Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin

D. L. Laskin, D. E. Heck, C. R. Gardner, L. S. Feder, J. D. Laskin

Research output: Contribution to journalArticle

60 Citations (Scopus)

Abstract

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-γ (IFN-γ) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, N(G)-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-γ than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-γ was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-γ was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-γ. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages. Taken together, our data demonstrate that exposure of rats to endotoxin augments the capacity of both hepatic macrophages and endothelial cells to produce nitric oxide but that this response is regulated distinctly in these cells. Increased sensitivity of nonparenchymal cells to inflammatory mediators may be important in the response of the liver to excess quantities of this bacterially derived product.

Original languageEnglish (US)
Pages (from-to)751-758
Number of pages8
JournalJournal of Leukocyte Biology
Volume56
Issue number6
StatePublished - Dec 1 1994

Fingerprint

Endotoxins
Hepatocytes
Nitric Oxide
Endothelial Cells
Macrophages
Interferons
Nitric Oxide Synthase Type II
Tetradecanoylphorbol Acetate
Arginine
Liver
Canavanine
Buthionine Sulfoximine
Pronase
Proteins
Phorbol Esters
Collagenases
Centrifugation
Nitric Oxide Synthase
Superoxides
Northern Blotting

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology
  • Cell Biology

Keywords

  • Endothelial cells
  • Endotoxemia
  • Glutathione
  • Kupffer cells
  • Nitric oxide

Cite this

@article{232a6097bc3e404d9ad0f49654c681ba,
title = "Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin",
abstract = "Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-γ (IFN-γ) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, N(G)-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-γ than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-γ was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-γ was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-γ. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages. Taken together, our data demonstrate that exposure of rats to endotoxin augments the capacity of both hepatic macrophages and endothelial cells to produce nitric oxide but that this response is regulated distinctly in these cells. Increased sensitivity of nonparenchymal cells to inflammatory mediators may be important in the response of the liver to excess quantities of this bacterially derived product.",
keywords = "Endothelial cells, Endotoxemia, Glutathione, Kupffer cells, Nitric oxide",
author = "Laskin, {D. L.} and Heck, {D. E.} and Gardner, {C. R.} and Feder, {L. S.} and Laskin, {J. D.}",
year = "1994",
month = "12",
day = "1",
language = "English (US)",
volume = "56",
pages = "751--758",
journal = "Journal of Leukocyte Biology",
issn = "0741-5400",
publisher = "FASEB",
number = "6",

}

Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin. / Laskin, D. L.; Heck, D. E.; Gardner, C. R.; Feder, L. S.; Laskin, J. D.

In: Journal of Leukocyte Biology, Vol. 56, No. 6, 01.12.1994, p. 751-758.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin

AU - Laskin, D. L.

AU - Heck, D. E.

AU - Gardner, C. R.

AU - Feder, L. S.

AU - Laskin, J. D.

PY - 1994/12/1

Y1 - 1994/12/1

N2 - Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-γ (IFN-γ) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, N(G)-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-γ than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-γ was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-γ was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-γ. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages. Taken together, our data demonstrate that exposure of rats to endotoxin augments the capacity of both hepatic macrophages and endothelial cells to produce nitric oxide but that this response is regulated distinctly in these cells. Increased sensitivity of nonparenchymal cells to inflammatory mediators may be important in the response of the liver to excess quantities of this bacterially derived product.

AB - Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-γ (IFN-γ) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, N(G)-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-γ than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-γ was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-γ was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-γ. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages. Taken together, our data demonstrate that exposure of rats to endotoxin augments the capacity of both hepatic macrophages and endothelial cells to produce nitric oxide but that this response is regulated distinctly in these cells. Increased sensitivity of nonparenchymal cells to inflammatory mediators may be important in the response of the liver to excess quantities of this bacterially derived product.

KW - Endothelial cells

KW - Endotoxemia

KW - Glutathione

KW - Kupffer cells

KW - Nitric oxide

UR - http://www.scopus.com/inward/record.url?scp=0028575633&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028575633&partnerID=8YFLogxK

M3 - Article

C2 - 7527831

AN - SCOPUS:0028575633

VL - 56

SP - 751

EP - 758

JO - Journal of Leukocyte Biology

JF - Journal of Leukocyte Biology

SN - 0741-5400

IS - 6

ER -