Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages

O. Bloom, H. Wang, J. Vishnubhakat, M. Meistrall, Kevin Cockroft, G. A. Zimmerman, K. J. Tracey

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

TNF, a cytokine mediator of septic shock and tissue injury, is overproduced in endotoxemic hypophysectomized animals. We recently identified and isolated a serum protein from hypophysectomized animals that significantly increased TNF production In LPS-stimulated macrophages. Amino acid sequence analysis identified the protein as hemoglobin A. Here we investigate the direct activity of hemoglobin as an enhancer of TNF production. Murine macrophage-like RAW 264.7 cells were plated at 2×106 cells/ml in 100 λ of serum-free RPMI medium (+pen/strep & L-glutamine) per well in a 96-well plate and incubated at 37°C for 3 hrs. In a second 96-well plate, IPS (10 ng/ml, 0111:B4, Sigma) and hemoglobin (Hb A, Sigma) were preincubated in the concentrations shown at 37°C for 30 min. At t=0 the media was aspirated from the cultures and replaced with the preincubated experimental media. After a 4 hr incubation (37°C), the conditioned supernatants were collected and assayed for TNF by ELISA. TNF production (pg/ml) in the cultures is shown In the Table (mean ±s.e., n=6/condition) [Hemoglobin] (μ/ml) Addition 0 12.5 125 500 HbA Alone 24±12 10±5 94±21 255±42 HbA + LPS 3662±450*5086±213*6311± 503*14472±689**P<0.05 vs HbA-alone by ANOVA It is now plausible to investigate the result of inhibiting HbA enhancement of TNF effect in diseases complicated by hemolysis and hemoglobinemia, such as trauma, cardiopulmonary bypass, and critical illness.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number3
StatePublished - 1997

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tumor necrosis factors
Macrophages
production economics
hemoglobin
Hemoglobins
macrophages
Tumor Necrosis Factor-alpha
Animals
septic shock
Hemoglobin A
hemolysis
glutamine
Serum-Free Culture Media
Protein Sequence Analysis
Wounds and Injuries
tumor necrosis factor-alpha
blood proteins
Septic Shock
Analysis of variance (ANOVA)
Hemolysis

All Science Journal Classification (ASJC) codes

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Bloom, O., Wang, H., Vishnubhakat, J., Meistrall, M., Cockroft, K., Zimmerman, G. A., & Tracey, K. J. (1997). Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages. FASEB Journal, 11(3).
Bloom, O. ; Wang, H. ; Vishnubhakat, J. ; Meistrall, M. ; Cockroft, Kevin ; Zimmerman, G. A. ; Tracey, K. J. / Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages. In: FASEB Journal. 1997 ; Vol. 11, No. 3.
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abstract = "TNF, a cytokine mediator of septic shock and tissue injury, is overproduced in endotoxemic hypophysectomized animals. We recently identified and isolated a serum protein from hypophysectomized animals that significantly increased TNF production In LPS-stimulated macrophages. Amino acid sequence analysis identified the protein as hemoglobin A. Here we investigate the direct activity of hemoglobin as an enhancer of TNF production. Murine macrophage-like RAW 264.7 cells were plated at 2×106 cells/ml in 100 λ of serum-free RPMI medium (+pen/strep & L-glutamine) per well in a 96-well plate and incubated at 37°C for 3 hrs. In a second 96-well plate, IPS (10 ng/ml, 0111:B4, Sigma) and hemoglobin (Hb A, Sigma) were preincubated in the concentrations shown at 37°C for 30 min. At t=0 the media was aspirated from the cultures and replaced with the preincubated experimental media. After a 4 hr incubation (37°C), the conditioned supernatants were collected and assayed for TNF by ELISA. TNF production (pg/ml) in the cultures is shown In the Table (mean ±s.e., n=6/condition) [Hemoglobin] (μ/ml) Addition 0 12.5 125 500 HbA Alone 24±12 10±5 94±21 255±42 HbA + LPS 3662±450*5086±213*6311± 503*14472±689**P<0.05 vs HbA-alone by ANOVA It is now plausible to investigate the result of inhibiting HbA enhancement of TNF effect in diseases complicated by hemolysis and hemoglobinemia, such as trauma, cardiopulmonary bypass, and critical illness.",
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Bloom, O, Wang, H, Vishnubhakat, J, Meistrall, M, Cockroft, K, Zimmerman, GA & Tracey, KJ 1997, 'Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages', FASEB Journal, vol. 11, no. 3.

Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages. / Bloom, O.; Wang, H.; Vishnubhakat, J.; Meistrall, M.; Cockroft, Kevin; Zimmerman, G. A.; Tracey, K. J.

In: FASEB Journal, Vol. 11, No. 3, 1997.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages

AU - Bloom, O.

AU - Wang, H.

AU - Vishnubhakat, J.

AU - Meistrall, M.

AU - Cockroft, Kevin

AU - Zimmerman, G. A.

AU - Tracey, K. J.

PY - 1997

Y1 - 1997

N2 - TNF, a cytokine mediator of septic shock and tissue injury, is overproduced in endotoxemic hypophysectomized animals. We recently identified and isolated a serum protein from hypophysectomized animals that significantly increased TNF production In LPS-stimulated macrophages. Amino acid sequence analysis identified the protein as hemoglobin A. Here we investigate the direct activity of hemoglobin as an enhancer of TNF production. Murine macrophage-like RAW 264.7 cells were plated at 2×106 cells/ml in 100 λ of serum-free RPMI medium (+pen/strep & L-glutamine) per well in a 96-well plate and incubated at 37°C for 3 hrs. In a second 96-well plate, IPS (10 ng/ml, 0111:B4, Sigma) and hemoglobin (Hb A, Sigma) were preincubated in the concentrations shown at 37°C for 30 min. At t=0 the media was aspirated from the cultures and replaced with the preincubated experimental media. After a 4 hr incubation (37°C), the conditioned supernatants were collected and assayed for TNF by ELISA. TNF production (pg/ml) in the cultures is shown In the Table (mean ±s.e., n=6/condition) [Hemoglobin] (μ/ml) Addition 0 12.5 125 500 HbA Alone 24±12 10±5 94±21 255±42 HbA + LPS 3662±450*5086±213*6311± 503*14472±689**P<0.05 vs HbA-alone by ANOVA It is now plausible to investigate the result of inhibiting HbA enhancement of TNF effect in diseases complicated by hemolysis and hemoglobinemia, such as trauma, cardiopulmonary bypass, and critical illness.

AB - TNF, a cytokine mediator of septic shock and tissue injury, is overproduced in endotoxemic hypophysectomized animals. We recently identified and isolated a serum protein from hypophysectomized animals that significantly increased TNF production In LPS-stimulated macrophages. Amino acid sequence analysis identified the protein as hemoglobin A. Here we investigate the direct activity of hemoglobin as an enhancer of TNF production. Murine macrophage-like RAW 264.7 cells were plated at 2×106 cells/ml in 100 λ of serum-free RPMI medium (+pen/strep & L-glutamine) per well in a 96-well plate and incubated at 37°C for 3 hrs. In a second 96-well plate, IPS (10 ng/ml, 0111:B4, Sigma) and hemoglobin (Hb A, Sigma) were preincubated in the concentrations shown at 37°C for 30 min. At t=0 the media was aspirated from the cultures and replaced with the preincubated experimental media. After a 4 hr incubation (37°C), the conditioned supernatants were collected and assayed for TNF by ELISA. TNF production (pg/ml) in the cultures is shown In the Table (mean ±s.e., n=6/condition) [Hemoglobin] (μ/ml) Addition 0 12.5 125 500 HbA Alone 24±12 10±5 94±21 255±42 HbA + LPS 3662±450*5086±213*6311± 503*14472±689**P<0.05 vs HbA-alone by ANOVA It is now plausible to investigate the result of inhibiting HbA enhancement of TNF effect in diseases complicated by hemolysis and hemoglobinemia, such as trauma, cardiopulmonary bypass, and critical illness.

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