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Infection and Immunity, March 2001, p. 1273-1279, Vol. 69, No. 3
Department of Immunopathology, The Women's
and Children's Hospital, North Adelaide, South Australia 5006, and Department of Paediatrics, University of Adelaide, Adelaide,
South Australia 5000, Australia
Received 19 June 2000/Returned for modification 18 September
2000/Accepted 22 November 2000
One of the recognized associations of bacterial infection with
cardiovascular events is the activation of endothelium and upregulation
of adhesion molecules. The two major proinflammatory mediators
implicated in the causation of cardiovascular events, bacterial
lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF), were
found to cooperate to enhance the adhesive properties of endothelial
cells. These caused synergistic upregulation of intercellular adhesion
molecule-1, E-selectin, and vascular cell adhesion molecule-1 in human
umbilical vein endothelial cells as determined by flow cytometry
analysis and enzyme-linked immunosorbent assay. This synergism was not
due to TNF causing an upregulation of CD14 expression. Treatment with
both LPS and TNF resulted in a marked increase in the translocation of
NF- Bacterial lipopolysaccharide (LPS),
a major component of the cell wall of gram-negative bacteria, is a
highly biologically active molecule which stimulates macrophages to
produce and release tumor necrosis factor (TNF) (14). One
of the consequences of TNF production is the upregulation of vascular
endothelial cell adhesion molecule expression. Apart from this indirect
effect on endothelial cells via TNF, LPS per se has the ability to
upregulate endothelial cell adhesion molecules directly (3,
32). Endothelial adhesion molecules play an important role in
regulating the movement of leukocytes from the blood to foci of
inflammation (8, 10, 12).
E-selectin, a member of the selectin family of adhesion molecules, not
normally present on the surface of unstimulated endothelial cells,
appears on the cell surface within a few hours after exposure to a
number of proinflammatory stimuli. Intercellular adhesion molecule 1 (ICAM-1), constitutively expressed at low levels on endothelial cells
and vascular cell adhesion molecule-1 (VCAM-1), not expressed on
resting endothelial cells, are members of the immunoglobulin
superfamily and can be upregulated many fold by proinflammatory
mediators including TNF and LPS (3, 4, 9, 20).
In addition to conventional risk factors for coronary events it has
been recognized that there is an association between bacterial infection and myocardial infarction (23, 33). Indeed, the addition of inflammatory markers to screening based on lipid levels is
a stronger mediator of cardiovascular events (27).
Atherosclerotic disease is now viewed as an inflammatory disease
(29). In an attempt to investigate the links between
infection and vascular disease the gram-negative organism
Chlamydia pneumoniae has been found to be present in
atheromatous lesions (6). It has been demonstrated in
vitro that this organism invades and activates endothelial cells
(11). The resulting upregulation of endothelial adhesion
molecules promotes leukocyte infiltration and atherosclerotic lesions
(29). However, the contribution of this organism to ischemic heart disease is still not clear (34).
In infection-induced inflammation both exogenous and endogenous
mediators are present, forming a proinflammatory network which is
likely to operate by cross talk (13, 15). The manner in which these mediators act within the network remains poorly defined, and we have investigated this concept by studying the relationship between LPS and TNF with respect to expression of the adhesion molecules E-selectin, ICAM-1, and VCAM-1, on endothelial cells.
Materials.
Human recombinant TNF alpha (TNF-
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1273-1279.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Bacterial Lipopolysaccharide and Tumor Necrosis
Factor Alpha Synergistically Increase Expression of Human Endothelial
Adhesion Molecules through Activation of NF-
B and p38
Mitogen-Activated Protein Kinase Signaling Pathways
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
B into the nucleus. The activity of p38 mitogen-activated protein
kinase was also synergistically enhanced, while the activity of
c-jun N-terminal kinase was increased in an additive
manner. The results demonstrate that LPS and TNF act synergistically to
upregulate the expression of endothelial cell adhesion molecules,
possibly by amplification of signaling pathways upstream of
transcription. These findings have implications for the understanding
of the acceleration of atherosclerotic events seen in low-grade
infections with gram-negative organisms.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
) was a gift
from G. R. Adolf, Ernst Boehringer Institute, Vienna, Austria. The
activity was 6 × 107 U/mg, and the preparation was
>99% pure. The endotoxin contamination was less than 0.125 endotoxin
units/ml as assessed by the Limulus lysate assay. LPS from
Escherichia coli O127:B8, chromatographically purified by
gel filtration, was purchased from Sigma Chemical Co., St. Louis, Mo.
B
p65 (rabbit anti-human), extracellular signal-related kinase (ERK), and
p38 (C-20)-G (polyclonal goat anti-human immunoglobulin G) were
purchased from Santa Cruz Biotechnology, Santa Cruz, Calif.
Culture of human umbilical vein endothelial cells (HUVEC).
Human umbilical cords were collected immediately after delivery and
stored in sterile containers at 4°C for a maximum period of 36 h. The veins were cannulated, washed with Hanks balanced salt solution
(HBSS), and filled with collagenase (37°C) (0.4 mg/ml; activity, 219 U/mg; collagenase type II; Worthington). After incubation in a
waterbath (37°C for 2 min) the content of each vein was collected.
The veins were washed once with HBBS to harvest any remaining cells.
Cells from each cord were centrifuged separately (450 × g
for 5 min); the supernatant was discarded; and the pellet was
resuspended in RPMI 1640 supplemented with penicillin (80 U/ml),
streptomycin (80 µg/ml), L-glutamine (3.2 mmol/liter)
(all from ICN-Flow), and pooled, heat-inactivated human group AB serum
(20%). Cells were grown to confluence in 75-cm2 culture
flasks (Corning) which were precoated with 0.2% gelatin (Multicel;
TRACE Biosciences, Melbourne, Australia). Endothelial cells were
identified by their characteristic monolayer cobblestone appearance and
positive staining for factor VIII-related antigen using
peroxidase-conjugated rabbit immunoglobulin G antibody to human von
Willebrand factor (DAKO) and 3,3'-diaminobenzidine. Only first-passage
cells from one cord were used for one experiment. Prior to conducting
experiments, cells were harvested from culture flasks with trypsin
(0.05 mg/ml) and EDTA (0.02 mg/ml) (both from ICN-Flow). For FACS
analysis of adhesion molecule expression, cells were plated at
105 cells/well in 24-well plates (2.0-cm2
wells; Linbro, Flow, McLean, Va.). For estimation of adhesion molecule
mRNA levels, cells were plated at 5 × 105 cells/well
in six-well plates (9.4-cm2 wells; Linbro, Flow). For ERK,
p38, c-jun N-terminal kinase (JNK), and NF-B assays,
cells were plated at 2.5 × 106 cells per dish in
tissue culture plates (54-cm2 dishes; Corning). All plates
and dishes were precoated with 0.2% gelatin as described above. Cells
were used when confluent, usually 24 to 72 h after plating. Prior
to treatment the culture medium was removed from the wells and
treatments were added in fresh medium containing 5% human group AB serum.
ELISA. HUVEC (5 × 104/well) were plated into 96-well plates which had been precoated with 0.2% gelatin. The cells reached confluence within 1 to 3 days and were treated in the wells. At the end of any treatment period, the wells were washed twice with warm 0.1% bovine serum albumin (BSA) in HBSS, 0.2 ml per well, and then fixed overnight with glutaraldehyde (0.025%; 20°C; 0.2 ml/well). The monolayers were then washed twice with BSA and incubated with blocking buffer (0.1% BSA in HBSS in 0.1 M glycine; 20°C for 2 h). The ELISA was performed with three washes with 0.2 ml of 0.1% BSA per well between each step. HUVEC were incubated with 50 µl of primary monoclonal antibody per well and 70 µl of secondary HRP-conjugated antibody per well 1 h each at 37°C. Finally, 100 µl of enzyme substrate, consisting of 0.55 mg of 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate-(6)] per ml and 0.012% hydrogen peroxide in citrate-phosphate buffer, pH 4.2, was added per well, and color was developed until cell-alone wells gave a standardized absorbance reading at 410 nm (optical density, 0.3 units) using an ELISA plate reader (Dynatech MR 7000).
Flow cytometry. Endothelial cells were treated with various concentrations of agonists and for the times indicated in the figure legends. At the end of the treatment period the cells were washed twice with warm HBSS and then enzymatically detached with 0.35 ml of trypsin-EDTA per well. The cell suspension was collected into FACS tubes (Becton Dickinson), transferred onto ice, washed with 0.5 ml of ISOTONE II solution (4°C; Coulter Electronics), and then resuspended in 50 µl of primary antibody solution (1:100) and incubated on ice for 30 min. After three washes with ISOTONE II (0.5 ml) the secondary antibody was added. After 30 min on ice the cells were washed and fixed with formaldehyde-ISOTONE II (1:33; 0.3 ml). The fluorescence intensity of the cell population was analyzed by flow cytometry on a FACScan (Becton Dickinson). Ten thousand cells per sample were counted, and the data were processed using Lysis II software (Becton Dickinson). The fluorescence values of isotype-matched negative controls were substracted from fluorescence values of the treatments.
Measurement of mRNA.
The human E-selectin cDNA probe was a
3.8-kb insert cloned into the XhoI site of a pB-SK vector, a
gift from J. Gamble, Hanson Centre for Cancer Research, Institute of
Medical and Veterinary Science, Adelaide, Australia. The GAPDH
(glyceraldehyde-3-phosphate dehydrogenase) probe cocktail was purchased
from Clontech. HUVEC were treated as described in the legend of Fig. 2.
RNA for the slot blots was isolated by the RNAzolB method
(Cinna/Biotrex) by direct addition of 0.4 ml of RNAzolB per
106 cells and lysis on the tissue culture plate. Chloroform
(50 µl) was added to the lysates, and each tube was vigorously shaken for 15 s and placed on ice for 5 min. After centrifugation (14,000 × g for 15 min at 4°C) the upper phase was carefully
removed and the RNA was precipitated by the addition of an equal volume
of isopropanolol by incubation on ice for 15 min. The RNA was pelleted by centrifugation at 14,000 × g (15min, 4°C). The RNA
samples were diluted in a solution containing formaldehyde, SSC (1×
SSC is 0.15 M NaCl plus 0.015 M sodium citrate), sodium dodecyl sulfate (SDS), and diethyl-pyrocarbonate and heated (65°C for 5 min). The
samples were applied to a Gene Screen Plus nylon membrane using a slot
blot vacuum manifold (Hoeffer). The filters were baked (2 h at 80°C),
and prehybridization was performed for 16 to 24 h in a solution
containing formamide, dextran sulfate, SDS, SSC, Denhardt's solution,
and denatured herring sperm DNA (NEN, Du Pont). DNA probes were
oligolabeled using [-32P]dCTP (Amersham) as described
previously (17) and used for hybridization. The extent of
hybridization was subjected to quantitative analysis by Instant Imager
(Packard Instruments). The quantitations for the blots were calculated
as the value obtained for the E-selectin probing of that blot divided
by the value of its GAPDH reprobing and expressed as a normalized ratio.
Nuclear extracts.
Nuclear extracts were prepared as
described by others (21) with slight modifications.
Briefly, confluent endothelial monolayers were stimulated with diluent,
TNF, LPS, or TNF and LPS. After incubation cells were washed twice with
cold phosphate-buffered saline (8 ml; 4°C), harvested with a cell
scraper, and centrifuged (175 × g for 5 min at 4°C). The
pellet was resuspended in 1 ml of phosphate-buffered saline cold.
Following centrifugation (14,000 × g at 4°C for 15 s) the
cells were lysed (10 min at 4°C) by adding 80 µl of lysis buffer
(pH 7.8) (HEPES [10 mmol/liter], MgCl2 [1.5 mmol/liter], KCl [10 mmol/liter], sucrose [300 mmol/liter 0.5% [vol/vol] Nonidet P-40, dithiothreitol [1 mmol/liter],
phenylmethylsulfonyl fluoride [1 mmol/liter], leupeptin [10
µg/ml], aprotinin [10 µg/ml], pepstatin A [10 µg/ml]
benzamidine [10 µg/ml]). The tubes were washed a second time, the
pellets were resuspended in 60 µl of lysis buffer, and the nuclei
were disrupted by sonication. After centrifugation (14,000 × g for 30 s) an aliquot was kept aside for protein
determination while the remaining supernatants were collected, mixed
with Laemmli buffer (100°C for 5 min), and stored at 
70°C until
analyzed by Western blotting.
Western blotting. The Western blotting assay was carried out as described previously (16). Briefly, equal amounts of nuclear proteins (usually 80 to 100 µg) were separated by SDS-12% polyacrylamide gel electrophoresis (SDS-12% PAGE). After transfer, equal loading per lane was confirmed by staining the intracellular membrane with Ponceau S. The blots were then blocked and incubated with primary antibody, washed (six times), and subsequently incubated with secondary antibody (both antibodies at 1:1,000; 1 h at 37°C). After six washes immunocomplexes were visualized by enhanced chemiluminescence. The blots were scanned with an ImageQuant scanner and quantified with ImageQuant software version 3.3 (Molecular Dynamics, USA).
ERK, p38, and JNK assays.
After exposure of HUVEC to the
various agonists for 15 min incubations were terminated by removing the
culture medium and washing the cells twice (8 ml of HBSS at 4°C). Per
dish, 200 µl of lysis buffer (HEPES [20 mmol/liter] [pH 7.4],
0.5% [vol/vol] Nonidet P-40, NaCl [100 mmol/liter], EDTA [1
mmol/liter], Na3VO4 [2 mmol/liter],
dithiothreitol [2 mmol/liter], phenylmethyl sulfonyl fluoride [1
mmol/liter], leupeptin [10 µg/ml], aprotinin [10 µg/ml], pepstatin A [10 µg/ml], benzamidine [10 µg/ml]) was added, and the cells were harvested with a cell scraper and incubated with constant mixing (4°C for 2 h). After centrifugation (14,000 × g for 20 s) an aliquot of each supernatant was taken
for protein determination while the remaining supernatants were
collected and stored at 70°C until assayed for kinase activity.
Equal amounts of ERK and p38 were immunoprecipitated before
determination of kinase activity as described elsewhere
(16). Briefly, lysates (usually 800 µg of protein) were
precleared with protein A-Sepharose. Anti-p38 or anti-ERK antibody (3 µg/sample) was added, and tubes were incubated with constant mixing
(90 min at 4°C). The antigen-antibody complexes were precipitated by
the addition of protein A-Sepharose. The immunoprecipitates were washed
twice at 4°C, first with lysis buffer and then with assay buffer
(HEPES [20 mmol/liter] [pH 7.2], 
-glycerophosphate [20
mmol/liter], p-nitrophenylphosphate [3.8 mmol/liter],
MgCl2 [10 mmol/liter], dithiothreitol [1 mmol/liter], Na3VO4 [50 µmol/liter], ATP [20
µmol/liter]). The assay was started by adding 30 µl of assay
buffer containing 6 µCi of [
-32P]ATP per sample and
35 µg of myelin basic protein per ml. After a 20-min incubation at
30°C, the assay was terminated by the addition of Laemmli buffer and
the samples were boiled (100°C for 5 min). Phosphorylated myelin
basic protein was resolved by SDS-16% PAGE and was detected and
quantified using an Instant Imager and Imager software (Packard
Instruments). JNK was assayed by a solid-phase method as described
previously (16). Briefly, glutathione
S-transferase-Jun (1-79) fusion protein was purified from
bacterial lysates using glutathione-Sepharose 4B (Pharmacia Biotech) at
4°C with gentle rocking. Lysate protein (1,000 µg),
MgCl2 (15 mmol/liter), and ATP (10 µmol/liter) were added
to 25 µl (packed volume) of glutathione S-transferase-Jun
(1-79) coupled to glutathione-Sepharose beads. The mixtures were
incubated with gentle rocking (2 h at 4°C). After centrifugation
(14,000 × g at 4°C for 5 min) the beads were washed once
with lysis buffer, once with wash buffer (pH 7.0) [piperazine-N,
N'-bis(2-ethanesulfonic acid) (10 mmol/liter), NaCl (100 mmol/liter)] and once with assay buffer (as above). The assay was
started by adding 30 µl of assay buffer containing 6 µCi of
[-32P]ATP/sample (30°C for 20 min) and terminated by
the addition of Laemmli buffer (100°C for 5 min). Samples were
resolved by SDS-12% PAGE, and detection and quantification of
phosphorylated glutathione S-transferase-Jun (amino acid
residue 1-79) were carried out as above.
Statistical analysis. Data were processed by either analysis of variance or the nonparametric Kruskal-Wallis test with Dunne's posttest if the data were not normally distributed.
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RESULTS |
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Materials and Methods
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Discussion
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TNF and LPS act synergistically to upregulate HUVEC adhesion
molecule expression.
Concentration-response studies were performed
to determine the lowest concentrations of TNF and LPS which produced a
response. These were found to be 5 U of TNF per 105 cells
and 0.1 ng of LPS per ml (Fig. 1).
Consistent with previous studies our results demonstrated that
E-selectin began to appear on the cell surface 2.5 to 3 h
following TNF or LPS addition and peaked at 6 h. VCAM-1 expression
was evident at 7 to 8 h and was maximal at 12 h. ICAM-1 was
constitutively expressed on resting cells, and its upregulation
followed a similar time course to that of VCAM-1 (Fig.
2).
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) or LPS (0.1 ng/ml), (
), or TNF plus LPS (
) for the times
indicated. The results are the means ± standard errors of the
means (error bars) of five experiments with cells from five cords. (A)
E-selectin was measured by ELISA. The value for untreated cells was
0.009 and was subtracted from the treatment results. Each experiment
was run in triplicate. *, for TNF versus TNF plus LPS or LPS versus
TNF plus LPS, P < 0.001; for the sum of TNF and LPS
versus TNF plus LPS, P < 0.01. OD, optical density.
(B) E-selectin was measured by flow cytometry. Cells were treated with
diluent (
), TNF (5 U/105 cells) (
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Effects on NF-B accumulation in the nucleus.
NF-B is
activated by proinflammatory cytokines and bacterial products.
Activation is achieved by the phosphorylation and subsequent
degradation of NF-B's naturally occurring cytosolic inhibitor,
IB. p65 rather than p50 was examined because it is the dominant,
transcriptionally active subunit of NF-B (30). The
activated NF-B then translocates into the nucleus, where it binds to
the promoters of adhesion molecule genes (21, 25). This is
a transient phenomenon that peaks between 1 and 4 h after a
stimulus and diminishes thereafter, coinciding with the reconstitution of IB (26). Experiments were set up in which
endothelial cells were treated with diluent, TNF, LPS, or TNF and LPS
for 2.5 h and the quantity of NF-B in nuclear extracts was
assayed by Western blotting. The amount of NF-B that had accumulated
in the nucleus after the combined treatment with TNF and LPS was
significantly greater than that obtained with either TNF or LPS alone,
as well as greater than the sum of these responses (Fig.
7). When nuclear extracts of HUVEC were
assayed after 2.5, 8, 20, or 28 h of incubation in the presence of
both TNF and LPS, there was still an increase in NF-B after 28 h (Fig. 8).
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Effects of TNF and LPS on MAP kinases.
Besides NF-B, the
transcriptional factors c-Jun and ATF2 are also required for E-selectin
transcription. The transcriptional activities of c-Jun and ATF2 are
regulated via their phosphorylation by JNK and/or p38 mitogen-activated
protein (MAP) kinase (20, 26). JNK, p38, and ERK are
members of three distinct MAP kinase cascades, each consisting of a
series of kinases in which upstream kinases activate downstream kinases
by phosphorylation (31). While there is no conclusive
evidence that ERK is important in TNF-mediated endothelial cell
activation, studies have demonstrated that inhibition of the activities
of kinases within the JNK and/or p38 cascades results in the inhibition
of the transcriptional activity of the E-selectin promoter in
endothelial cells (20). LPS also has the capability to
stimulate the activities of p38, JNK, and ERK (1, 27).
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DISCUSSION |
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Materials and Methods
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The data demonstrate that TNF and LPS act synergistically on endothelial cells to increase the adhesive properties of these cells. When added together at concentrations encountered in bacterium-induced inflammation, these mediators caused significantly greater expression of the adhesion molecules E-selectin, ICAM-1, and VCAM-1 than the sum of either alone. Importantly, at these concentrations TNF or LPS per se caused little or no upregulation of endothelial adhesion molecule expression.
The induction of endothelial adhesion molecules is an important early step in the development of an inflammatory reaction; thus, the synergism described is likely to be relevant in immunity to bacteria. Promoting the infiltration of circulating leukocytes to foci of infection may enhance the resolution of an infection; however, promoting the infiltration of leukocytes into atherosclerotic plaques is likely to contribute to an exacerbation of such lesions and possibly to the initiation cardiovascular events. E-selectin and VCAM-1 are instrumental in recruiting monocytes and lymphocytes, which are implicated in atheromatous lesions (28).
Our findings of synergism between mediators demonstrate the importance
of cross talk in the regulation of the inflammatory response. To
understand how this interaction may operate, we focused on the effects
of TNF and LPS on E-selectin mRNA expression since the intracellular
signaling pathways that regulate E-selectin expression are known.
Consistent with the data on E-selectin protein expression, TNF and LPS
were synergistic in upregulating the expression of E-selectin mRNA. TNF
and LPS individually have been shown to activate NF-B in endothelial
cells (20), and our data demonstrate that TNF and LPS
caused a synergistic activation of NF-B. Although the precise
mechanism by which TNF and LPS caused the synergistic activation of
NF-B is not known, it is possible that the signaling cascade due to
TNF and LPS converged at NF-B-inducing kinase. Consistent with this
synergism, it has been shown independently that TRAF2 (activated by
TNF) and TRAF6 (activated by LPS) directly interact with
NF-B-inducing kinase (23, 24).
The importance of the NF-B system for endothelial adhesion molecule
expression is illustrated by the fact that overexpression of IB
blocks TNF-induced VCAM-1 transcriptional activity (35), inhibition of NF-B reduces E-selectin expression (5),
and overexpression of the p65 subunit of NF-B results in activation of E-selectin promoter activity without stimuli (7). The
observation that in the presence of both TNF and LPS the usually
transient activation of NF-B persisted for as long as 28 h is
consistent with the role played by this transcription factor in the
amplification and perpetuation of the inflammatory response
(2). Furthermore, activated NF-B has been found to be
present in atherosclerotic lesions (5).
When the activities of other relevant intracellular signaling
molecules, p38, JNK, and ERK, were investigated, TNF and LPS were shown
to be synergistic in the upregulation of the activity of p38. The
activity of JNK was increased in an additive manner. While both
mediators individually increased the activity of ERK, there was no
enhancement with costimulation. These data suggest that NF-B, and
possibly p38, and JNK but not ERK are likely to contribute to the TNF
and LPS synergism on the upregulation of E-selectin expression.
Recently we have demonstrated the presence of the LPS receptor, CD14, on HUVEC (17a). Previously we also demonstrated that TNF upregulated CD14 expression on human neutrophils (18). However, it is unlikely that this is a mechanism of synergism between the cytokine and LPS in HUVEC since we found that treatment of HUVEC with 10 to 100 U of TNF did not increase the expression of CD14 (data not presented).
During infection immune cells as well as other cell types are exposed concomitantly to a variety of mediators, including cytokines and bacterial products. Synergism between mediators as demonstrated by us for TNF and LPS in terms of endothelial adhesion molecule expression may influence the course of bacterial infection and endothelial pathophysiology such as atherosclerosis. In addition to TNF, LPS is capable of inducing a range of inflammatory mediators such as interleukin 1 and activating complement components. It would be interesting to compare other mediators with the effects of TNF on the LPS responses, taking into consideration those mediators which use intracellular signaling molecules (e.g., interleukin 1) (36) very similar to and those which use signaling pathways (complement) significantly different from LPS. The understanding of these interactions and their mechanisms is pivotal to the development of new strategies to manipulate the intercellular mediator network for therapeutic advantages.
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ACKNOWLEDGMENTS |
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We are indebted to the birthing mothers, who donated their cords, and the midwives of the labor ward of the Women's and Children's Hospital, North Adelaide, Australia, who provided them to us 24 h a day. We are grateful to Ian Bates from the Red Cross, Adelaide, South Australia, Australia, for the generous supply of human group AB serum.
This work received support from the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Immunopathology, The Women's and Children's Hospital, 72 King William Rd., North Adelaide, South Australia 5006, Australia. Phone: 61 8 8204 7216. Fax: 61 8 8204 6046. E-mail: aferrant{at}medicine.adelaide.edu.au.
Present addresses: Respiratory Medicine Unit, Rayne Laboratory,
University of Edinburgh Teviot Place, Edinburgh EH8 9AG, United Kingdom.
Editor: R. N. Moore
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References
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Infection and Immunity, March 2001, p. 1273-1279, Vol. 69, No. 3
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.3.1273-1279.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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