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Infection and Immunity, May 2000, p. 2854-2862, Vol. 68, No. 5
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Capsular Polysaccharide and O-Specific Antigen
Divergently Modulate Pulmonary Neutrophil Influx in an
Escherichia coli Model of Gram-Negative Pneumonitis in
Rats
Thomas A.
Russo,1,2,3,*
Bruce A.
Davidson,3,4
Roger L.
Priore,5
Ulrike B.
Carlino,1,3
Jadwiga D.
Helinski,3,4 and
Paul R
Knight III2,3,4
Department of
Medicine,1 Department of
Microbiology,2 The Center for Microbial
Pathogenesis,3 Department of
Anesthesiology,4 and Department of
Social and Preventative Medicine,5 State
University of New York at Buffalo, Buffalo, New York 14214
Received 3 September 1999/Returned for modification 10 January
2000/Accepted 27 January 2000
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ABSTRACT |
Enteric gram-negative bacilli cause a severe, often
life-threatening pneumonia. An improved understanding of the
pathogenesis of this infection may lead to improved treatment. Nearly
all of the responsible gram-negative bacilli possess capsular
polysaccharides and/or an O-specific antigen as part of their
lipopolysaccharide (LPS). We hypothesized that these surface
polysaccharides may modulate the pulmonary host response. To
investigate this, a rat pneumonitis model was used, and pulmonary
neutrophil influx, a critical aspect of host defense, was measured. To
assess for the effect of the capsule and O-specific antigen on this
host response, three proven, isogenic derivatives that are deficient in
capsular polysaccharide alone (CP9.137), the O-specific antigen moiety of the LPS alone (CP921), and both the capsular polysaccharide and
O-specific antigen (CP923), as well as their wild-type parent (CP9),
were used as challenge strains at various intratracheal challenge
inocula (CI). Total lung myeloperoxidase (MPO), a surrogate marker for
neutrophils, was measured for 15 h post-bacterial challenge. To
determine the effect of capsule and the O-specific antigen on the
measured MPO levels, a mathematical model was developed and used to
describe the MPO levels as a function of time for each CI of each of
the four strains. The results from this analysis demonstrated that in
the absence of the K54 capsule, 80.7 times the CI is necessary to
achieve the same maximum MPO level relative to K54 positive strains
(P < 0.0001). In contrast, a diametric effect was
observed in the absence of the O-specific antigen, where 0.13 times the
CI was necessary to achieve the same maximum MPO level relative to
O4-positive strains (P = 0.0032). No interactive effect was observed between the capsule and the O-specific antigen. These findings demonstrate that these surface polysaccharides modulate
pulmonary neutrophil influx and suggest that the K54 capsular
polysaccharide is a proinflammatory mediator and that the O4-specific
antigen attenuates the proinflammatory response. If these speculations
are substantiated, an understanding of how the capsule and the
O-specific antigen modulate host response could have significant
therapeutic implications. The potential use of biologic modulators
directed against the host response, as well as approaches based on
inactivating bacterial components (e.g., surface polysaccharides)
in attempts to modify sepsis syndromes, could be developed.
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INTRODUCTION |
Enteric gram-negative bacilli are a
group of pathogens that are capable of causing severe, life-threatening
pneumonia (8, 24, 26). Despite the availability of active
antimicrobial agents, there has been little improvement in outcome from
this infection over the last 10 to 15 years. As a result, this syndrome continues to cause significant morbidity and mortality and strongly contributes to the economic burden of our national health care system.
An improved understanding of the pathogenesis of this infection may
result in improved treatment or prevention of infection.
The importance of neutrophils in protecting against infection in the
lung is as great as any other site in the body (23). The
increased susceptibility and severity of infection that occurs in the
setting of chemotherapy-induced neutropenia or genetically inherited
disorders of neutrophil function (e.g., chronic granulomatous disease)
are unequivocal proof. We are now capable of modulating the host
response to bacterial infection, including both neutrophil numbers and
their state of activation. However, as is true for many biologic
response systems, an appropriately balanced response, which in this
case is maximal bacterial clearance while minimizing pulmonary damage,
is needed for optimal outcome (4, 5). Prior to considering
manipulating the host biologic response as a treatment modality in
gram-negative pneumonitis, it is critical to understand the mechanism
by which the host responds to bacterial challenge and how certain
bacterial components modulate this response. Although our understanding
of the pulmonary inflammatory response, which leads to the influx of
neutrophils, into the lung has significantly progressed (13, 23,
25), other than studies on lipid A there is little information
available on other gram-negative factors that may affect this crucial
host response. This information is needed for the logical development
of rapid diagnostic tests, which in turn will enable the clinician to
effectively utilize a variety of novel therapeutics, including immune modulators.
We hypothesized that bacterial surface polysaccharides may modulate the
host inflammatory response in gram-negative pneumonitis. To test this
hypothesis, we utilized a rat model of acute pulmonary infection and
measured pulmonary neutrophil influx over the course of infection. An
extraintestinal human isolate of Escherichia coli (CP9,
O4/K54/H5) was used as a model pathogen, in part because E. coli is one of the commonly isolated agents in nosocomial
gram-negative pneumonia (2) but more importantly because
three proven isogenic derivatives have been generated that are
deficient in capsular polysaccharide alone (CP9.137), the O-specific
antigen moiety of the lipopolysaccharide alone (CP921), and both the
capsular polysaccharide and the O-specific antigen (CP923). Challenge
with these strains and subsequent measurement of myeloperoxidase (MPO), a surrogate marker for neutrophils, and bacterial growth enabled us to
evaluate the ability of these defined bacterial traits to modulate
neutrophil influx.
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MATERIALS AND METHODS |
Bacterial strains.
A human bacteremic isolate of E. coli (CP9, O4/K54/H5) and isogenic derivatives have been used as
model pathogens for these studies (19, 21). CP9 is well
characterized and is virulent in a variety of in vivo infection models
(18, 20). It possesses a group 3 capsule (K54), O4-specific
antigen, alpha-hemolysin, cytotoxic necrotizing factor, P pilus (class
I PapG adhesin), Prs pilus (class III PapG adhesin), and type 1 pilus
and is complement resistant. Transposon mutagenesis and transduction
was used to generate proven isogenic derivatives of CP9 deficient in
the K54 antigen alone (CP9.137), the O4 antigen alone (CP921), or both the K54 and O4 antigens (CP923) (19, 21). Previous studies established that the LPS in O4 antigen deficient mutants consists of
intact lipid A and core polysaccharide moieties plus either a single or
a portion of a single O4 antigen pentasaccharide (21).
Pulmonary infection model.
An established rat (Long-Evans)
model for studying pulmonary damage was used and modified for these
studies (9-11, 14). Long-Evans rats (250 to 300 g)
were anesthetized with 5% halothane in 100% oxygen until unconscious
and then maintained at 2% halothane. The animals were suspended from
the front teeth via a suture on a 60°C incline board. The trachea was
exposed surgically, and a 4-in. piece of 1-0 silk was slipped under the
trachea using a curved hemostat. The challenge inoculum (1.2 ml/kg) was
introduced intratracheally via a 1-ml syringe and 26-gauge needle, and
the incision was closed with surgical staples. Experimental animals were challenged with various challenge inocula (CI; 1.3 × 106 to 3.0 × 108 CFU) of CP9 (wild type
[wt]), CP9.137 (capsule deficient), CP921 (O-specific antigen
deficient), and CP923 (capsule and O-specific antigen deficient) to
establish the consequences of infection for each of these strains.
Control animals were anesthetized identically to experimental animals
but did not receive a bacterial challenge. Animals that died within 15 min after bacterial challenge were excluded from the analysis since
these deaths were attributed to the challenge procedure and not to the
infecting bacteria themselves. Each experimental animal was
prospectively assigned a time for subsequent sampling. At harvest the
animal was anesthetized as described above, and betadine was used to
sterilize its abdomen and chest. The descending aorta and vena cava
were exposed, and appropriate samples were removed (e.g., blood gas,
blood culture, blood for serum, etc.). For removal of the lungs, the
thoracic cavity was exposed, and 10 ml of 1× phosphate-buffered saline (PBS; pH 7.4) was injected into the right ventricle to "flush" the
lung vasculature. The trachea, lungs, and heart were removed en bloc,
and the lungs were subsequently dissected free and weighed. If
appropriate, sterile saline was added to yield a total weight of
10 g. The lung tissue was then homogenized (Polytron PT200; Brinkmann Instruments, Westbury, N.Y.) for 3 s three times on ice
(setting 6). Pilot experiments have demonstrated that homogenization resulted a 100% yield of the bacterial challenge inoculum.
(i) Assessment of neutrophil influx.
Total lung MPO activity
was measured at 1, 3, 6, 9, 12, and 15 h postchallenge for a given
strain and CI. Groups of three animals were preassigned to each of the
six harvest times; therefore, in each experiment the MPO activity was
determined in 18 animals. CP9 (wt), CP9.137 (capsule deficient), CP921
(O-specific-antigen deficient), and CP923 (capsule and
O-specific-antigen deficient) were each evaluated at several different
CI as follows: for CP9 (four experiments), 1.3 × 106,
5.6 × 106, 1.2 × 107, and 5.3 × 107; for CP9.137 (three experiments), 1.2 × 107, 7.7 × 107, and 2.3 × 108; for CP921 (five experiments), 3.0 × 106, 9.6 × 106, 3.4 × 107, 1.3 × 108, and 2.1 × 108; and for CP923 (three experiments), 1.3 × 107, 6.7 × 107, and 3.0 × 108. The lung MPO levels provide a precise means of
measuring organ content of recruited neutrophils. Total lung MPO
activity was quantified by the method of Goldblum et al.
(7). Lung homogenates were assayed for MPO activity using a
spectrophotometer reading with 0-dianisidine hydrochloride (Sigma) at
460 nm. Units were calculated based on the rate of increase over time.
(ii) Determination of bacterial titers.
Dissected total lung
tissue was weighed, and sterile normal saline was added to yield a
total weight of 10 g. The lung tissue was homogenized as described
above, and serial 10-fold dilutions were performed in 1× PBS for
determination of the CFU per milliliter. These titers were multiplied
by 10 to determine total lung titers.
(iii) Analysis of the effect of capsular polysaccharide and
O-specific antigen on MPO.
To quantify the effects of capsular
polysaccharide and O-specific antigen on MPO activity, the effects were
modeled using functions presented in the Appendix (see below). As a
first approximation, the effects of CP9.137, CP921, and CP923 were
modeled as if each were a dilution or concentration of CP9. For each
strain, the model for maximum MPO activity was a linear function of the
common logarithm of the challenge inoculum. The function used to
describe the increase in MPO activity before the maximum is reached was modeled using a logistic function of the logarithm of time. The model
for MPO activity was fitted using nonlinear regression by using SPSS
for Windows. The effects of deleting the capsule and deleting the
O-specific antigen and the synergy between these two effects were
estimated by reparameterizing the model. These effects were tested for
statistical significance by dividing each estimate by its standard
error and comparing the resulting Z score to a standard normal distribution.
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RESULTS |
Establishing relevance of the rat pneumonia model.
The
pertinent features of gram-negative pneumonia include bacterial growth,
pulmonary damage, and an ensuing inflammatory response. To establish
whether these characteristics pertained to our rat pneumonitis model,
experimental animals were challenged with various CI (1.3 × 106 to 5.3 × 107 CFU) of CP9 (wt). Groups
of three animals were preassigned to each of the six harvest times (1, 3, 6, 9, 12, and 15 h), and the consequences of infection were measured.
At a CI of 5.3 × 107 CFU of CP9, all 18 animals were
either premoribund or dead by 3 h (Fig.
1). A CI of 1.2 × 107
CFU (two experiments, 35 animals) resulted in proliferation at 
6 h in
88% (21 of 24) of the animals. Although in some animals there was a
transient decrease in bacterial titer at 3 h, the subsequent
growth trend was upward (Fig. 1). Blood cultures were positive in only
one animal (1 of 35, 2.8%). In two additional experiments (CI of
approximately 1.0 × 107 CFU) with death as the
endpoint (18 animals), the mean bacterial titer of CP9 at death was
1.1 × 1010 CFU, and deaths occurred over the
timeframe of 8.5 to 24 h in >90% of the animals that died. At
a CI of 5.6 × 106 CFU (one experiment, 17 animals)
clearance was observed in 13 of 17 animals, and at a CI of 1.3 × 106 CFU (one experiment, 18 animals) clearance occurred in
all 18 animals (Fig. 1). Therefore, a CI of approximately 1.0 × 107 CFU was most appropriate for a relevant bacterial
pneumonia model. A higher CI resulted in fulminant death, and a lower
CI generally resulted in bacterial clearance.
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FIG. 1.
Growth of CP9 in vivo at various CI in the rat
pneumonitis model. Intratracheal challenge was performed as described
in Materials and Methods. Each color represents animals given the same
indicated CI. Each point represents the bacterial titer from a single
animal that was harvested at a prospectively assigned time (closed
symbols) or was harvested immediately at the time of death (open
symbols).
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After challenge with 1.2 × 10
7 CFU of CP9, a
significant increase in pulmonary damage occurred over time as
manifested by increases
in lung weight, bronchoalveolar lavage protein
content, the endothelial
permeability index, and a decrease in
oxygenation compared to
the controls (Table
1). A significant inflammatory response,
as measured by neutrophil influx, also occurred (Table
1). These
parameters demonstrated that this model represented a progressive
bacterial proliferation and pulmonary damage with subsequent death
due
to respiratory failure, a result similar to untreated gram-negative
pneumonitis in humans.
Initial neutrophil influx is dependent on the bacterial CI.
The measurement of pulmonary MPO was used as a surrogate marker for the
influx of neutrophils into the lung. Baseline MPO levels (0 h) were
consistently low (34.0 ± 4.6). After bacterial challenge, MPO
levels increased, reaching a plateau level 6 to 8 h postchallenge.
Maximal MPO activity increased approximately 10- to 25-fold, depending
on the challenge strain and the CI utilized.
Bacterial growth or clearance varied depending on the challenge strain
(Fig.
2) and the CI. Therefore, it was
critical to
establish whether MPO activity (over the 15-h timeframe of
these
experiments) was dependent on the initial CI or the bacterial
load over time (the total number of bacteria over the course of
the
experiment which is dependent on the degree of subsequent
growth or
clearance of the bacteria). The latter measurement is
conceptually
equivalent to the area under the curve. Analysis
of MPO activity from
CI in which both clearance and growth occurred
for a given strain lent
significant insight into this matter.
This occurred for CP9 at CI of
5.6 × 10
6 and 1.2 × 10
7 (Fig.
1)
and for CP9.137 at a CI of 7.7 × 10
7. Presumably,
these CI were on the threshold at which the resident
host defenses were
able to contain the infecting bacterium. Comparison
of samples in which
bacterial clearance was achieved versus those
in which the challenge
strain was able to proliferate demonstrated
no difference in MPO
activity (Table
2). Therefore, this
analysis
demonstrated that MPO activity, at least for the 15-h time
course
of these experiments, depended primarily on the initial CI.
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FIG. 2.
Growth of CP9, CP9.137 (capsule deficient), CP921
(O-specific antigen deficient), and CP923 (capsule and O-specific
antigen deficient) in vivo in the rat pneumonitis model. Each color
represents animals challenged with approximately 1.0 × 107 CFU a given bacterial strain. Each point represents the
bacterial titer from a single animal that was harvested at a
prospectively assigned time (closed symbols) or was harvested
immediately at the time of death (open symbols).
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Assessing the effect of the K54 capsular polysaccharide and the
O4-specific antigen on neutrophil influx.
MPO levels were measured
and analyzed after challenge with CP9 in four experiments, CP9.137 in
three experiments, CP921 in five experiments, and CP923 in three
experiments. A modeling function was developed to describe the MPO
data, expressing the lung MPO level as a function of basal lung MPO
level (Y0), duration of E. coli challenge
(t), and the log of the challenge inoculum (X). Of the
parameters utilized to define this function (b, c,
Rn, Qn; see Appendix)
only Rn, which describes the strain-specific
effect on maximal lung MPO, was found to be different between strains
CP9, CP9.137, CP921, and CP923. Since these strains differ solely in
whether they produce capsule, O-specific antigen, or both it was
decided to further analyze these data in terms of the effect of capsule
and/or the O-specific antigen on the maximal lung MPO. The usefulness
of this approach was that (i) analysis was more powerful since data
obtained for all of the strains were utilized and (ii) interactions
(synergism, antagonism) between capsule and O-specific antigen could be
identified. These advantages would be lost by utilizing a
strain-to-strain comparison. To perform the analysis in this manner,
Rn and Qn were
reparameterized to rn and
qn and then reanalyzed (see Appendix).
Statistical tests were performed to formally test (i) the effect of
capsule and the O-specific antigen on the kinetics of increase of MPO
production, (ii) the effect of capsule and the O-specific antigen on
the maximal MPO levels, and (iii) whether an interaction between
capsule and O-specific antigen occurs. A summary of these data is
presented in Table 3. These tests compared (i) a grouping of strains that were deficient in the capsule
to the grouping of strains that contained the capsule (i.e., a
comparison of CP9.137 and CP923 to CP9 and CP921), (ii) a grouping of
strains that were deficient in the O-specific antigen to the grouping
of strains that contained the O-specific antigen (i.e., a comparison of
CP921 and CP923 to CP9 and CP9.137), and (iii) a grouping of strains
that are deficient in either the capsule or the O-specific antigen to
the grouping of strains that were deficient in neither or both (i.e., a
comparison of CP9.137 and CP921 to CP9 and CP923). The results in Table
3 indicate that there was no statistically significant effect on the
kinetics of increase in MPO activity in the presence or absence of
capsule. However, the K54 capsule did exert a statistically significant effect on the maximal MPO level. In the absence of the K54 capsule, 80.7 times (101.9071 = 80.7) the challenge inoculum is
necessary to achieve the same maximum MPO level, relative to a
K54-positive strains (P < 0.0001). The results in
Table 3 also indicate that there was no statistically significant
effect on the kinetics of increase in MPO activity in the presence or
absence of O-specific antigen. However, the O4-specific antigen did
exert a statistically significant effect on the maximal MPO level. In
the absence of the O-specific antigen, 0.13 times
(10
0.8787 = 0.13) the challenge inoculum was
necessary to achieve the same maximum MPO level, relative to a
O4-positive strains (P = 0.0032). The absence of the
O-specific antigen had an opposite effect on neutrophil influx as the
loss of the capsule. No synergy was observed between effects of the
capsule and the O-specific antigen. These findings are graphically
depicted in Fig. 3.
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TABLE 3.
Capsule and O-specific antigen significantly alter
maximum MPO levels but do not significantly change time to 50% of
maximum MPO increases
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FIG. 3.
The effect of deleting the K54 capsular polysaccharide
or the O4-specific antigen on pulmonary neutrophil influx (determined
by both maximum MPO level and the time to 50% maximum MPO level) in
the rat pneumonitis model. The CI multiplier is the factor by which the
challenge inoculum must be multiplied to achieve the same response that
occurred in the presence of the K54 capsule or O4-specific antigen
(e.g., in the absence of the K54 capsule, 80.7 times the challenge
inoculum is necessary to achieve the same maximum MPO level relative to
a K54 positive strains). A potential interaction between the K54
capsule and O4 specific antigen was also assessed. *, P < 0.005.
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These results can also be expressed in terms of the CI of each strain
which is required to produce a given maximum MPO level.
For example,
when comparing strains differing in only the presence
or absence of
capsule, the CI required to produce a maximum of
500 U of total lung
MPO would be 1.34 × 10
6 CFU for CP9 and 1.15 × 10
8 CFU for CP9.137 (86 times as high relative to CP9), and
the CI
required would be 1.88 × 10
5 CFU for CP921 and
1.43 × 10
7 CFU for CP923 (76 times as high relative
to CP921). Similarly,
when comparing strains differing in only the
presence or absence
of O-specific antigen, the CI required to produce a
maximum of
500 U of total lung MPO would be 1.34 × 10
6 CFU for CP9 and 1.88 × 10
5 CFU for
CP921 (0.14 times as high relative to CP9), and the CI
required would
be 1.15 × 10
8 CFU for CP9.137 and 1.43 × 10
7 CFU for CP923 (0.12 times as high relative to CP9.137).
An illustration of this fitted model for the MPO activity of each
strain at approximately equivalent CI (1.0 × 10
7 CFU)
is shown in Fig.
4. Since there were no
effects of capsule
or the O-specific antigen on the kinetics of
increase in MPO activity,
this illustration uses a reduced model with
those effects removed.
A qualitative assessment of the effect of
capsule can be appreciated
from this graph. Diminished MPO levels were
observed in the absence
of the K54 capsule, and increased MPO levels
were observed in
the absence of the O-specific antigen.
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FIG. 4.
An example of the fitted model for the MPO activity of
CP9 (wt), CP9.137 (capsule deficient), CP921 (O-specific-antigen
deficient), and CP923 (capsule and O-specific-antigen deficient) at an
approximate CI of 1.0 × 107 CFU. The derivation of
this model is described in detail in Materials and Methods and The
Appendix.
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DISCUSSION |
The findings from this study demonstrate that the effect of the
K54 capsule is an increase and the effect of the O4-specific antigen is
a decrease in pulmonary neutrophil influx. These data strongly support
the hypotheses that the K54 capsular polysaccharide is a
proinflammatory mediator that stimulates the host defense response and
that the O4-specific antigen attenuates the proinflammatory response
and downregulates the host defense response. Confirmation of these
hypotheses will require additional studies on host defense factors and
studies of the mechanism by which capsule and O-specific antigen
modulate pulmonary neutrophil influx. In addition, further experiments
are needed to establish whether these results are relevant to other
gram-negative surface polysaccharides. However, if this is the case,
our findings will be of considerable importance since capsular
polysaccharide and the O-specific antigen are nearly ubiquitous surface
components of the gram-negative bacilli capable of causing pneumonia
and a variety of other extraintestinal infections.
We chose to use whole live organisms in this study for several reasons.
Most importantly, a live-organism challenge will present the bacterial
component in question in the most physiologic manner and mimics the
clinical reality of gram-negative pneumonitis. The host will be exposed
to both shed and attached surface polysaccharides. Further, the host
will be exposed to the native form of these components. It has been
previously demonstrated that the form of bacterial components affects
the host response (6). The process of killing whole
organisms or the purification of individual components will likely
result in alterations to the native form. Lastly, the use of purified
components always raises the problem of potential contamination by
other bacterial products. The use of a live set of defined, isogenic
mutants that are deficient in various components of interest avoids
these potential problems which otherwise is very difficult to control for.
Previously published data from our laboratory indirectly supports the
hypothesis that the K54 capsule is proinflammatory. Mice were injected
intraperitoneally with killed CP9 (wt) or two independent isogenic
capsule-deficient derivatives (CP9.108 and CP9.C56). Since killed
organisms were utilized, subsequent death was likely due to a
bacterial-product-mediated host response. At high CI (7.0 × 109 and 7.7 × 109 CFU, respectively) the
mean survival of the capsule-deficient strains (2.44 and 2.0 days,
respectively) was significantly longer than that of CP9 (1.39 days)
(P < 0.001) (20).
Although it is generally conceded that multiple microbial determinants
activate the host pulmonary response, most of the available data
involves the role of lipid A or peptidoglycan. Bacterial endotoxin has
been shown to be a potent proinflammatory mediator with resultant
activation of alveolar macrophages and the complement cascade, and
subsequent recruitment and activation of neutrophils and monocytes
(1). The signal transduction pathway of the proinflammatory effects of lipid A has been relatively well worked out and is mediated
in part through CD14 which is present on macrophages and other lipid
A-responsive cells (15). A number of studies on both
gram-positive and gram-negative pathogens have demonstrated that
peptidoglycan, alone or in a polysaccharide-peptidoglycan complex
(e.g., lipoteichoic acid but not the capsular polysaccharides), is able
to stimulate a proinflammatory response. Studies have focused on
chronic noninfectious inflammatory processes (e.g., arthritis)
(22) and on mediators of gram-positive related septic shock
(3), although some infectious processes have also been evaluated (12, 17). Various studies have demonstrated
activation of macrophages, release of various proinflammatory mediators
including cytokines (e.g., tumor necrosis factor alpha and
interleukin-1
), nitric oxide, prostaglandins (e.g., prostaglandin
E2), and leukotrienes (e.g., leukotriene B4) and a subsequent influx of
neutrophils (3, 12, 17, 22). The CD14 signal transduction
pathway has also been implicated as effecting these responses
(16). However, we are unaware of data that demonstrate that
capsule increases pulmonary neutrophil influx or stimulates the host
proinflammatory response. Data have been published on
Cryptococcus neoformans capsule which, in contrast, reduces
tumor necrosis factor alpha and interleukin-1 production from human
monocytes in vitro (27).
Previously published results from our laboratory also support the
hypothesis that the O-specific antigen attenuates the host proinflammatory response. Mice were challenged with CP9 (wt), CP920 (O4
deficient), and CP921(O4 deficient) via intraperitoneal injection, and
the 50% lethal doses (LD50s) were determined. Contrary to
predictions, the LD50s of CP920 (3.2 × 106 CFU) and CP921 (5.2 × 106 CFU) were
significantly lower than that of CP9 (1.7 × 107 CFU)
(P < 0.05) (21).
To date, the O-specific antigen is usually thought of as a bacterial
component that protects against various host defense factors. Findings
from this model system have substantiated that fact, as demonstrated by
the increased clearance of CP921 from the lung compared to CP9 (Fig.
2). However, in addition to this important role, an effect of the
O-specific antigen is to decrease pulmonary neutrophil influx. This
finding, in combination with the LD50 data described above,
suggests that the O-specific antigen attenuates the host inflammatory
response. The mechanism by which this effect is exerted will be
interesting and is presently being evaluated. It may or may not be
independent of lipid A. It is possible that the O-specific-antigen
effect is mediated by altering the biologic activity of lipid A. Previous studies have demonstrated that the form of lipid A is
important in its localization within the host (6).
Alternatively, the O-specific antigen may act independently of lipid A
but through the same signal transduction pathway (e.g., a lipid A
binding protein/CD14 pathway) or through a completely independent
pathway. It is interesting to note that most gram-negative systemic
pathogens possess an O-specific antigen, whereas mucosal gram-negative
pathogens generally do not. It is tempting to speculate as to whether
it is advantageous for a systemic pathogen to possess a component that
attenuates the host response. Potential benefits include a reduction in
bactericidal activity generated by the host and/or a lower probability
that the host response becomes unregulated (e.g., septic shock). Such a
response may lead to premature death of the host, thereby decreasing
the likelihood of bacterial transmission to a new individual. If these speculations are substantiated, an understanding of how the O-specific antigen attenuates host response could have significant implications for both the potential use of biologic modulators directed against the
host response and approaches based on inactivating bacterial components
(e.g., lipid A) in attempts to modify sepsis syndromes.
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APPENDIX |
Description of the methods used to develop and fit the
model for MPO activity. To describe the effects of the four strains on MPO levels, a empirical model was constructed. As a first
approximation, the effects of CP9.137, CP921, and CP923 were modeled as
if each were a dilution or concentration of CP9. For each strain, the
MPO levels observed appeared to increase until about 6 h after
administration of the CI and then remain at similar levels for several
hours (Fig. 3). The maximum MPO level for each strain was modeled as a
linear function of the common logarithm of CI. The function used was:
Ymax = Y0 + b(X 
Rn) (for n = 1, 2, 3, and 4),
where Ymax is the maximum MPO level, Y0 is the
MPO level at time zero, X is the common logarithm of the CI,
n = 1 for strain 1 (CP9), n = 2 for
strain 2 (CP9.137), n = 3 for strain 3 (CP921),
n = 4 for strain 4 (CP923), b is a slope parameter used
to estimate the relationship of maximum MPO level to the common
logarithm of the CI, and Rn is a parameter used
to estimate a strain-specific effect on the maximum MPO level for each strain.
In this model, the maximum MPO level is thus Ymax = Y0 + b(X R1) for CP9,
Ymax = Y0 + b(X R2) for CP9.137, Ymax = Y0 + b(X R3) for CP921, and
Ymax = Y0 + b(X R4) for CP923.
To explore whether it was reasonable to describe the maximum MPO level
as a linear function of the common logarithm of the CI for each strain,
the relationship between the CI and the MPO level was first studied for
all observations using the MPO levels for all animals sacrificed at
6 h or later after administration of the CI. For each strain,
these MPO levels and their means were graphed against the common
logarithm of the CI to see if the function appeared to be linear, and
the fitting linear regression was fitted. For each strain, this
relationship appeared to be reasonably described by a linear function
(Fig. A1). To test the goodness of fit
of the linear relationship between maximum MPO and logarithm of the CI,
additional parameters were added to allow for a separate maximum MPO at
each CI of each strain (with the relationship between maximum MPO and
the common logarithm of the CI not necessarily being linear). When the
additional parameters were added and the relationship was thus not
assumed to be linear, the fit was not significantly improved (F = 0.11513, df = 4,98, P = 0.97).
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|
FIG. A1.
Demonstration that maximum MPO level can be described
as a linear function of the common logarithm of the CI for each strain.
This illustration uses CP921 as the challenge strain. The × symbols represent MPO activity from a single animal at the given CI.
The closed circles are the mean of MPO activity at the indicated CI.
|
|
The next step in building the model was to describe the pattern by
which the MPO levels were increasing from the time of the CI until the
plateau was reached. For each strain, the MPO levels observed appear to
increase over time in a way which resembles a logistic function of the
logarithm of time. The relationship of MPO level and time was thus
modeled as:
where Y
t is the MPO level at time
t, Y
max is the maximum MPO level, Y
0
is the MPO level at time zero,
t is the time
(in hours)
after CI instillation,
n = 1 for strain 1 (CP9),
n = 2 for strain 2 (CP9.137),
n = 3 for
strain 3 (CP921),
n = 4
for strain 4 (CP923), c is a
parameter used to estimate the slope
constant of the logistic function,
and Q
n is a parameter
used for each strain to
estimate the log time at which the MPO
level reaches 50% of its
maximum
increase.
Combining the two parts of the model above and solving for
Yt, the full model used was:
where the constants and variables are as described
above.
The parameters R1, R2, R3, and
R4 represent the strain-specific effect on the maximum MPO
level for strains CP9, CP9.137, CP921, and CP923, respectively. The
parameters Q1, Q2, Q3, and Q4 represent the log time at which the MPO level reaches
50% of its maximum increase for strains CP9, CP9.137, CP921, and
CP923, respectively.
To test the effects on the maximum MPO level of deleting the capsule,
of deleting the O-specific antigen, and of the synergy between these
two effects, the model was reparameterized. The parameters
R1, R2, R3, and R4 were
replaced by the parameters r1, r2,
r12, and r0, where r1 = [(R2 R1] + (R4 R3)]/2 is the average effect of deleting the capsule,
r2 = [(R3 R1) + (R4 R2)]/2 is the average effect of
deleting the side chain, r12 = [(R1 R2) + (R4 R3)]/2 = [(R1 R3) + (R4 R2)]/2 is
the synergy between the effects of deleting the capsule and deleting the side chain, and r0 is the value of R averaged over all observations.
To test the effects of deleting the capsule, of deleting the side
chain, and of the synergy between these two effects on the time to 50%
of the maximum MPO increase, the model was reparameterized. The
parameters Q1, Q2, Q3, and
Q4 were replaced by the parameters q1,
q2, q12, and q0, where
q1 = [(Q2 Q1) + (Q4 Q3)]/2 is the average effect of
deleting the capsule, q2 = [(Q3 Q1) + (Q4 Q2)]/2 is
the average effect of deleting the side chain, q12 = [(Q1 Q2) + (Q4 Q3)]/2 = [(Q1 Q3) + (Q4 Q2)]/2 is
the synergy between the effects of deleting the capsule and deleting the side chain, and q0 is the value of Q averaged over all observations.
The model was fitted using nonlinear regression using SPSS for Windows.
The significance of the effects of deleting the capsule, of deleting
the side chain, and of the synergy between these two events were tested
by dividing each estimate by its standard error and then comparing the
resulting Z score to a standard normal distribution.
| |
ACKNOWLEDGMENTS |
This work was supported by grants AI 42059 (T.A.R.) and HL 48889 (P.R.K.) from the National Institutes of Health, a Merit Review Grant
(T.A.R.) from the Department of Veterans Affairs, and from the Lucille
P. Markey Charitable Trust.
We appreciate the continued support of Tim Murphy and Bruce Holm.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Division of Infectious Diseases, 3435 Main St., Biomedical Research Building, Rm. 141, Buffalo, NY 14214. Phone: (716) 829-2674. Fax: (716) 829-3889. E-mail: trusso{at}acsu.buffalo.edu.
Editor:
R. N. Moore
| |
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Infection and Immunity, May 2000, p. 2854-2862, Vol. 68, No. 5
0019-9567/00/$04.00+0
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Top
Abstract
Introduction
![<FR><NU><UP>Y</UP><SUB>t</SUB> − <UP>Y</UP><SUB>0</SUB></NU><DE><UP>Y<SUB>max</SUB></UP> − <UP>Y<SUB>0</SUB></UP></DE></FR><UP> = </UP><FR><NU><UP>1</UP></NU><DE><UP>1 + e</UP><SUP>−c[ln (t) − Qn]</SUP></DE></FR>](/content/vol68/issue5/fulltext/2854/img001.gif)
![<UP>Y</UP><SUB>t</SUB> = <UP>Y</UP><SUB>0</SUB> + <FR><NU><UP>b</UP>(<UP>X − R</UP><SUB>n</SUB>)</NU><DE>1 + <UP>e</UP><SUP>−c[<UP>ln</UP> (t) − <UP>Q</UP><SUB>n</SUB>]</SUP></DE></FR>](/content/vol68/issue5/fulltext/2854/img002.gif)
