Inflammation, Vol. 27, No. 4, August 2003 (C ? 2003)
Nitric Oxide Modulates MCP-1 Expression in Endothelial
Cells: Implications for the Pathogenesis of Pulmonary
Anjali Desai,1Mark J. Miller,1Xiaodong Huang,1and Jeffrey S. Warren1,2
Abstract—Monocyte chemoattractant protein-1 (MCP-1) is a pivotal mediator of angiocentric gran-
uloma formation in glucan-induced pulmonary granulomatous vasculitis. Based on the rationale that
mononuclear phagocytes retrieved from granulomas are rich sources of nitric oxide (NO) and that the
recruitment of mononuclear phagocytes into lesions abates as granuloma formation slows, we tested
the hypothesis that MCP-1 gene expression is regulated by a NO-sensitive mechanism. Preexposure
of endothelial cell (EC) monolayers to NO donor compounds markedly reduced cytokine-induced
fluctuations in endothelial reduced glutathione (GSH) pools but did not affect cGMP concentrations.
The lungs of mice bearing targeted disruptions of the inducible nitric oxide synthase (iNOS) gene
exhibited significantly higher concentrations of MCP-1 following glucan infusion than did those of
wild-type mice. Cumulatively, these data suggest that NO suppresses MCP-1 expression by blunting
the redox changes associated with cytokine-induced EC activation.
KEY WORDS: monocyte chemoattractant protein-1; pulmonary granulomatous vasculitis; nitric oxide; endothe-
lial cells; iNOS knockout mice.
Pulmonary granulomas develop in response to a va-
riety of microbial agents (eg. Mycobacterium tuber-
culosis), inhaled foreign particulates (e.g. metal dusts
such as beryllium), and etiologically unknown factors
(e.g. Wegener’s granulomatosis, sarcoidosis) (1). Sev-
eral forms of pulmonary granulomas are characterized
by monocyte/macrophage-rich collections of inflamma-
tory cells centered around blood vessels (i.e. angiocen-
tric). Monocyte chemoattractant protein-1 (MCP-1), a
member of the C-C or beta subfamily of chemokines
2To whom correspondence should be addressed at Department of
Pathology-Box 0602, University of Michigan Medical School, 1301
Catherine Road, Ann Arbor, Michigan 48109-0602. E-mail: warren@
has been identified as a pivotal mediator of granu-
loma formation. Secreted by a variety of cell types, in-
cluding endothelial cells (ECs), smooth muscle cells,
fibroblasts, and monocytes, MCP-1 induces monocyte re-
cruitment and activation, and can upregulate adhesion
molecule expression and cytokine production by mono-
cytes (2). Monocyte chemoattractant protein-1 can acti-
MCP-1 expression is obligatory for lesion development
in a rodent model of pulmonary granulomatous vasculitis
(4). In this model, the intravenous infusion of particulate
yeast cell wall glucan results in the rapid, synchronous
fully mature, are composed primarily of monocytes and
macrophages. Recent studies indicate that the upregu-
lation of MCP-1 is, in part, regulated by the cytokines
TNF-α and IL-1β (5). MCP-1 expression during the
C ? 2003 Plenum Publishing Corporation
Desai, Miller, Huang, and Warren
pathogenesis of glucan-induced granuloma formation is
biphasic with an early (1 h) blood-vessel wall associated
rise in MCP-1 and a later (6–24 h) rise in MCP-1
associated with granuloma cells per se (6). Experiments
in which neutrophils have been either selectively de-
pleted or their adherence to vascular wall endothelium
inhibited by means of infused sialyl-Lewis glycomimetic
P-selectin antagonists have revealed that neutrophils
are obligatory for full granuloma development even
though they are not present in definitive lesions (48–96 h)
(7, 8). These observations, coupled with evidence that
the infusion of catalase inhibits granuloma formation
and that pharmacologic modulation of intracellular
glutathione redox status modulates MCP-1 expression
in granulomatous vasculitis, support the hypothesis that
neutrophils and locally produced H2O2 are important
mediators of monocyte recruitment (9).
While there is extensive evidence suggesting that
the expression of proinflammatory mediators including
MCP-1, is redox sensitive, the intracellular mediators and
transduction pathway(s) are complex and incompletely
locally produced NO regulates the expression of endothe-
of glucan-induced granulomatous vasculitis rests on evi-
dence that mononuclear phagocytes in pulmonary lesions
release NO discretely at sites of granuloma formation. In
this scenario, NO may temporally and anatomically de-
limit the extent of an evolving granulomatous lesion by
either down-regulating further MCP-1 elaboration and/or
by preventing MCP-1 expression in vessel wall cells ad-
jacent to the lesion. In the present context, “delimitation”
indicates the cessation of net “growth” of a granuloma.
ment of an individual granuloma must reach a maximum
size before it begins to then “resolve”. The in vitro data
presented suggest that locally produced NO may regu-
late endothelial MCP-1 expression by blunting cytokine-
and/or reactive oxygen intermediate-induced changes in
cellular redox status.
MATERIALS AND METHODS
Unless otherwise specified, materials were pur-
chased from Sigma Chemical Company (St. Louis, MO).
NONOates were purchased from Cayman Chemicals
(Ann Arbor, MI). Mice were obtained from Jackson Lab-
oratories (Bar Harbor, ME).
Endothelial Cell Culture
Human umbilical vein endothelial cells (HUVECs)
were isolated from umbilical cords as previously
Treatment of HUVECs for Enzyme Immunoassay
Aqueous solutions of NO were generated using
NONOate compounds. NONOates are nucleophilic NO
adducts that spontaneously decompose at neutral pH to
release NO (12). As indicated in the results, we em-
ployed combinations of two short half-life NO donors,
Methylamine hexamethylene methylamine (MAHMA)
NONOate (t1/2= 1 min at 37◦C) and Diethylamine
(DEA) NONOate (t1/2= 2 min at 37◦C), or Dipropy-
lenetriamine (DPTA) NONOate (t1/2= 3 h at 37◦C) for
longer sustained release. NONOate stock solutions were
prepared in 0.01 N NaOH and then diluted into M199
medium to initiate NO release. After a 30-min exposure
to NO, HUVECs were stimulated with either TNF-α or
IL-1β(1 ng/mL) for 8 h. The conditioned media collected
at the end of this 8-h incubation period were subjected to
the EIA experiment in a cytotoxicity detection (LDH) as-
MCP-1 Enzyme Immunoassay (EIA)
MCP-1 EIAs were carried out as previously
Ribonuclease (RNase) Protection Assay
Total RNA was extracted from endothelial cells
using Tri Reagent according to manufacturer’s in-
structions. The RNase protection assays were per-
formed using the RiboQuant Multi-probe kit from
Extraction of Nuclear Protein
Nuclear extracts were prepared and protein concen-
trations determined as previously decribed (9, 14).
Nitric Oxide Modulates MCP-1 Expression in Endothelial Cells
tous vasculitis. Lab. Invest. 79(7):837–847.
transcription factors, and inflammation. Adv. Pharmacol. 38:403–
11. Desai, A., M. J. Miller, H. F. Gomez, and J. S. Warren. 1999. Lox-
production by endothelial cells. Clin. Toxical. 37(4):447–456.
12. Hrabie, J. A., J. R. Klose, and D. A. Wink. 1993. New nitric-
oxide releasing zwitterions derived from polyamines. J. Org. Chem.
13. Desai, A., H. A. Lankford, and J. S. Warren. 2001. Homocysteine
augments cytokine-induced chemokine expression in human vascu-
lar smooth muscle cells: Implications for atherogenesis. Inflamma-
14. Smith, P. K., R. I. Krohn, G. T. Hermanso, A. K. Mallia, A. K.
and D. C. Klenk. 1985. Measurement of protein using bicinchoninic
acid. Anal. Biochem. 150:76–85.
15. Linton, M. and P. S. Gallo Jr. 1975. The Practical Statistician: Sim-
plified Handbook of Statistics. Brooks/Cole, Monterey, CA.
16. Martin, T., P. M. Cardarelli, G. C. N. Parry, K. A. Felts, and
R. R. Cobb. 1997. Cytokine induction of monocyte chemoattrac-
tant protein-1 gene expression in human endothelial cells depends
on the cooperative action of NF-κB and AP-1. Eur. J. Immunol.
17. Murad, F. 1994. Regulation of cytosolic guanylyl cyclase by nitric
oxide: the NO-cyclic GMP signal transduction system. Adv. Phar-
18. Zeiher, A. M., B. Fisslthaler, B. Schray-Utz, and R. Busse. 1995.
Nitric oxide modulates the expression of monocyte chemoattractant
protein 1 in cultured human endothelial cells. Circ. Res. 76:980–
19. Tsao, P. S., B. Wang, R. Buitrago, J. Y. Shyy, and J. P. Cooke. 1997.
Nitric oxide regulates monocyte chemotactic protein-1. Circulation
20. Khan B. V., D. G. Harrison, M. T. Olbrych, R. W. Alexander, and
R. M. Medford. 1996. Nitric oxide regulates vascular cell adhe-
sion molecule 1 gene expression and redox-sensitive transcriptional
events in human vascular endothelial cells. Proc. Natl. Acad. Sci.
21. Katsuyama, K., M. Shichiri, F. Marumo, and Y. Hirata. 1998. NO
inhibits cytokine-induced iNOS expression and NF-κB activaton by
interfering with phosphorylation and degradation of IκB-α. Arte-
rioscler. Thromb. Vasc. Biol. 18:1796–1802.
23. Janssen-Heininger, Y. M., N. E. Poynter, and P. A. Baeuerle. 2000.
Recent advances towards understanding redox-mechanisms in the
activation of nuclear factor κB. Free Radic. Biol. Med. 28(9):1317–
24. Umansky, V., S. P. Hehner, A. Dumont, T. G. Hofmann,
V. Schirrmacher, W. Droge, and M. Lienhard Schmitz. 1998.
Co-stimulatory effect of nitric oxide on endothelial NF-κB im-
plies a physiological self-amplifying mechanism. Eur. J. Immunol.
tion factor NF-kappa B/Rel in induction of nitric oxide synthase. J.
Biol. Chem. 269(7):4705–4708.
26. Niu, X. F., C. W. Smith, and P. Kubes. 1994. Intracellular oxidative
lial cell adhesion to neutrophils. Circ. Res. 74(6):1133–1140.