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Infection and Immunity, February 2001, p. 751-757, Vol. 69, No. 2
Department of Basic Medical Sciences,
University of Missouri-Kansas City,1 and
Saint Luke's Hospital-Kansas City,2
Kansas City, Missouri
Received 9 August 2000/Returned for modification 4 October
2000/Accepted 2 November 2000
Lipoteichoic acids (LTA), cell wall components of gram-positive
bacteria, have been reported to induce various inflammatory mediators
and to play a key role in gram-positive-microbe-mediated septic shock.
In a large number of these studies, investigators used commercially
available LTA purified from a variety of gram-positive bacteria,
including Staphylococcus aureus, Bacillus
subtilis, and Streptococcus sanguis. We report here
that, although these commercially available LTA could be readily shown
to stimulate production of nitric oxide (NO) in RAW 264.7 mouse
macrophages, the activity was dramatically inhibited by polymyxin B, a
relatively specific inhibitor of endotoxin biological activity.
One-step purification of the commercially available S. aureus LTA using hydrophobic interaction chromatography resulted
in two well-separated peak fractions, one highly enriched for LTA and a
second highly enriched for endotoxin. The LTA-enriched fractions did
not induce production of NO in RAW 264.7 macrophages, although they
caused a dose-dependent induction of NO in the presence of low
concentrations of gamma interferon (IFN-
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.2.751-757.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Commercial Preparations of Lipoteichoic Acid
Contain Endotoxin That Contributes to Activation of Mouse Macrophages
In Vitro
) (which by itself induced
little NO), regardless of the presence of polymyxin B. In contrast, the
endotoxin-enriched fractions by themselves inhibited in high levels of
NO in RAW 264.7 macrophages but activity was almost completely
inhibited in the presence of polymyxin B. Consistent with these
findings, our data also indicate that commercial LTA preparations from
S. aureus, B. subtilis, and S. sanguis were not able to induce NO from
lipopolysaccharide-hyporesponsive C3H/HeJ mouse peritoneal macrophages,
but in the presence of IFN-
, these LTA preparations were able to
induce relatively high levels of NO from C3H/HeJ macrophages. These
results indicate that commercially available LTA can contain
contaminating and potentially significant levels of endotoxin that can
be expected to contribute to the putative macrophage-stimulating
effects of LTA as assessed by NO production. The fact that the purified
LTA, by itself, was not able to induce significant levels of NO
secretion in RAW 264.7 macrophages supports the conclusion that caution
in attributing high-level biological activity to this microbial cell
wall constituent should be exercised.
*
Corresponding author. Mailing address: Office of
Research Administration, Room 3112 Main Hospital, Saint Luke's
Hospital of Kansas City, 4401 Wornall Rd., Kansas City, MO 64111. Phone: (816) 932-9844. Fax: (816) 932-6091. E-mail:
dmorrison{at}saint-lukes.org.
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