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Immunology Research Group,
* Departments of Microbiology and Infectious Disease,
Physiology and Biophysics, and
Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Alberta, Canada; and
Biochemical Pharmacology, University of Konstanz, Konstanz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expresses the adhesion molecules that
mediate tethering, rolling, and adhesion. Therefore, a localized
response to LPS or infection will induce brisk recruitment of
leukocytes without undue damage to the surrounding tissue or organs. In
contrast, an inappropriate inflammatory response such as systemic
sepsis will cause leukocytes to localize to the lung, where they become
physically trapped and are not able to recirculate (3, 4, 5, 6).
Indeed, a key feature in human septic shock from Gram-negative or
Gram-positive bacteria is inappropriate leukocyte recruitment
predominantly in lung with a tendency for impaired lung function
(7).
Gram-negative septic shock is believed to result from the effects of
LPS (8, 9). Recent evidence has established a model for
LPS recognition that involves both CD14, a
glycosylphosphatidylinositol-linked protein, and an associate signal
transducer Toll-like receptor 4
(TLR4)3 (10, 11). Therefore, TLR4 is a critical receptor involved in the LPS
detection system (12) that confers responsiveness to
circulating and tissue leukocytes as well as endothelium
(13). By contrast, lipoteichoic acid (LTA), the
Gram-positive cell membrane equivalent to LPS, does not appear to
activate TLR4, but rather functions through a second TLR, namely TLR2,
to activate immune responses. Nevertheless, in macrophages or cell line
equivalents, the in vitro work has suggested very similar mechanisms of
action for the two lipoproteins. Both LPS and LTA act by binding a TLR
(14, 15, 16, 17, 18), activating various tyrosine kinases, and
translocating the transcription factor NF-
B (19),
resulting in the production and release of numerous proinflammatory
mediators, including TNF-, IL-6, IL-1
, IL-12, and NO
(20, 21, 22, 23, 24). However, two very recent in vitro studies
reported some significant differences in mediator release in response
to TLR2 vs TLR4 activation (25, 26). Although in vivo
studies have suggested that both LPS and LTA can induce the same end
result, namely shock (8, 27, 28), at least for
leukocyte-endothelial cell interactions LPS and LTA may elicit very
different responses. For example, TLR4, the receptor for LPS, was
abundant on endothelium, whereas TLR2, the receptor for LTA, could not
be detected (13). Since the endothelium affects leukocyte
trafficking by expressing adhesion molecules in lungs as well as
peripheral microvasculature, LPS vs LTA responses may be quite
different.
We have investigated leukocyte recruitment in response to local and systemic LPS or LTA by directly visualizing leukocyte behavior in vivo, further characterizing the direct effects of each lipoprotein on various microvascular beds. Our findings demonstrate that LTA and LPS induce profoundly different responses in terms of leukocyte recruitment. Whereas LPS elicited profound leukocyte recruitment, LTA had no noticeable effects in vivo or in a human endothelial-leukocyte assay in vitro. Our results provide direct evidence that the in vitro macrophage activity of LTA is not predictive of its physiological effects upon leukocyte-endothelial cell interactions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents for tissue culture, unless specified, were
purchased from Life Technologies (Gaithersburg, MD). LTA from
Staphylococcus aureus (Sigma-Aldrich, Oakville, Ontario,
Canada) was reconstituted in sterile PBS, filtered, and stored at
-20°C. Highly purified LTA, which was >99% pure and contained <6
pg LPS/mg LTA, as assessed by Limulus amebocyte assay, was
isolated using a novel isolation procedure (24).
Peptidoglycan (PepG) from S. aureus (Fluka, Oakville,
Ontario, Canada) was reconstituted in sterile PBS and sonicated for
1 h before use. Smooth LPS from E. coli was
provided by S. Goyert (North Shore University Hospital/New York
University School of Medicine, Manhasset, NY.). rTNF- was purchased
from R&D Systems (Minneapolis, MN).
In vivo experiments
Male BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) weighing between 20 and 25 g were used at 6–10 wk of age and were housed in a double barrier pathogen-free facility. One postcapillary venule was visualized per mouse; therefore, "n" refers to the total number of mice. For consistency, we chose unbranched vessels that were 25–35 µm in diameter, consistent with postcapillary venules.
Intravital microscopy
The mouse cremaster preparation, which was approved by the animal care committee, University of Calgary, was used to study the behavior of leukocytes in the microcirculation (29). Mice were anesthetized by i.p. injection of a mixture of xylazine hydrochloride (10 mg/kg; MTC Pharmaceuticals, Cambridge, Ontario, Canada) and ketamine hydrochloride (200 mg/kg; Rogar/STB, London, Ontario, Canada). The jugular vein was cannulated and used to administer additional anesthetic. The cremaster muscle was dissected free of tissues and exteriorized onto an optically clear viewing pedestal. The muscle was cut longitudinally with a cautery and held flat against the pedestal by attaching silk sutures to the corners of the tissue. The muscle was then superfused with bicarbonate-buffered saline.
An intravital microscope (Axioskop; Carl Zeiss Canada, Don Mills,
Ontario, Canada) with a x25 objective lens (Wetzlar L25/0.35; E.
Leitz, Munich, Germany) and a x10 eyepiece was used to examine the
cremasteric microcirculation. A video camera (Panasonic 5100 HS, Osaka,
Japan) was used to project the images onto a monitor, and the images
were recorded for playback analysis using a videocassette recorder.
Single unbranched cremasteric venules (25–40 µm in diameter) were
selected, and to minimize variability, the same section of cremasteric
venule was observed throughout the experiment. The number of rolling
and adherent leukocytes was determined off-line during video playback
analysis. Rolling leukocytes were defined as those cells moving at a
velocity less than that of erythrocytes within a given vessel.
Leukocyte rolling velocity was determined by measuring the time
required for a leukocyte to roll along a 100-µm length of venule.
Rolling velocity was determined for 20 leukocytes at each time
interval. Leukocytes were considered adherent to the venular
endothelium if they remained stationary for 30 s or longer.
Leukocyte emigration was defined as the number of extravascular
leukocytes per microscopic field of view (x25 objective lens), and was
determined by averaging the data derived from four to five fields
adjacent to postcapillary venules. Venular diameter (Dv) was
measured on-line using a video caliper (Microcirculation Research
Institute, Texas A&M University, College Station, TX). Centerline RBC
velocity (VRBC) was also measured on-line
using an optical Doppler velocimeter (Microcirculation Research
Institute), and mean RBC velocity (Vmean)
was determined as VRBC/1.6. Venular wall
shear rate (
) was calculated based on the Newtonian definition:
= 8 (Vmean/Dv).
Determination of tissue myeloperoxidase activity
At the end of each experiment, samples of the lung were weighed, frozen on dry ice, and processed for determination of myeloperoxidase (MPO) activity. MPO is an enzyme found in cells of myeloid origin, and has been used extensively as a biochemical marker of granulocyte (mainly neutrophil) infiltration into the lung (3, 30). The samples were stored at -20°C for no more than 1 wk before the MPO assay was performed. MPO activity was determined using an assay described previously (31), but with the volumes of each reagent modified for use in 96-well microtiter plates. Change in absorbance at 450 nm over a 90-s period was determined using a kinetic microplate reader (Molecular Devices, Sunnyvale, CA).
Circulating and peritoneal neutrophil counts
At the end of each experiment, whole blood was collected via cardiac puncture. Total leukocyte counts were performed, using a Bright-line hemocytometer (Hausser Scientific, Horsham, PA). Mice were sacrificed, and the peritoneal cavity was washed with 10 ml HBSS. The cells were sedimented by centrifugation at 260 x g for 5 min. The pellet was resuspended in DMEM containing 5% FCS. The total number of cells in the lavage fluid was counted using a hemocytometer.
Quantitation of expression of P-selectin
Expression of the adhesion molecules P-selectin was quantified
using a modified dual-radiolabeled Ab technique (32, 33).
The Abs RB40.34 (against P-selectin) and A110-1 (a rat IgG1, 
isotype standard) were labeled with either 125I
(RB40.34) or 131I (A110-1) using the iodogen
method, as previously described (32, 33). A110-1 was used
to detect nonspecific binding in the murine system.
To study P-selectin, animals were injected i.v. with a mixture of 10 µg 125I-anti-labeled P-selectin (RB40.34), and a variable dose of 131I-labeled A110-1. The Abs were allowed to circulate for 5 min, then the animals were heparinized. A blood sample was obtained from a carotid artery catheter, then the mice were exsanguinated by blood withdrawal through the carotid artery catheter and simultaneous i.v. infusion with bicarbonate-buffered saline. The lung, heart, pancreas, mesentery, small intestine, colon, skin, and muscle were harvested and weighed. Both 131I and 125I activities were measured in plasma and tissue samples.
P-selectin expression was calculated per gram of tissue, by subtracting the accumulated activity of the nonbinding Ab (131I-labeled A110-1) from the accumulated activity of the binding Ab (125I-labeled RB40.34). Data for P-selectin were represented as the percentage of the injected dose of Ab per gram of tissue. It has been demonstrated previously that this approach provides reliable quantitative values of adhesion molecule expression, and that radiolabeled binding Ab can be displaced specifically with sufficient amounts of unlabeled Ab. The technique is sufficiently sensitive that very small, basal levels of P-selectin can be detected in wild-type mice relative to P-selectin-deficient mice, in which values are zero (32).
Experimental protocol
To determine the in vivo effect of LTA, PepG, and LPS, we examined the local and systemic responses in BALB/c mice. Our initial experiments were performed using LTA, PepG, or LPS concentrations that were similar to the concentrations of LPS used in previous studies.
Local LTA or LPS administration
The local response to LTA was examined by injecting varying concentrations of LTA in 200 µl sterile saline s.c. beneath the scrotal skin using a 30-gauge needle. The animals were prepared for intravital microscopy at 3.5 or 23.5 h after LTA administration, and the microvasculature was observed for 60 min. At 4.5 or 24.5 h, the animals were sacrificed, the lungs were harvested for MPO assay, and peripheral blood was drawn for circulating leukocyte counts. Preliminary experiments with LPS demonstrated a dramatic effect of 0.05 µg/kg at 4 h on the local microvasculature; therefore, our initial experiments with LTA were performed under the same conditions. Increased concentrations and prolonged times were subsequently examined.
Systemic LTA, PepG, LPS, or live S. aureus administration
The systemic response was examined by injecting 500 µl of varying concentrations of LTA, PepG, or LPS i.p. for 4 and/or 24 h. The microvasculature was directly observed at 3.5 or 23.5 h for 60 min. At the end of the intravital observations, the lungs from each animal were immediately harvested and frozen at -70°C, peripheral blood was drawn, and a peritoneal lavage was performed. The initial concentration of 500 µg/kg for 4 h was chosen to make a direct comparison between LTA and LPS. Subsequent experiments with LTA determined the effects of increasing concentrations and longer response times. S. aureus ATCC strain 25923 was grown on blood agar plates (Difco, Detroit, MI) for 18 h, harvested, and washed twice with saline. Mice were injected i.p. with 1010 CFU/g body weight.
In vitro experiments
As in vivo studies revealed very minor leukocyte-endothelium response to LTA, we performed in vitro studies: 1) in macrophage cell lines to determine whether our LTA had biological activity, and 2) in a human endothelial-leukocyte system to ensure that the lack of biological activity of LTA was not restricted to mice.
Activation of macrophage cell lines
THP-1 (American Type Culture Collection, Manassas, VA) and RAW 264.7 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1 mM sodium pyruvate at 37°C in a 5% CO2 atmosphere. To induce the differentiation of the THP-1 cells to the more mature macrophage-like phenotype, cells were suspended to a cell density of 5 x 105 cells/ml in fresh medium containing 5 x 10-5 mM 1,25-dihydroxyvitamin D3 and incubated for 72 h (34). Both cell lines were stimulated with either LPS (100 ng/ml) or LTA (100 ng/ml) for the indicated times. Western blots were performed on cell-free lysates (35) with the phospho-specific mitogen-activated protein kinase (MAPK) Ab (Promega, Madison, WI), phospho-specific p38 Ab (New England Biolabs, Beverly, MA), or the total p38 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) as a loading control, according to manufacturer’s specification.
Endothelial cell isolation
Microvascular endothelium was isolated using a previously established protocol (36). Discarded neonatal human foreskin was cut into 2- to 3-mm2 pieces and digested with 0.5 mg/ml collagenase type A1 (Boehringer Mannheim Biochemicals, Indianapolis, IN) in supplemented M199 for 16 h at 4°C. Each segment of skin is placed keratinized side down, and microvessels were obtained by compressing the tissue with a spatula. The collected endothelial cells were cultured in EBM-2 with supplements (Clonetics, San Diego, CA) on 35-mm gelatin-coated tissue culture dishes. Human dermal microvascular endothelial cell (HDMEC) was grown to confluence for each flow chamber experiment, and cells were used before the seventh passage. Adhesion molecule expression was previously shown to be stable up to and including the seventh passage. This protocol was approved by the Ethics Committee of the University of Calgary.
Leukocyte recruitment under flow conditions
Leukocyte recruitment was determined using a parallel plate flow chamber (36). All experiments were performed with heparinized whole blood obtained from healthy human donors. The blood was diluted (10%) in sterile HBSS at 37°C. The human blood was drawn through the flow chamber and over the HDMEC at a rate of 2 dynes/cm2 using an infusion pump. Experiments were recorded and analyzed off-line. Leukocyte rolling flux was determined by counting the number of leukocytes that roll past a given plane that is perpendicular to the direction of the flow. Leukocytes that stayed stationary for at least 10 s were counted as adherent cells. For each condition, at least six fields were visualized.
Statistical analysis
Data are shown as mean ± SEM. The mean for intravital experiments was determined by combining the data from three time points (3.5, 4, and 4.5 h postinjection) for each vessel. All data were analyzed using one-way ANOVA, and Bonferroni correction was applied where multiple comparisons were necessary.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Under normal conditions, baseline leukocyte rolling flux within a
postcapillary venule was approximately 75 cells/min (Fig. 1
A). None of these cells
adhered (Fig. 1B) or emigrated out of the microvessels (Fig. 1C). Four hours after local administration of LPS, the
rolling flux ranged between 100 and 200 cells/min (Fig. 1A).
Leukocyte adhesion was apparent, with an average of 12.5 cells/100 µm
(Fig. 1B). Additionally, an average of 20 cells emigrated
into the surrounding tissue (Fig. 1C). In contrast, LTA did
not have any effect on leukocyte rolling, adhesion, or emigration (Fig. 1, A–C). It was conceivable that the effective dose of LTA
may be higher than LPS, so we decided to increase the dose by 10- and
100-fold (0.5 and 5 µg/kg) (Fig. 1). Even under these
ultrapathological concentrations of LTA, no increase in
leukocyte-endothelial cell interactions was observed. Local LPS
administration at these concentrations causes stasis of the local
microcirculation (P. Kubes, unpublished observation). No vascular
changes, including decreased blood flow or a change in vessel diameter,
were noted with LTA (data not shown). As the effect of local LTA
administration may not follow the same time course as local LPS
administration, we examined the response 24 h after the initial
administration of LTA. LTA did not have any effect on leukocyte rolling
(Fig. 1A), adhesion (Fig. 1B), or emigration
(Fig. 1C) at 24 h.
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Although the local administration of LPS had no apparent systemic
effects (e.g., no change in circulating leukocyte counts), systemic
administration of LPS induced a striking decline in peripheral
circulating leukocytes (Fig. 2A). Concurrent with a
reduction in circulating leukocytes was a dramatic increase of
neutrophil accumulation in the lung (Fig. 2B).
Interestingly, addition of LPS i.p. at concentrations that had systemic
effects did not recruit any leukocytes to the site of injection, as
there was no change in peritoneal leukocyte numbers (Fig. 2C). In direct contrast, LTA did not cause a drop in the
circulating leukocyte counts (Fig. 2A) nor an increase in
neutrophil sequestration to the lung within the same 4-h time frame
(Fig. 2B). Moreover, administration of LTA for 24 h did
not have any effect on the circulating leukocyte counts, lung MPO
values, or peritoneal leukocyte counts (Fig. 2, A–C). We
examined systemic LTA at 10 times (5,000 µg/kg) and 100 times (50,000
µg/kg) the LPS dose. High doses of LTA did not have any effect on the
number of peripheral circulating leukocytes, neutrophil sequestration
to the lung, or peritoneal sequestration (Fig. 2, A–C). We
administered the highly purified LTA systemically, but did not observe
any effect on leukocyte rolling, adhesion, or emigration (Table I
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The decrease in circulating leukocyte counts may have been
responsible for the lack of recruitment into the peritoneum. To further
explore leukocyte-endothelial cell interactions in the periphery
following systemic LPS administration, we exteriorized the cremaster
muscle. Remarkably, the number of white blood cells rolling through the
vessels dropped from 50–100 cells/min in the control group to less
than 5 cells/min in the LPS group (Fig. 3A). Due to the 95% reduction
in rolling cells, very few leukocytes adhered (Fig. 3B) and
emigrated (Fig. 3C) out of the vessels compared with local
LPS administration and consistent with the observation in the
peritoneum. Fig. 3 highlights that neither LTA (500 µg/kg) nor
concentrations of LTA 10- or 100-fold higher lowered the number of
rolling leukocytes (Fig. 3A), but did have a minor effect on
leukocyte adhesion (Fig. 3B), which did not result in
emigration (Fig. 3C). Twenty-four-hour administration of LTA
did not have any effects on leukocyte-endothelial cell interactions
(Fig. 3). Therefore, whereas systemic LPS caused profound changes in
leukocyte trafficking, systemic LTA had very minor effect.
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summarizes quantitative P-selectin
expression in various murine vascular beds. All tissues had minimal
basal P-selectin expression and responded very significantly to LPS.
The pulmonary, stomach, and cremaster muscle microvasculature revealed
minimal P-selectin expression (Fig. 4, A–C) following LTA
administration. Similar lack of effect was noted in heart, other parts
of the gastrointestinal tract (small and large intestine), skin, and
spleen (data not shown). By contrast, the mesentery (Fig. 4D) and pancreas (data not shown) demonstrated some
significant increase in P-selectin in response to LTA, but the values
were still much lower than with LPS.
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To ensure that these observations were not related to species
resistance to LTA, we examined leukocyte-endothelial cell interactions
in surrogate human blood vessels (flow chamber seeded with human
endothelium). Perfusion of leukocytes across unstimulated endothelium
caused no rolling or adhesion. Addition of LPS for 4 h caused a
profound increase in rolling and adhesion that was comparable with
values observed with our most potent inducer (TNF-) of
leukocyte-endothelium interactions in human systems (Fig. 5). In direct contrast, LTA at similar
concentrations to LPS caused no leukocyte rolling or adhesion (Fig. 5).
Twenty-four-hour incubations with LTA had no effect on leukocyte
rolling and adhesion (data not shown). The concentration of LTA was
examined at 0.1, 1, 10, and 100 µg/ml. None of the LTA concentrations
induced leukocyte rolling and adhesion. The highly purified form of LTA
failed to induce leukocyte-endothelial cell interactions (data not
shown). Each concentration was tested at least four times. These data
are consistent with the observations that LPS and TNF- induced
E-selectin and VCAM-1 on human endothelium, but LTA failed to induce
any response (data not shown).
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To ensure that our LTA was active, we used a monocyte cell line
THP-1 that is known to respond to LTA. Unstimulated THP-1 cells did not
adhere to plastic, whereas when LPS (100 ng/ml) was added to the cell
suspension, essentially all THP-1 cells firmly adhered. LTA (100 ng/ml)
induced an identical proadhesive phenotype (Fig. 6A), but was quantitatively
lower than with LPS (Fig. 6B). In addition, 100 ng/ml of
both LPS and LTA induced the phosphorylation of MAPK (p42/p44, also
known as extracellular signal-related kinase 1/2) (Fig. 6C). Finally, message for various chemokines was detected
with both lipoproteins (data not shown).
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D). Interestingly, in
both cell lines, there was a noticeable difference between LPS and LTA
stimulation in terms of amplitude and timing. LPS induced a more rapid
and robust kinase activation, which was easily detected within 15 min
of stimulation compared with the weaker activation by LTA, which was
first detected at 30 min. This difference in signaling between LPS and
LTA was also consistent when highly purified LTA was used (data not
shown). Gram-positive bacteria induce leukocyte-endothelial cell interactions
To ensure that Gram-positive bacteria could induce
leukocyte-endothelial cell interactions, animals received an i.p.
injection of live S. aureus (1010
CFU/g). In 4 h, the bacteria caused a 50% decrease in circulating
leukocyte counts (Fig. 7A).
The MPO assay showed that a significant number of neutrophils had
sequestered in the lung (Fig. 7B). Interestingly, the
numbers of neutrophils sequestered in the lung were similar between
S. aureus- and LPS-treated animals. The smaller drop in
circulating leukocyte counts allowed for some infiltration into the
peritoneum (Fig. 7C). Intravital microscopy revealed that
S. aureus induced the characteristic drop in the number
of rolling leukocytes that was seen with LPS, but not LTA (Fig. 7D). Much like LPS, which essentially eliminated all
rolling, only a few leukocytes rolled and adhered with S.
aureus (Fig. 7, D and E) with some
emigration into the surrounding tissue (Fig. 7F).
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A) or the number of leukocytes that
sequestered into the lung (Fig. 7B), there were some effects
on leukocyte behavior in the periphery. A subtle increase in peritoneal
leukocyte recruitment was noted with PepG (Fig. 7C). Whereas
a very profound drop in rolling cells was seen in the peripheral
muscle microcirculation with LPS or Gram-positive bacteria, the PepG
did not decrease rolling leukocytes, and in fact an increase was noted
(Fig. 7D). Moreover, a very significant rise in adhesion
(Fig. 7E) and some increase in emigration were also noted
(Fig. 7F). Addition of LTA to PepG in no way enhanced the
PepG response (data not shown). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we directly visualized the activation of the innate immune system in the microvasculature in response to local or systemic LTA or LPS administration in an attempt to establish the importance of these two molecules as inducers of leukocyte-endothelium interactions in vivo. Our in vivo data reveal very profound differences between LPS and LTA with respect to leukocyte-endothelial cell interactions. Whereas local LPS induced a cascade of events, including leukocyte rolling, firm adhesion, and migration of leukocytes into the surrounding tissue, LTA had a very limited impact on any of these parameters. Neither extremely high concentrations of LTA, different preparations of LTA, nor longer times of exposure to LTA could induce leukocyte-endothelial cell interactions. These data are not consistent with the many studies that have demonstrated that LTA, devoid of other contaminants, could stimulate various macrophage cell lines to produce and release cytokines and chemokines in vitro (21, 22, 23, 24, 38). Clearly, our data would suggest that if indeed these cells were activated in vivo, the amounts of cytokine and chemokine were simply not sufficient to induce leukocyte-endothelial cell interactions.
An alternative explanation is that the Gram-positive and Gram-negative sepsis has very different pathological mechanisms, with only the latter recruiting neutrophils to the sites of infection. However, this does not appear to be the case. The inappropriate inflammatory immune responses observed with Gram-positive and Gram-negative bacteria are strikingly similar and are in fact indistinguishable clinically (39). Comparing experiments with live S. aureus (Gram positive) or E. coli (Gram negative) reveal endpoint similarities, including similar cardiovascular dysfunction (40), rapid neutrophil infiltration into lungs, and increased cytokine and adhesion molecule expression (41). In fact, in our study, administration of S. aureus induced a similar pattern of leukocyte responses, as did LPS, but not LTA, suggesting perhaps that the latter molecule does not account for the leukocyte responses observed in Gram-positive sepsis.
At odds with our results are reports that LTA has been able to induce E-selectin, VCAM-1, and ICAM-1 expression on human endothelium from both umbilical vein (42) and lung (43), leading to leukocyte adhesion. Although it is tempting to speculate that our data differ due to human vs mouse sensitivity to LTA, when we stimulated human endothelium with LTA, no notable increase in leukocyte rolling and adhesion was observed. The other difference between in vivo and previously published in vitro systems is that the in vitro work was done under static conditions. This is not entirely reflective of the dynamic shear conditions that limit leukocyte-endothelium interactions. Indeed, our in vitro work was also performed under shear through flow chambers, but when we arrested flow and allowed the cells to settle onto the endothelium, leukocyte adhesion was still not observed (data not shown). An alternative explanation is that some LTA preparations have been demonstrated to contain sufficient LPS to account for cellular activation (44). Although the aforementioned studies that observed endothelial responses to LTA also included studies that attempted to discount potential LPS-contaminating effects, LPS as a potential synergistic or priming factor for LTA remains a serious concern. Consistent with this concern is the fact that the receptor essential for LTA effects, namely TLR2, was not detected on endothelium (13). Presently, the work as a whole suggests that LTA does not directly stimulate endothelium sufficiently in vivo or under shear conditions in vitro to induce leukocyte-endothelial cell interactions. Our only caveat at this stage is the small increase in P-selectin in mesentery and pancreas, which raises some minor tissue specificities.
Our data also provide some interesting new observations with respect to leukocyte-endothelial cell responses with systemic LPS. A number of investigators have reported that leukocyte recruitment is reduced to the primary site of infection in systemic septic response (45). Lack of adhesiveness or responsiveness perhaps due to shedding of critical adhesion molecules or internalization of chemokine receptors has been proposed as an explanation for the lack of leukocyte recruitment (46, 47, 48). Our data suggest another response to systemic LPS worth noting. Addition of LPS to the peritoneal cavity at concentrations that had systemic effects caused a very profound drop in circulating leukocytes and dramatic sequestration of leukocytes in the lung. Interestingly, this leukocytopenia in wild-type animals translated into fewer leukocyte-endothelial cell interactions in peripheral vasculature and an inability to recruit leukocytes (adhesion and emigration) into peripheral microcirculations, including the peritoneum and skeletal muscle. This delay in leukocyte recruitment into nonpulmonary tissues in septic humans has been appreciated for many years and may account for the multiorgan dysfunction associated with septic shock (49). Although LPS has been shown to increase endothelial adhesion molecule expression in all tissues (32), the preferential recruitment of leukocytes into lungs may not be related to molecular adhesive events, but rather due to physical trapping of activated (rigid) leukocytes within narrow architecture of pulmonary capillaries (3, 6, 30), which precedes the adhesion molecule expression in other tissues. In this regard, our PepG data are consistent with the importance of the lung in systemic endotoxemia. PepG did not increase leukocyte trapping in the lung; hence, no reduction in circulating counts and no decrease in leukocyte rolling in the periphery were observed, allowing for substantial leukocyte adhesion and recruitment.
In some respects, it is not entirely surprising that LTA is not the activator of leukocyte-endothelial cell interactions. First, LTA represents a very small fraction of the outer membrane of Gram-positive bacteria, whereas LPS makes up a substantial amount of Gram-negative outer membrane. Second, Gram-positive bacteria are often coated with capsules so that from a teleological standpoint, evolving recognition receptors against a reasonably rare outer membrane molecule that is often not seen by the innate detection system may not be likely. This of course begs the question as to which Gram-positive molecules do activate leukocyte-endothelial cell interactions? Certainly, there are many potential candidates. PepG have been postulated as important inducers of the immune system. Indeed, our data do suggest that PepG may be responsible for the adhesion of leukocytes in peripheral microvasculatures with Gram-positive bacteria. However, the trapping of leukocytes in the lung and the drop in leukocyte numbers (leading to fewer rolling leukocytes) clearly require some molecule other than PepG or LTA or PepG together with LTA (P. Kubes, M. Ho, and B. G. Yipp, unpublished data).
Many groups have demonstrated dramatic LTA responses via TLR2 in numerous macrophage cell lines. Although the tendency is to assume that the detection systems on macrophage cell lines and endothelium/neutrophils are the same, our data suggest that only macrophages are activated via LTA and this is not sufficient to induce an inflammatory response similar to that seen with LPS or S. aureus in vivo. Indeed, a recent study has reported ample TLR4, the LPS receptor, but minimal TLR2, the LTA receptor, on endothelium, suggesting that endothelium may use other mechanisms to detect Gram-positive bacteria.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Paul Kubes, Health Sciences Center, 3330 Hospital Drive NW, Calgary, Alberta, Canada, T2N 4N1. E-mail address: pkubes{at}ucalgary.ca
3 Abbreviations used in this paper: TLR, Toll-like receptor; HDMEC, human dermal microvascular endothelial cell; LTA, lipoteichoic acid; MAPK, mitogen-activated protein kinase; MPO, myeloperoxidase; PepG, peptidoglycan.
Received for publication October 22, 2001. Accepted for publication February 26, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B through Toll-like receptor 4 (TLR-4) in cultured human dermal endothelial cells: differential expression of TLR-4 and TLR-2 in endothelial cells. J. Biol. Chem. 275:11058.B translocation. J. Biol. Chem. 276:22041., interleukin 6 (IL-6), and IL-10 production in both T cells and monocytes in a human whole blood model. Infect. Immun. 68:3965.4-integrin in acute, subacute, and chronic leukocyte recruitment in vivo. J. Immunol. 159:4514.[Abstract]
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 S. M. Kerfoot and P. Kubes Local coordination verses systemic disregulation: complexities in leukocyte recruitment revealed by local and systemic activation of TLR4 in vivo J. Leukoc. Biol., June 1, 2005; 77(6): 862 - 867. [Abstract] [Full Text] [PDF]
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 A. S. Damazo, S. Yona, F. D'Acquisto, R. J. Flower, S. M. Oliani, and M. Perretti Critical Protective Role for Annexin 1 Gene Expression in the Endotoxemic Murine Microcirculation Am. J. Pathol., June 1, 2005; 166(6): 1607 - 1617. [Abstract] [Full Text] [PDF]
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 M. K. Dahle, G. Overland, A. E. Myhre, J. F. Stuestol, T. Hartung, C. D. Krohn, O. Mathiesen, J. E. Wang, and A. O. Aasen The Phosphatidylinositol 3-Kinase/Protein Kinase B Signaling Pathway Is Activated by Lipoteichoic Acid and Plays a Role in Kupffer Cell Production of Interleukin-6 (IL-6) and IL-10 Infect. Immun., October 1, 2004; 72(10): 5704 - 5711. [Abstract] [Full Text] [PDF]
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 S. C. Mullaly and P. Kubes Toll Gates and Traffic Arteries: From Endothelial TLR2 to Atherosclerosis Circ. Res., October 1, 2004; 95(7): 657 - 659. [Full Text] [PDF]
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S. Dunzendorfer, H.-K. Lee, and P. S. Tobias Flow-Dependent Regulation of Endothelial Toll-Like Receptor 2 Expression Through Inhibition of SP1 Activity Circ. Res., October 1, 2004; 95(7): 684 - 691. [Abstract] [Full Text] [PDF]
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M. Triantafilou, M. Manukyan, A. Mackie, S. Morath, T. Hartung, H. Heine, and K. Triantafilou Lipoteichoic Acid and Toll-like Receptor 2 Internalization and Targeting to the Golgi Are Lipid Raft-dependent J. Biol. Chem., September 24, 2004; 279(39): 40882 - 40889. [Abstract] [Full Text] [PDF]
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L. Sharma, E. Melis, M. J. Hickey, C. D. Clyne, J. Erlich, L. M. Khachigian, P. Davenport, E. Morand, P. Carmeliet, and P. G. Tipping The Cytoplasmic Domain of Tissue Factor Contributes to Leukocyte Recruitment and Death in Endotoxemia Am. J. Pathol., July 1, 2004; 165(1): 331 - 340. [Abstract] [Full Text] [PDF]
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G. G. Neely, S. Epelman, L. L. Ma, P. Colarusso, C. J. Howlett, E. K. Amankwah, A. C. McIntyre, S. M. Robbins, and C. H. Mody Monocyte Surface-Bound IL-15 Can Function as an Activating Receptor and Participate in Reverse Signaling J. Immunol., April 1, 2004; 172(7): 4225 - 4234. [Abstract] [Full Text] [PDF]
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S. Fernandez, P. Jose, M. G. Avdiushko, A. M. Kaplan, and D. A. Cohen Inhibition of IL-10 Receptor Function in Alveolar Macrophages by Toll-Like Receptor Agonists J. Immunol., February 15, 2004; 172(4): 2613 - 2620. [Abstract] [Full Text] [PDF]
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J. G. Moreland, G. Bailey, W. M. Nauseef, and J. P. Weiss Organism-Specific Neutrophil-Endothelial Cell Interactions in Response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus J. Immunol., January 1, 2004; 172(1): 426 - 432. [Abstract] [Full Text] [PDF]
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 K. Peters, R. E. Unger, J. Brunner, and C.J. Kirkpatrick Molecular basis of endothelial dysfunction in sepsis Cardiovasc Res, October 15, 2003; 60(1): 49 - 57. [Abstract] [Full Text] [PDF]
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