| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Cutting Edge |
Department of Academic Surgery, National University of Ireland, Cork University Hospital, Cork, Ireland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
III/IIR on neutrophils (polymorphonuclear neutrophils)
and peritoneal macrophages, with increased bacterial recognition and
bactericidal activity, whereas LPS-tolerized mice exhibit an impaired
ability to ingest and to kill bacteria. These results indicate that BLP
tolerance is a novel adaptive host response associated with a unique
protective effect during septic shock. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bacterial lipoprotein (BLP),3 characterized by a unique NH2-terminal lipo-amino acid, N-acyl-S-diacylglyceryl cysteine, is the most abundant protein in the outer membrane of both Gram-positive and Gram-negative bacteria. Like LPS, BLP can be released from proliferating Escherichia coli, and treatment of bacteria with antibiotics significantly enhances BLP release (4). BLP is known to activate monocytes/macrophages to produce inflammatory cytokines and to induce lethal shock in both LPS-responsive C3H/HeN mice and LPS-hyporesponsive C3H/HeJ mice (5, 6, 7). Pre-exposure of murine macrophages (8) and human THP-1 monocytes (9) to BLP induces tolerance to the stimulatory effects of BLP but also a cross-tolerance to LPS, which leads us to investigate the protective effect of BLP tolerance in septic shock and to compare this with LPS tolerance.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
LPS from E. coli O55:B5 (Sigma-Aldrich, St. Louis, MO) and BLP, a synthetic bacterial lipopeptide (Pam3Cys-Ser-Lys4-OH; Boehringer Mannheim Biochemica, Mannheim, Germany) that was endotoxin-free as confirmed by limulus amebocyte lysate assay (Charles River Endosafe, Charleston, SC), were dissolved in PBS (Life Technologies, Paisley, Scotland, U.K.). Staphylococcus aureus 14458 and Salmonella typhimurium were obtained from American Type Culture Collection (Manassas, VA) and the National University of Ireland culture collection, respectively. Bacteria were cultured at 37°C in trypticase soy broth (Merck, Darmstadt, Germany), harvested at the mid-logarithmic growth phase, washed twice, and resuspended in PBS for in vivo use. The concentration of resuspended bacteria was determined and adjusted spectrophotometrically at 550 nm.
Septic shock models
Pyrogen-free male MF-1, C57BL/6, and CD-1 mice (8- to 10-wk old and 18–22 g) were purchased from Harlan (Oxon, U.K.). All animal procedures were conducted under a license from the Department of Health and Children (Republic of Ireland). Tolerance in mice was induced by i.p. injection of 10 mg/kg BLP (BLP tolerance), 10 mg/kg LPS (LPS tolerance), or an equal volume (200 µl) of PBS (no tolerance) 24 h before septic challenges. Nontolerized, BLP-tolerized, and LPS-tolerized male MF-1 mice were injected i.p. with BLP (45 mg/kg), LPS (45 mg/kg), or their combination (30 plus 30 mg/kg). Survival was monitored for at least 10 days. Male C57BL/6 mice were tolerized to BLP or LPS and received an i.p. injection of 200 µl PBS containing live S. aureus and S. typhimurium (2 x 107 plus 2 x 106 CFU/mouse). Survival was monitored for at least 14 days. Following induction of BLP or LPS tolerance, male CD-1 mice were subjected to polymicrobial sepsis induced by cecal ligation and puncture (CLP) (10). Briefly, the cecum was exposed through a 1.0–1.5 cm abdominal midline incision in anesthetized mice, ligated at its base with 3-0 silk suture, and punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity and the abdominal incision was closed. Survival was monitored for at least 10 days.
Cytokine measurements
We performed additional experiments in mice as described above.
Blood samples from nontolerized, BLP-tolerized, and LPS-tolerized mice
were collected at different time points after septic challenges. Serum
TNF-
, IL-6, IL-10, and IL-12 were determined by ELISA (R&D Systems,
Minneapolis, MN).
FACScan analysis of immunofluorescence
Heparinized blood and peritoneal lavage were collected from
nontolerized, BLP-tolerized, and LPS-tolerized mice and dual-stained
with anti-Ly-6G (BD PharMingen, San Diego, CA), anti-F4/80 Ag
(Serotec, Oxford, U.K.), anti-complement receptor type 3 (CR3; BD
PharMingen), and anti-FcIII/IIR (BD PharMingen) mAbs conjugated
with PE or FITC. PE- or FITC-conjugated anti-mouse isotype-matched
mAbs (BD PharMingen) were used as negative controls. Erythrocytes were
lysed using lysis buffer (BD Biosciences, Mountain View, CA). FACScan
analysis was performed from at least 5000 events for detecting the
expression of CR3 and FcIII/IIR on polymorphonuclear neutrophils
(PMN; Ly-6G-positive cells) and macrophages (F4/80-positive cells)
using CellQuest software (BD Biosciences).
Bacterial uptake, ingestion, and intracellular killing
S. aureus and S. typhimurium were heat-killed at 95°C for 20 min and labeled with 0.1% FITC (Sigma-Aldrich). Heparinized blood and peritoneal lavage collected from nontolerized, BLP-tolerized, and LPS-tolerized mice were incubated with 1 x 106 CFU/ml of heat-killed, FITC-labeled S. aureus or S. typhimurium at 37°C for 15 min. Bacterial uptake by PMN and peritoneal macrophages was assessed by FACScan analysis. Bacterial ingestion was further determined after the external fluorescence of the bound, but noningested, bacteria was quenched with 0.025% crystal violet (Sigma-Aldrich). Intracellular bacterial killing was determined as previously described (11). Briefly, peritoneal macrophages were incubated with live S. aureus or S. typhimurium (macrophage:bacteria = 1:20) at 37°C for 60 min, in the presence or absence of cytochalasin B (5 µg/ml; Sigma-Aldrich). After macrophages were lysed, total and extracellular bacterial killing were determined by incubation of serial 10-fold dilutions of the lysates on tryptone soya agar (Merck) plates at 37°C for 24 h. Intracellular bacterial killing was calculated according to the total and extracellular bacterial killing.
Statistical analysis
All data are presented as the mean ± SD. Statistical analysis was performed using the log rank test for survival studies and the Mann-Whitney U test for all others. Differences were judged statistically significant when p < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Induction of BLP tolerance completely protected mice against
BLP-induced lethality with 100% survival as compared with 40%
survival in nontolerized mice (p = 0.0000; Fig. 1
a). Furthermore, BLP
tolerance significantly improved survival in endotoxic shock, from 20%
in nontolerized mice to 60% (p = 0.0152; Fig. 1b), indicating a cross-tolerance to LPS-induced lethality.
Complete protection was also conferred by BLP tolerance in mice
challenged with a combination of BLP and LPS (p
= 0.0000 vs nontolerized mice; Fig. 1c). LPS tolerance
protected against its own lethality by reducing the mortality rate from
80% in nontolerized mice to 20% (p = 0.0002;
Fig. 1b). However, it failed to protect mice against lethal
BLP challenge and a combined BLP plus LPS challenge (Fig. 1, a and c).
|
d). BLP tolerance-afforded protection
was also observed in mice challenged with CLP-induced polymicrobial
sepsis in which survival was significantly improved from 3.6% in
nontolerized mice to 43% in BLP-tolerized mice
(p = 0.0013; Fig. 1e). However,
induction of LPS tolerance offered no protection against either lethal
bacterial infection or polymicrobial sepsis (Fig. 1, d and
e). Furthermore, tolerance induced by other forms of LPS
derived from E. coli O111:B4 and S. typhimurium
did not protect mice against lethal BLP challenge, or against microbial
sepsis (data not shown). Attenuated inflammatory cytokine production in both BLP- and LPS-tolerized mice
We measured serum levels of TNF- and IL-6 in BLP-tolerized
mice following lethal BLP and LPS challenges and compared them to
levels encountered in LPS-tolerized mice. As shown in Fig. 2a, induction of BLP tolerance
resulted in a near complete attenuation of TNF- and IL-6 release in
mice challenged with lethal BLP and also resulted in a significant
reduction in peak serum levels of these two cytokines in mice
challenged with lethal LPS. LPS tolerance significantly attenuated the
release of TNF- and IL-6 in LPS-challenged mice as well as IL-6 in
BLP-challenged mice, but had no inhibitory effect on TNF- production
induced by lethal BLP challenge (Fig. 2a).
|
, IL-6, and IL-12 as well as anti-inflammatory
cytokine IL-10. These increases were significantly attenuated in both
BLP-tolerized and LPS-tolerized mice (Fig. 2b). Similar
results were found in BLP-tolerized and LPS-tolerized mice subjected to
CLP-induced polymicrobial sepsis (Fig. 2c). Notably, there
was a more profound inhibition of TNF- production observed in
LPS-tolerized mice following challenges with lethal bacterial infection
or polymicrobial sepsis (Fig. 2, b and c). Enhanced PMN and macrophage activation in BLP-tolerized mice
We determined CR3 and FcIII/IIR expression on PMN and
peritoneal macrophages in whole blood and peritoneal lavage collected
from nontolerized, BLP-tolerized, and LPS-tolerized mice. Induction of
BLP tolerance resulted in an increase in the circulating PMN population
and a recruitment of PMN into the peritoneal cavity, with an increased
surface expression of CR3 and FcIII/IIR (Fig. 3, a and b). In
addition, BLP tolerance significantly up-regulated the expression of
these two receptors on peritoneal macrophages (Fig. 3c). In
contrast, although LPS tolerance resulted in an increase in PMN numbers
in the circulation and peritoneal cavity, there was a down-regulation
of CR3 and FcIII/IIR expression on PMN and macrophages in
LPS-tolerized mice (Fig. 3).
|
We next assessed the ability of PMN and peritoneal macrophages
to recognize, phagocytose, and kill bacteria. PMN and macrophages from
BLP-tolerized mice had significantly increased uptake and ingestion of
both S. aureus and S. typhimurium, compared with
cells from nontolerized mice (Fig. 4, a and b). Furthermore, intracellular killing of
S. aureus and S. typhimurium by peritoneal
macrophages was significantly enhanced in BLP-tolerized mice (Fig. 4c). In contrast, although there was an increased uptake of
S. aureus and S. typhimurium by PMN and
macrophages in LPS-tolerized mice, LPS tolerance did not augment
bacterial ingestion as seen with BLP tolerance (Fig. 4, a
and b). Indeed, peritoneal macrophages from LPS-tolerized
mice showed a significant reduction in intracellular killing of
S. aureus when compared with nontolerized mice (Fig. 4c).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In response to bacterial invasion, mononuclear phagocytes produce
inflammatory cytokines including TNF-, IL-1
, IL-6, and IL-12
(14). Although appropriate amounts of these cytokines are
essential for cell-mediated microbicidal activity, excessive production
can lead to an uncontrolled inflammatory response, multiple organ
failure, and ultimately death (14, 15, 16). As the protective
effect of LPS tolerance in endotoxin shock is associated with
attenuation of proinflammatory cytokine production, in particular
TNF- (1, 2), we compared serum levels of TNF- and
IL-6 following lethal BLP or LPS challenge in BLP-tolerized and
LPS-tolerized mice. Induction of BLP tolerance not only blunted the
release of TNF- in mice challenged with lethal BLP, but also reduced
peak serum level of TNF- in mice challenged with lethal LPS. In
contrast, LPS tolerance failed to inhibit TNF- production in
BLP-challenged mice, which may partly explain why LPS tolerance
conferred no protection against a lethal BLP challenge. In mice
challenged with microbial sepsis, both BLP and LPS tolerance
significantly reduced the serum levels of TNF-, IL-6, IL-10, and
IL-12. LPS tolerance resulted in a much greater inhibition of TNF-
production, an effect that may be deleterious to host defense against
invading bacteria, as certain amounts of endogenous TNF- are almost
certainly required for effective eradication of bacterial infection
(17, 18), and blocking TNF- accelerates death of mice
challenged with bacteria (19, 20). These results, in
keeping with the survival data, suggest that induction of BLP tolerance
inhibits inflammatory responses but, in contrast to LPS tolerance, may
simultaneously prevent uncontrolled bacterial infection.
PMN are the first line of host defense against bacterial infection.
Invading bacteria are ingested and killed by PMN through activation of
CR3 and FcIII/IIR which are also involved in macrophage-mediated
bacterial phagocytosis (21, 22). Overexpression of these
two phagocytic receptors is associated with enhanced bacterial
clearance (23). To investigate possible mechanisms
contributing to enhanced host defense against microbial sepsis observed
in BLP-tolerized mice, we assessed CR3 and FcIII/IIR expression on
PMN and peritoneal macrophages. Induction of BLP tolerance resulted in
an increased PMN population in the circulation and peritoneal cavity,
with overexpression of CR3 and FcIII/IIR on PMN as well as on
peritoneal macrophages. In contrast, LPS tolerance led to a
suppressed expression of these two phagocytic receptors. To
determine whether this altered phenotype of PMN and peritoneal
macrophages is associated with BLP tolerance-afforded protection
against lethal bacterial infection, we further assessed the ability of
these cells to recognize, ingest, and kill S. aureus
and S. typhimurium. PMN and peritoneal macrophages from
BLP-tolerized mice, in keeping with the activation of phagocytic
receptors on these cells, exhibited increased bacterial recognition,
phagocytosis, and intracellular killing. The enhanced bacterial
recognition and microbicidal activity observed in BLP-tolerized mice
may contribute to their improved survival over LPS-tolerized mice
following lethal microbial sepsis.
Microbial sepsis can lead to an uncontrolled inflammatory response characterized by the excessive release of proinflammatory cytokines, which is host self-destructive. Our findings provide evidence that the development of tolerance to the stimulatory effects of the bacterial cell wall component BLP is host self-controlled and self-protective, and represents a beneficial and not a harmful adaptive host response to bacterial infection. Secondly, we provide evidence that the mechanism by which BLP tolerance protects mice against clinically relevant microbial sepsis directly relates to enhanced bacterial recognition, ingestion, and intracellular killing, demonstrating how BLP-tolerized cells, though hyporesponsive in one sense are also primed for an increased bactericidal activity. Finally, our disparate findings in relation to BLP- and LPS-tolerized mice illustrate the markedly variable effects that different bacterial Ags have on cells of the innate immune system. Though sepsis research has focused for many years on the immune stimulatory actions of Gram-negative endotoxin, our work clearly points toward other bacterial Ags and in particular BLP, a more ubiquitously abundant component of both Gram-negative and Gram-positive bacteria, as a potential target for study in sepsis immunology and therapeutic development.
|
Footnotes |
|---|
2 J.H.W. and M.D. contributed equally to this work.
3 Abbreviations used in this paper: BLP, bacterial lipoprotein; CLP, cecal ligation and puncture; CR3, complement receptor type 3; PMN, polymorphonuclear neutrophil.
Received for publication September 13, 2002. Accepted for publication November 7, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-interferon, tumor necrosis factor, and interleukin-6 in Staphylococcus aureus infection in mice. Infect. Immun. 63:1165.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  R. F. Wideman, O. T. Bowen, and G. F. Erf Broiler pulmonary hypertensive responses during lipopolysaccharide-induced tolerance and cyclooxygenase inhibition Poult. Sci., January 1, 2009; 88(1): 72 - 85. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kumar, L. D. Hazlett, and F.-S. X. Yu Flagellin Suppresses the Inflammatory Response and Enhances Bacterial Clearance in a Murine Model of Pseudomonas aeruginosa Keratitis Infect. Immun., January 1, 2008; 76(1): 89 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Coffey, J. H. Wang, R. Kelly, L. Romics Jr., A. O'Callaghan, C. Fiuza, and H. P. Redmond Tolerization with BLP down-regulates HMGB1 a critical mediator of sepsis-related lethality J. Leukoc. Biol., October 1, 2007; 82(4): 906 - 914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Buckley, J. H. Wang, and H. P. Redmond Cellular reprogramming by gram-positive bacterial components: a review J. Leukoc. Biol., October 1, 2006; 80(4): 731 - 741. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Romics Jr, G. Szabo, J. C. Coffey, J. H. Wang, and H. P. Redmond The Emerging Role of Toll-Like Receptor Pathways in Surgical Diseases Arch Surg, June 1, 2006; 141(6): 595 - 601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. H. Li, J. H. Wang, and H. P. Redmond Bacterial lipoprotein-induced self-tolerance and cross-tolerance to LPS are associated with reduced IRAK-1 expression and MyD88-IRAK complex formation J. Leukoc. Biol., April 1, 2006; 79(4): 867 - 875. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Feterowski, A. Novotny, S. Kaiser-Moore, P. F. Muhlradt, T. Rossmann-Bloeck, M. Rump, B. Holzmann, and H. Weighardt Attenuated pathogenesis of polymicrobial peritonitis in mice after TLR2 agonist pre-treatment involves ST2 up-regulation Int. Immunol., August 1, 2005; 17(8): 1035 - 1046. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. H. Elsasser, J. W. Blum, and S. Kahl Characterization of calves exhibiting a novel inheritable TNF-{alpha} hyperresponsiveness to endotoxin: associations with increased pathophysiological complications J Appl Physiol, June 1, 2005; 98(6): 2045 - 2055. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. von Knethen, A. Tautenhahn, H. Link, D. Lindemann, and B. Brune Activation-Induced Depletion of Protein Kinase C{alpha} Provokes Desensitization of Monocytes/Macrophages in Sepsis J. Immunol., April 15, 2005; 174(8): 4960 - 4965. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. C. O'Brien, J. H. Wang, and H. P. Redmond Bacterial Lipoprotein Induces Resistance to Gram-Negative Sepsis in TLR4-Deficient Mice via Enhanced Bacterial Clearance J. Immunol., January 15, 2005; 174(2): 1020 - 1026. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. P. Power, J. H. Wang, B. Manning, M. R. Kell, N. F. Aherne, Q. D. Wu, and H. P. Redmond Bacterial Lipoprotein Delays Apoptosis in Human Neutrophils through Inhibition of Caspase-3 Activity: Regulatory Roles for CD14 and TLR-2 J. Immunol., October 15, 2004; 173(8): 5229 - 5237. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Gould, J. A. Greene, V. Bhoj, J. L. DeVecchio, and F. P. Heinzel Distinct Modulatory Effects of LPS and CpG on IL-18-Dependent IFN-{gamma} Synthesis J. Immunol., February 1, 2004; 172(3): 1754 - 1762. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), LPS tolerance (•), or no tolerance (
), male MF-1 mice
(n = 20 per group) were challenged with BLP (45
mg/kg) (a), LPS (45 mg/kg) (b), or their
combination (30 plus 30 mg/kg) (c), male C57BL/6 mice
(n = 25 per group) were challenged with a
combination of live S. aureus and S.
typhimurium (2 x 107 plus 2 x
106 CFU/mouse) (d), and male CD-1 mice
(n = 28 per group) were challenged with CLP-induced
polymicrobial sepsis (e). Survival was monitored for at
least 14 days in animals challenged with live bacteria or at least 10
days in other groups.
