Endotoxin-induced nitric oxide production in pulmonary artery endothelial cells is regulated by cytokines
J. C. Cendan, L. L. Moldawer, W. W. Souba, E. M. Copeland 3rd and D. S. Lind
Department of Surgery, University of Florida, Gainesville.
BACKGROUND: L-Arginine is the sole precursor of nitric oxide (NO).
Bacterial lipopolysaccharide (endotoxin) (LPS) stimulates carrier-mediated
L-arginine transport in porcine pulmonary artery endothelial cells (PAECs)
through an autocrine pathway that involves interleukin-1 alpha (IL-1 alpha)
and tumor necrosis factor alpha (TNF-alpha). OBJECTIVES: To determine if
Escherichia coli LPS stimulates NO synthesis in PAECs and, if so, if LPS
stimulation of NO production is also mediated by autocrine secretion of
IL-1 alpha and TNF-alpha. DESIGN: Monolayers of PAECs were incubated with
various concentrations of LPS, recombinant human TNF-alpha, or IL-1 alpha,
and total nitrate-nitrite accumulation was measured at different time
points with the Greiss reagent following cadmium reduction. Release of
TNF-alpha and IL-1 alpha release by LPS-stimulated PAECs were measured
using the WEHI (for TNF-alpha) and A375.S2 (for IL-1 alpha) bioassays. The
PAECs were then incubated with saline solution or LPS in the presence or
absence of either a polyclonal antibody to human TNF or IL-1 receptor
antagonist, and nitrate-nitrite accumulation was measured at 48 hours.
RESULTS: Production of NO by PAECs was increased 230% by LPS (1
microgram/mL), 350% by TNF-alpha (1000 U/mL), and 240% by IL-1 alpha (1000
U/mL) (P < .05 vs control). The LPS-stimulated NO production was
inhibited by IL-1 receptor antagonist (100 micrograms/mL) or antibody to
TNF (10 micrograms/mL) to control levels (P < .05 vs LPS; difference vs
saline solution was not significant). The LPS-stimulated TNF-alpha
secretion by PAECs and TNF-alpha activity were maximal at 6 hours (400 +/-
42 pg/mL). The IL-1 alpha activity was not detectable in LPS-stimulated
PAECs by the A375.S2 bioassay. CONCLUSIONS: Endotoxin, TNF-alpha, and IL-1
alpha stimulated NO synthesis in PAECs. Endotoxin-stimulated NO synthesis
through an autocrine pathway involving the cytokines TNF-alpha and IL-1
alpha.
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Willuweit et al.
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Freyer et al.
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Hu et al.
Blood 1998;92:2759-2765.
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Xu et al.
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