and IL-8 and IL-6 expression in endothelial cells, we used gel shift analyses to test whether methemoglobin results in activation of NF-
B. TNF-
treatment in parallel incubations and analyses served as positive control. Methemoglobin treatment increased NF-
B binding in endothelial cells (Fig. 6A). However, the oligonucleotide-protein complex showed a faster migration pattern than samples from TNF-
-treated controls (Fig. 6B, lane 5 vs. lane 3). Preincubation of cell protein extracts with the mixture of the antibodies against the p50 and p65 subunits of the NF-
B complex resulted in a supershift in methemoglobin- and TNF-
-treated cells (Fig. 6B, lane 3 vs. lane 4 and lane 6 vs. lane 5). Use of antibodies against the p50 and p65 subunits individually also resulted in shifts (not shown), providing additional evidence that the proteins interacting with the NF-
B consensus sequence contain the p50-p65 heterocomplex after methemoglobin treatment.
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Because activation of NF-B is partially mediated through redox alterations, we tested the effects of the antioxidant NAC on methemoglobin- and TNF-
-induced endothelial cell responses. NAC had a concentration-dependent inhibitory effect on the methemoglobin- or TNF-
-induced E-selectin membrane content and IL-6 and IL-8 production (Fig. 7). The inhibitory effect of NAC showed a similar pattern of concentration dependence on the methemoglobin- and TNF-
-induced E-selectin and IL-8 responses.
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Finally, we tested the effects of SN50, a specific cell-permeable peptide that interacts with the NF-B translocation sequence (26), as well as CAPE, an inhibitor of NF-
B activation and translocation (30), on the methemoglobin-induced response. CAPE (Fig. 8A) and SN50 (Fig. 8B) inhibited methemoglobin- or TNF-
-induced E-selectin membrane content and IL-6 and IL-8 production in a concentration-dependent manner.
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To assess potential toxic effects of the treatment regimens, lactate dehydrogenase content in the collected cell medium was determined. The treatment protocols did not result in cell detachment, elevated lactate dehydrogenase release, or noticeable changes in cell morphology in the incubation times employed in this study (data not shown).
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DISCUSSION |
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Methemoglobin represents 1-1.5% of total hemoglobin within RBCs under normal conditions (45). Thus it is estimated that hemolysis of 10% of the circulating RBCs would result in a methemoglobinemia in the micromolar concentration range. Such a degree of hemolysis, although rare, may occur after genetic or acquired RBC diseases (6, 9). Additionally, under a variety of pathological conditions, methemoglobinemia becomes pronounced, approaching 20% of total hemoglobin (45). After a hemolytic crisis, circulating haptoglobin is unlikely to prevent methemoglobin-induced responses, because free hemoglobin at
15 µM exhausts plasma haptoglobin (21). Nevertheless, a micromolar concentration range of methemoglobin is likely to develop at inflammatory sites or wound environments accompanied by capillary damage or blood clotting, especially when activated phagocytes releasing reactive oxygen or nitrogen species accumulate at these sites. Under these conditions, free hemoglobin from damaged RBCs is readily converted to methemoglobin, which can target endothelial cells in the vicinity.
Our observations may also explain some of the adverse effects of stroma-free hemoglobin solutions. These preparations may contain 5-15% methemoglobin, which increases further in the circulation after infusion (24). This condition may overload the hepatic capacity to catabolize hemoglobin, methemoglobin, and its derivatives via HO (14, 25), resulting in methemoglobinemia and consequent activation of the vascular endothelium. Consistent with this conclusion, human HO deficiency was shown to be accompanied by endothelial cell activation and injury, increased sensitivity to oxidative stress, and elevated blood cytokines (47).
The fact that methemoglobin markedly increased cellular mRNA levels of E-selectin, IL-6, and IL-8, together with the observation that actinomycin D inhibited the response, suggests that the elevated mRNA levels are the result of increased gene transcription, rather than increased mRNA stability, after methemoglobin administration. It is noteworthy that the general pattern of methemoglobin effects on E-selectin and IL-8 expression is similar to that observed after treatment with TNF- or IL-1
. These observations and the fact that NAC or inhibitors of NF-
B resulted in a similar inhibitory pattern on methemoglobin- and TNF-
-induced responses suggest that the signal transduction pathways resulting in endothelial cell activation are shared and/or converge after methemoglobin, TNF-
, or IL-1
treatments. This finding is supported further by the observation that combined treatments with methemoglobin and TNF-
or IL-1
were additive only when they were administered at submaximal concentrations.
This is the first report indicating that methemoglobin activates NF-B in endothelial cells. Although the rate of migration of the NF-
B complex after methemoglobin treatment was different from that observed after TNF-
, the fact that antibodies against the p50 and p65 subunits resulted in a similar supershift after TNF-
and methemoglobin treatments confirmed that NF-
B becomes activated under these conditions. I
B kinases, as well as the p65 subunit, are subject to redox-dependent regulation by specific intracellular kinases (36). Several independent studies in a variety of cell types demonstrated that NAC treatment prevents TNF-
-induced activation of I
B kinases and also inhibits serine phosphorylation and translocation of p65 (31, 32, 37). The findings that treatment with NAC, as well as SN50 and CAPE (26, 30), caused a similar inhibition pattern on the TNF-
- and methemoglobin-induced increased E-selectin membrane expression and IL-6 and IL-8 production further support the potential involvement of NF-
B activation in the methemoglobin-induced response.
It is also known that elevated gene expression of HO-1 is not dependent on NF-B, inasmuch as the promoter for HO-1 lacks an NF-
B consensus sequence (13). These facts suggest that the signaling pathway of hemin, hemoglobin, and methemoglobin resulting in elevated HO-1 expression is distinct from the mechanisms causing elevated cytokine, chemokine, or adhesion molecule expression after methemoglobin treatment. It remains to be determined whether the faster migration of the NF-
B complex after methemoglobin treatment is an artifact associated with the interference of trace amounts of methemoglobin in cellular extracts or is a unique characteristic of methemoglobin-mediated NF-
B activation with functional significance.
The fact that the inhibition of HO-1 by zinc protoporphyrin IX did not diminish the methemoglobin-induced responses indicates that carbon monoxide generation, free intracellular iron, or downstream intermediates of hemin catabolism are not required for mediation of the stimulatory effects of methemoglobin on E-selectin membrane expression and IL-6 and IL-8 production. In contrast, the observed stimulatory effects of HO-1 inhibition suggest that the accumulation of metabolites upstream of the HO-1 step may have contributed to the observed effects. Alternatively, under HO-1-inhibited conditions, the absence of carbon monoxide or intracellular iron may have had an augmenting effect on methemoglobin actions, in agreement with previous observations (40).
The exact mechanism of methemoglobin action causing the activation of endothelial cells remains to be further elucidated. However, the observations that hemoglobin or hemin had no stimulatory effects on IL-6, IL-8, or E-selectin expression, chelating iron in the cell medium had no inhibitory effects, and haptoglobin inhibited the methemoglobin-induced responses indicate that the presence of methemoglobin molecules per se is required for the activation of endothelial cells. It is well known that hemin is responsible for HO-1 induction in endothelial cells (1, 4, 8, 22, 48). The observation that hemoglobin, methemoglobin, or hemin caused elevated HO-1 expression indicates that hemin catabolism proceeded after all three of these treatments. The fact that only methemoglobin was efficient in stimulating IL-6, IL-8, and E-selectin expression suggests that the fine quaternary structure of methemoglobin plays a role in the observed effects. The quaternary structure of methemoglobin is different from that of oxy- or deoxyhemoglobin (21). Furthermore, the tetrameric form of hemoglobin or methemoglobin dissociates into dimers in dilute solutions, with midpoint equilibrium at 6 µM (21). Formation of methemoglobin by oxidants such as ferricyanide or nitrites also results in the appearance of hybrid intermediates (38). The fact that haptoglobin, which binds predominantly the
dimmers, but not the tetrameric form, resulted in 50% inhibition at near-midpoint equilibrium concentration of methemoglobin suggests that the dimeric configuration of methemoglobin is effective, but other biologically active forms may also be present. Exact elucidation of this question requires further studies.
The inhibitory effect of cytochalasin D on IL-6 and IL-8 release and E-selectin expression suggests that internalization of methemoglobin is necessary for its actions. However, cytochalasin D, in addition to inhibiting endocytosis, may also affect the processing and transport of de novo synthesized cellular proteins (44). This is presumably reflected in its inhibitory effects on IL-6 release and E-selectin membrane expression after TNF- or IL-1
treatments. Thus it remains to be elucidated whether endocytosis of methemoglobin by endothelial cells is an obligatory requirement for the observed effects.
Taken together, we conclude that the interaction between methemoglobin and endothelial cells results in increased production and release of IL-6 and IL-8 and elevated membrane expression of E-selectin. The methemoglobin-induced cell activation does not require the metabolism of hemin through the HO-1 pathway but, rather, is mediated through the activation of signaling mechanisms initiated at the cell membrane level or through intermediates of methemoglobin catabolism upstream of HO-1 action. These observations indicate that the presence of free methemoglobin in blood or interstitial fluid at inflammatory or injury sites may contribute to endothelial cell activation. Elevated chemokine release, together with an increased expression of adhesion molecules, by endothelial cells may promote phagocyte recruitment, whereas increased cytokine production can modulate cellular responses in an autocrine or a paracrine fashion. Methemoglobin-induced endothelial cell activation may be a clinically important event after intoxications accompanied by methemoglobinemia or during infections, especially when acquired or genetic conditions predisposing to hemolysis or diminished antioxidant activity are also present.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Balla J, Nath KA, Balla G, Juckett MB, Jacob HS, and Vercellotti GM. Endothelial cell heme oxygenase and ferritin induction in rat lung by hemoglobin in vivo. Am J Physiol Lung Cell Mol Physiol 268: L321-L327, 1995.
3. Bateman RM, Jagger JE, Sharpe MD, Ellsworth ML, Mehta S, and Ellis CG. Erythrocyte deformability is a nitric oxide-mediated factor in decreased capillary density during sepsis. Am J Physiol Heart Circ Physiol 280: H2848-H2856, 2001.
4. Bauer I, Wanner GA, Rensing H, Alte G, Miescher EA, Wolf B, Pannen BHJ, Clemens MG, and Bauer M. Expression pattern of heme oxygenase isoenzymes 1 and 2 in normal and stress-exposed rat liver. Hepatology 27: 829-838, 1998.[ISI][Medline]
5. Berger SP, Hunger R, Yard BA, Schnuelle P, and van der Woude FJ. Dopamine induces the expression of heme oxygenase-1 by human endothelial cells in vitro. Kidney Int 58: 2314-2319, 2000.[ISI][Medline]
6. Berkowitz FE. Hemolysis and infectioncategories and mechanisms of their interrelationship. Rev Infect Dis 13: 1151-1162, 1991.[ISI][Medline]
7. Bratosin D, Mazurier J, Tissier JP, Estaquier J, Huart JJ, Ameisen JC, Aminoff D, and Montreuil J. Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. Biochimie 80: 173-195, 1998.[ISI][Medline]
8. Choi AMK and Alam J. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am J Respir Cell Mol Biol 15: 9-19, 1996.[Abstract]
9. Coleman MD and Coleman NA. Drug-induced methaemoglobinaemiatreatment issues. Drug Saf 14: 394-405, 1996.[ISI][Medline]
10. Condon MR, Kim JE, Deitch EA, Machiedo GW, and Spolarics Z. Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis. Am J Physiol Heart Circ Physiol 284: H2177-H2184, 2003.
11. D'Agnillo F and Alayash AI. Interactions of hemoglobin with hydrogen peroxide alters thiol levels and course of endothelial cell death. Am J Physiol Heart Circ Physiol 279: H1880-H1889, 2000.
12. Davenport RD. Cytokines as intercellular signals in hemolytic transfusion reactions. Biol Signals 5: 240-245, 1996.[ISI][Medline]
13. De Maio A. Heat shock proteins: facts, thoughts, and dreams. Shock 11: 1-12, 1999.[ISI][Medline]
14. Goda N, Suzuki K, Naito M, Takeoka S, Tsuchida E, Tametani T, and Suematsu M. Distribution of heme oxygenase isoforms in rat livertopographic basis for carbon monoxide-mediated microvascular relaxation. J Clin Invest 101: 604-612, 1998.
15. Gow AJ, Luchsinger BP, Pawloski JR, Singel DJ, and Stamler JS. The oxyhemoglobin reaction of nitric oxide. Proc Natl Acad Sci USA 96: 9027-9032, 1999.
16. Graca-Souza AV, Arruda MAB, de Freitas MS, Barja-Fidalgo C, and Oliveira PL. Neutrophil activation by heme: implications for inflammatory processes. Blood 99: 4160-4165, 2002.
17. Hack CE and Zeerleder S. The endothelium in sepsis: source of and a target for inflammation. Crit Care Med 29: S21-S27, 2001.[ISI][Medline]
18. Hayashi S, Takamiya R, Yamaguchi T, Matsumoto K, Tojo SJ, Tamatani T, Kitajima M, Makino N, Ishimura Y, and Suematsu M. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress role of bilirubin generated by the enzyme. Circ Res 85: 663-671, 1999.
19. Hewett PW and Murray JC. Human lung microvessel endothelial cells: isolation, culture, and characterization. Microvasc Res 46: 89-102, 1993.[ISI][Medline]
20. Huang KT, Han TH, Hyduke DR, Vaughn MW, Van Herle H, Hein TW, Zhang C, Kuo L, and Liao JC. Modulation of nitric oxide bioavailability by erythrocytes. Proc Natl Acad Sci USA 98: 11771-11776, 2001.
21. Jandl JH. Physiology of red cells. In: Textbook of Hematology. New York: Little, Brown, 1996, p. 150-200.
22. Jeney V, Balla J, Yachie A, Varga Z, Vercellotti GM, Eaton JW, and Balla G. Pro-oxidant and cytotoxic effects of circulating heme. Blood 100: 879-887, 2002.
23. Jurado RL. Iron, infections, and anemia of inflammation. Clin Infect Dis 25: 888-895, 1997.[ISI][Medline]
24. Ketcham EM and Cairns CB. Hemoglobin-based oxygen carriers: development and clinical potential. Ann Emerg Med 33: 326-337, 1999.[ISI][Medline]
25. Kyokane T, Norimizu S, Taniai H, Yamaguchi T, Takeoka S, Tsuchida E, Naito M, Nimura Y, Ishimura Y, and Suematsu M. Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver. Gastroenterology 120: 1227-1240, 2001.[ISI][Medline]
26. Lin YZ, Yao SY, Veach RA, Torgerson TR, and Hawiger J. Inhibition of nuclear translocation of transcription factor NF-B by a synthetic peptide-containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem 270: 14255-14258, 1995.
27. Loegering DJ, Commins LM, Minnear FL, Gary LA, and Hill LA. Effect of Kupffer cell phagocytosis of erythrocytes and erythrocyte ghosts on susceptibility to endotoxemia and bacteremia. Infect Immun 55: 2074-2080, 1987.[ISI][Medline]
28. Madan B, Gade WN, and Ghosh B. Curcuma longa activates NF-B and promotes adhesion of neutrophils to human umbilical vein endothelial cells. J Ethnopharmacol 75: 25-32, 2001.[ISI][Medline]
29. McFaul SJ, Bowman PD, Villa VM, Gutierrez-Ibanez MJ, Johnson M, and Smith D. Hemoglobin stimulates mononuclear leukocytes to release interleukin-8 and tumor necrosis factor-. Blood 84: 3175-3181, 1994.
30. Natarajan K, Singh S, Burke TR, Grunberger D, and Aggarwal BB. Caffeic acid phenylethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-B. Proc Natl Acad Sci USA 93: 9090-9095, 1996.
31. Oka S, Kamata H, Kamata K, Yagisawa H, and Hirata H. N-acetylcysteine suppresses TNF-induced NF-B activation through inhibition of I
B kinases. FEBS Lett 472: 196-202, 2000.[ISI][Medline]
32. Pajonk F, Riess K, Sommer A, and McBride WH. N-acetyl-L-cysteine inhibits 26s proteasome function: implications for effects on NF-B activation. Free Radic Biol Med 32: 536-543, 2002.[ISI][Medline]
33. Pannen BHJ, Kohler N, Hole B, Bauer M, Clemens MG, and Geiger KK. Protective role of endogenous carbon monoxide in hepatic microcirculatory dysfunction after hemorrhagic shock in rats. J Clin Invest 102: 1220-1228, 1998.
34. Poss KD and Tonegawa S. Reduced stress defense in heme oxygenase 1-deficient cells. Proc Natl Acad Sci USA 94: 10925-10930, 1997.
35. Rodriguez-Juan C, Perez-Blas M, Suarez-Garcia E, Lopez-Suarez JC, Muzquiz M, Cuadrado C, and Martin-Villa JM. Lens culinaris, Phaseolus vulgaris, and Vicia faba lectins specifically trigger IL-8 production by the human colon carcinoma cell line Caco-2. Cytokine 12: 1284-1287, 2000.[ISI][Medline]
36. Schmitz ML, Bacher S, and Kracht M. IB-independent control of NF-
B activity by modulatory phosphorylations. Trends Biochem Sci 26: 186-190, 2001.[ISI][Medline]
37. Schubert SY, Neeman I, and Resnick N. Novel mechanism for inhibition of NFB activation in TNF-
-treated arterial endothelial cells by natural antioxidants (Abstract). FASEB J 16: A200, 2002.
38. Shih ML and Korte WD. Analysis of hemoglobin derivatives by capillary isoelectric focusing and its application in the antidotal research of cyanide poisoning. Anal Biochem 238: 137-144, 1996.[ISI][Medline]
39. Spolarics Z. Endotoxemia, pentose cycle, and the oxidant/antioxidant balance in the hepatic sinusoid. J Leukoc Biol 63: 534-541, 1998.[Abstract]
40. Suematsu M, Goda N, Sano T, Kashiwagi S, Egawa T, Shinoda Y, and Ishimura Y. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver. J Clin Invest 96: 2431-2437, 1995.[ISI][Medline]
41. Tchirkov A, Rolhion C, Bertrand S, Dore JF, Dubost JJ, and Verrelle P. IL-6 gene amplification and expression in human glioblastomas. Br J Cancer 85: 518-522, 2001.[ISI][Medline]
42. Titheradge MA. Nitric oxide in septic shock. Biochim Biophys Acta 1411: 437-455, 1999.[ISI][Medline]
43. Wagener FADT, deWitte T, and Abraham NG. Heme induces ICAM-1, VCAM-1 and E selectin expression in endothelial cells (Abstract). Hypertension 30: 47, 1997.
43. Wagener FE, de Witte T, and Abraham NG. Heme induces the expression of adhesion molecules ICAM-1, VCAM-1, and E selectin in vascular endothelial cells. Proc Soc Exp Biol Med 216: 456-463, 1997.[Abstract]
44. Wagner O, Schuler H, Hofmann P, Langer D, Dancker P, and Bereiter-Hahn J. Sound attenuation of polymerizing actin reflects supramolecular structures: viscoelastic properties of actin gels modified by cytochalasin D, profilin and -actinin. Biochem J 355: 771-778, 2001.[ISI][Medline]
45. Wright RO, Lewander WJ, and Woolf AD. Methemoglobinemia: etiology, pharmacology, and clinical management. Ann Emerg Med 34: 646-656, 1999.[ISI][Medline]
46. Wright RO, Woolf AD, Shannon MW, and Magnani B. N-acetylcysteine reduces methemoglobin in an in vitro model of glucose-6-phosphate dehydrogenase deficiency. Acad Emerg Med 5: 225-229, 1998.[Abstract]
47. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, and Koizumi S. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest 103: 129-135, 1999.
48. Yoshida T, Biro P, Cohen T, Muller RM, and Shibahara S. Human heme oxygenase cDNA and induction of its messenger RNA by hemin. Eur J Biochem 171: 457-461, 1988.[Abstract]
49. Zhu XL, Pacheco ND, Dick EJ, and Rollwagen FM. Differentially increased IL-6 mRNA expression in liver and spleen following injection of liposome-encapsulated haemoglobin. Cytokine 11: 696-703, 1999.[ISI][Medline]