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Infection and Immunity, March 1999, p. 1505-1507, Vol. 67, No. 3
Department of Medical Microbiology,
University of Wales College of Medicine, Heath Park, Cardiff CF4
4XN, United Kingdom
Received 15 June 1998/Returned for modification 26 August
1998/Accepted 19 November 1998
Whole cells and lipopolysaccharides (LPSs) extracted from
Burkholderia cepacia, Pseudomonas aeruginosa,
Stenotrophomonas maltophilia, and Escherichia
coli were compared in their ability to stimulate tumor necrosis
factor alpha (TNF- In recent years, Burkholderia
cepacia infection in cystic fibrosis (CF) patients has become a
clinical issue of increasing concern. B. cepacia is highly
transmissible from person to person (4, 9) and intrinsically
resistant to many antibiotics (3, 10). Once acquired,
B. cepacia infection is rarely eradicated from the lungs of
CF patients. In addition, a proportion of patients who acquire B. cepacia infection develop a rapidly fatal pneumonia and sepsis
with a high mortality rate, the so-called cepacia syndrome (6,
8). However, the pathogenic mechanisms involved in this syndrome
remain unclear. Indeed, the nature of the virulence factors that
contribute to the pathogenicity of B. cepacia in CF remains largely unknown. Due to its potent inflammatory activity and
association with sepsis, lipopolysaccharide (LPS) might be an important
virulence factor in B. cepacia infections. Elevated tumor
necrosis factor alpha (TNF- The human monocytic cell line MonoMac-6 (MM6) was obtained from the
German collection of microorganisms and cell cultures (Deutsche
Sammlung von Mikroorganismen), Braunschweig, Germany. In this study, we
used 13 B. cepacia clinical CF isolates (from 10 of which
LPS was purified), 7 B. cepacia environmental isolates (from
4 of which LPS was purified), 8 S. maltophilia isolates (from all 8 of which LPS was purified), and 7 P. aeruginosa
isolates (from all 7 of which LPS was purified). E. coli
O111:B4 LPS was purchased from Sigma Chemical Co., Poole, United
Kingdom, and LPS was also purified from E. coli NCTC 10418. LPSs were purified by the phenol-water extraction method (2)
and characterized by silver staining on a sodium dodecyl sulfate-14%
polyacrylamide gel. Coomassie blue staining of the gels did not reveal
any visible protein bands in the purified LPSs. The activity of
different doses of LPS to produce TNF- Statistical significance was calculated by the nonparametric
Mann-Whitney test by using Minitab release 10 software. The role of
CD14 in the LPS-stimulated TNF- Figure 1 shows the TNF-
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Lipopolysaccharide (LPS) from Burkholderia
cepacia Is More Active than LPS from Pseudomonas
aeruginosa and Stenotrophomonas maltophilia in
Stimulating Tumor Necrosis Factor Alpha from Human Monocytes
ABSTRACT
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) from the human monocyte cell line MonoMac-6.
B. cepacia LPS, on a weight-for-weight basis, was found to
have TNF--inducing activity similar to that of LPS from E. coli, which was approximately four- and eightfold greater than
the activity of LPSs from P. aeruginosa and S. maltophilia, respectively. The LPS-stimulated TNF- production
from monocytes was found to be CD14 dependent. These results suggest
that B. cepacia LPS might play a role in the pathogenesis
of inflammatory lung disease in cystic fibrosis, and in some patients
it might be responsible, at least in part, for the sepsis-like cepacia syndrome.
TEXT
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Abstract
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) levels have been implicated in both
systemic and local inflammations (7) and may play a role in
cepacia syndrome (5). In this report, we studied the
activity of B. cepacia whole cells as well as purified LPS
in stimulating human monocytes to release TNF- and compared it with
the activities of other gram-negative bacteria that also colonize the
lungs of CF patients, Pseudomonas aeruginosa and
Stenotrophomonas maltophilia. Patients carrying these
organisms may, over time, suffer a chronic deterioration of lung
function, but there are no reports that acquisition of these organisms
is followed by the acute pulmonary deterioration sometimes seen in
B. cepacia-infected patients. Escherichia coli was included in this study since the LPS from this organism is often
reported to represent the most biologically active form of LPS
(11), and it is used as a reference standard for the measurement of LPS activity by the Limulus assay.
was established by
stimulating MM6 cells with LPS concentrations ranging from 0.1 to 1000 ng/ml. However, at an LPS concentration above 500 ng/ml, the linearity of the TNF- response decreased, and thus 500 ng of LPS per ml was
used as the optimum concentration for this study. MM6 cells suspended
at 106/ml of RPMI 1640 medium containing 10% fetal calf
serum (Gibco) and 5 ng of phorbol myristate acetate per ml
(13) were stimulated with 500 ng of LPS or 106
CFU of whole bacteria (all bacterial isolates were grown under identical conditions). MM6 incubated with phorbol myristate acetate only (without LPS) did not produce any significant amounts of TNF-.
The cells were incubated overnight, and the supernatants were collected
after centrifugation at 3,000 × g for 3 min followed by filtration through a 0.2-µm-pore-size syringe filter (Acrodisc; Gelman Sciences, Ann Arbor, Mich.). Supernatants were stored at 
80°C until assayed. The TNF- enzyme-linked immunosorbent assay (ELISA) was performed with monoclonal anti-human TNF- antibody and
biotinylated anti-human TNF- antibody (R & D, Minneapolis, Minn.) in
accordance with the manufacturer's instructions.
production was investigated by using
monoclonal anti-human CD14 antibodies MY4 (Coulter Corporation, Miami,
Fla.) and UCHM-1 (Sigma). The monoclonal anti-human CD7 antibody 3A1
(Sigma) was used as an isotype control antibody for MY4 (immunoglobulin
G2b). Five microliters (2.5 µg) of monoclonal antibody was incubated
with 106 MM6 cells at room temperature for 30 min, and the
cells were then washed with RPMI 1640 medium to remove unbound
antibody. The MM6 cells were then stimulated with 500 ng of LPS. The
TNF- produced was measured by an ELISA as described above.
production in
MM6 cells stimulated with different doses of LPS. All of the LPS
preparations used gave a dose response for TNF- production, although
there was variation in the total TNF- stimulated by each type of
LPS. A 500-ng/ml dose of LPS was found to be optimal for each LPS type for TNF- production. Therefore, the different LPS types were compared at this dose. The ELISA results (Fig.
2) showed that B. cepacia LPS
from CF patients induced a concentration of TNF- similar to that
induced by E. coli LPS (mean TNF- concentration of 4,000 pg/ml). On the other hand, B. cepacia LPS from environmental isolates had half the activity (2,000 pg/ml) of that from CF isolates (Fig. 2). Moreover, LPS from B. cepacia CF isolates had
fourfold the activity of P. aeruginosa LPS (1,000 pg/ml) and
eightfold the activity of S. maltophilia LPS (500 pg/ml).
TNF- stimulated by the corresponding whole bacterial cells showed a
similar pattern to that produced by the extracted LPS (Fig. 2). If
LPS-mediated macrophage stimulation plays a role in the development of
inflammatory lung pathology, the differences in the TNF- stimulatory
activity between LPSs from B. cepacia, P. aeruginosa, and S. maltophilia may in part explain the
different pathogenic sequelae observed with lung infections due to
these organisms in CF patients. S. maltophilia, in
particular, is similar to B. cepacia in many aspects, such
as high patient-to-patient infection rate, resistance to most
antibiotics, and no significant exotoxin production. However, unlike
the case with B. cepacia, despite numerous investigations, there have yet to be reports of acute pulmonary inflammation occurring following S. maltophilia acquisition (1).
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FIG. 1.
Dose response of LPS-induced TNF- production from
stimulated MM6 cells. MM6 cells (106/ml) were incubated
with 0.1 to 500 ng of LPS per ml, and TNF- production was measured
by an ELISA. The TNF- produced was proportional to the LPS
concentration up to an LPS dose of 500 ng/ml. Bars represent the mean
(+ standard error) of four separate experiments.
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FIG. 2.
TNF- production by MM6 cells stimulated by whole
bacteria and the extracted LPS. The bars show the mean (+ standard
error) of TNF- production in response to the different LPS types
(number of different isolates = 4 to 10 in each group) from four
separate experiments. *, P = 0.021; **, P = 0.0054; ***, P = 0.00012 (all versus results for
B. cepacia [B.cep.] CF isolates). P.aerug, P. aeruginosa; S.malto, S. maltophila.
To study the role of CD14 in B. cepacia LPS-mediated TNF-
stimulation, monoclonal anti-human CD14 antibodies were used to block
CD14 binding sites and consequently inhibit TNF- production. When
monoclonal antibodies were added, TNF- production was significantly inhibited following B. cepacia LPS stimulation in comparison
with TNF- production in control cells or those cells incubated with the isotype antibody (Fig. 3). We found
similar results with S. maltophilia and P. aeruginosa. Contrary to a previous report by Shaw et al.
(12), our investigations suggest that B. cepacia LPS-mediated TNF- stimulation is almost completely mediated by CD14.
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We conclude that B. cepacia LPS is a potent immunostimulator
and that chronic colonization may lead to LPS shedding and TNF- production, resulting in tissue damage and lung inflammation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF4 4XN, United Kingdom. Phone: 44 1222 744725. Fax: 44 1222 742161. E-mail: JacksonSK{at}CF.AC.UK.
Editor: R. N. Moore
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Infection and Immunity, March 1999, p. 1505-1507, Vol. 67, No. 3
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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