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ARTICLE |
B in multiple organ injury and bacterial clearance in mouse models of sepsis
CORRESPONDENCE Shu Fang Liu: Sliu{at}lij.edu
 Top ABSTRACT RESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B (NF-B) activity in host defense and multiple organ injury in response to sepsis, we generated double transgenic (TG) mice (EC-rtTA/I-B
mt) that conditionally overexpress a degradation-resistant form of the NF-B inhibitor I-B (I-Bmt) selectively on vascular endothelium. The EC-rtTA/I-Bmt mice had no basal, but a relatively high level of doxycycline-inducible, I-Bmt expression. I-Bmt expression was detected in endothelial cells, but not in fibroblasts, macrophages, and whole blood cells, confirming that transgene expression was restricted to the endothelium. When subjected to endotoxemia, EC-rtTA/I-Bmt mice showed endothelial-selective blockade of NF-B activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infiltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, reduced systemic hypotension, and abrogated intravascular coagulation. When subjected to cecal ligation and puncture–induced sepsis, the TG mice had less severe multiple organ injury and improved survival compared with wild-type (WT) mice. WT and EC-rtTA/I-Bmt mice had comparable capacity to clear three different pathogenic bacteria. Our data demonstrate that endothelial NF-B activity is an essential mediator of septic multiple organ inflammation and injury but plays little role in the host defense response to eradicate invading pathogenic bacteria.
X. Ye and J. Ding contributed equally to this work.
© 2008 Ye et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
Sepsis and septic multiple organ dysfunction and injury (MOD/I) are leading causes of death among critically ill patients. During sepsis, bacterial pathogens trigger the release of hundreds of inflammatory mediators, including cytokines, chemokines, adhesion molecules, reactive oxygen species, and reactive nitrogen species (1–4). Although these molecules are important for host defense response against invading bacterial pathogens, excessive production of these mediators cause systemic inflammation and collateral tissue damage that lead to septic sequelaes, including coagulation, endothelial injury, microvascular leakage, and MOD/I.
Bacterial pathogens initiate systemic inflammation by activating cytokine networks and by inducing the expression of proinflammatory genes, a process that is principally mediated by activating an inducible transcription factor, such as NF-
Despite all of the evidence, the contribution of endothelial NF-
In this study, we sought to define the roles of endothelial-intrinsic NF-
We achieved endothelium-restricted rtTA expression using the vascular endothelial (VE)–cadherin-5 promoter, which has been shown to drive endothelial-specific transgene expression in widespread organs in TG mice (21). I-B. NF-B activation is a driving force in the initiation and progression of systemic inflammation and septic pathology (4, 5). NF-B is activated by a variety of bacteria and their products known to cause sepsis syndrome. NF-B activation appears to be a central common pathway, through which a variety of pathogens cause septic syndrome (4). NF-B regulates the expression of hundreds of proinflammatory genes, whose products mediate or contribute to the development of septic shock and septic MOD/I (4–6). Patients with septic shock showed an increased NF-B activity, which correlates with the severity of the disease and predicts clinical outcome (7–9). Animal studies have demonstrated that inhibition of NF-B activation decreases multiple inflammatory gene expression (4, 10), reverses systemic hypotension (4, 11), corrects myocardial dysfunction (4, 12), diminishes intravascular coagulation (4, 7), reduces tissue neutrophil influx and microvascular endothelial permeability (4, 13), prevents multiple organ injury, and reduces endotoxemic lethality (4, 14) in endotoxemic or septic animals. Mice deficient in NF-B–dependent genes are resistant to septic shock and sepsis-related death (4). B to the development of a septic phenotype remains unclear. The pathological process underlying sepsis and septic MOD/I involves complex cell–cell and mediator–mediator interactions. Each cell type and its NF-B system may play distinct roles in this process. The role of endothelial NF-B in the pathology of sepsis and septic MOD/I has not been well defined. Additionally, disruption of the NF-B signaling pathway or abolition of NF-B–dependent genes impairs the host defense capacity to eliminate invading bacteria and leads to a worsened outcome in a bacterial infection model of sepsis (4, 15–17), indicating that NF-B is also protective. The detrimental versus beneficial effects of NF-B activation at the organ level may depend on multiple factors, including the cell type in which NF-B activity is predominantly localized. Thus, elucidation of the specific contribution of each cellular NF-B to the overall detrimental or beneficial effect of NF-B activation at the organ level will help to better explain the complicated pathogenic roles of NF-B in sepsis, septic MOD/I, and other inflammatory conditions. B activity in multiple organ inflammation and injury (detrimental) and in bacterial clearance (beneficial) using both LPS and cecal ligation and puncture (CLP) models of sepsis. We generated double transgenic (TG) mice that conditionally overexpress a degradation-resistant form of I-B (I-Bmt), a superior inhibitor of NF-B, selectively on endothelium. Studies on these mice showed that endothelial-selective blockade of NF-B activation ameliorated multiple organ inflammation and injury in both LPS and CLP models of sepsis but had no effects on bacterial clearance capacity. Our results demonstrate that endothelial NF-B is an essential mediator of septic multiple organ inflammation and injury but plays little role in the host defense response to eliminate bacterial pathogens.
RESULTS
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ABSTRACT
RESULTS
DISCUSSION
MATERIALS AND METHODS
REFERENCES
Generating TG mice that conditionally overexpress I-Bmt selectively on endothelium
TG mice overexpressing the mutant I-B selectively on endothelium have been generated (18). Studies on those mice demonstrated an important role of NF-B in embryonic development of endothelial lineage (18). Adult mice of this strain have structural and functional defects in endothelial cells (ECs), as indicated by loss of endothelial tight junction, increased sensitivity to LPS-induced endothelial permeability, and enhanced susceptibility to tumor metastasis (18). A similar mouse strain with endothelium-restricted overexpression of I-B
N, an NF-B super repressor, has also been generated (19). Although the basal structure and function of endothelium were not examined, endothelial NF-B activity required for embryonic development of endothelial lineage is likely to be suppressed during embryonic development in this mouse strain (19). To avoid the embryonic side effects and examine the effects of endothelial-selective blockade of the NF-B pathway on septic response under a physiological setting, we generated TG mice that conditionally overexpress I-Bmt selectively on endothelium using a tetracycline-regulated gene expression system (20). Our TG mice do not express the I-Bmt gene until it is induced by feeding adult mice with doxycycline (Dox), and have normal NF-B activity that is critically required for embryonic development and for the development of normal endothelial lineage cells. Bmt expression was controlled by a TreCMV fusion promoter, whose activation requires the binding of rtTA and Dox (Fig. 1 A).
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Bmt single TG mouse lines. We intercrossed each of the EC-rtTA lines with each of the TreI-Bmt lines, which gave rise to 42 double TG mouse lines that carried both the rtTA and I-Bmt transgenes (EC-rtTA/I-Bmt mice). We performed RT-PCR analysis of basal and Dox-induced I-Bmt mRNA expression using transgene-specific primers and RNAs from 12 organs of each EC-rtTA/I-Bmt mouse line. Those analyses revealed that 8 out of the 42 EC-rtTA/I-Bmt mouse lines had high basal and low Dox-induced I-Bmt mRNA expression (leakiness), 31 out of the 42 lines had no basal but little Dox-induced I-Bmt mRNA expression (no expression), and 3 out of the 42 lines had no basal and relatively high level of Dox-induced I-Bmt mRNA expression. Among the 3 good lines, line 2/36 was the best and was further studied. Fig. 1, B and C shows I-Bmt mRNA expression in 12 organs of mouse line 2/36. After 35 cycles of PCR amplification, no I-Bmt mRNA was detected in any organ from a mouse that was not fed with Dox (Fig. 1 C). I-Bmt mRNA was detected in all organs examined, except spleen, from a mouse that was fed with Dox (Fig. 1 B). Further analysis of the spleen sample showed an I-Bmt mRNA expression, albeit at a low level (unpublished data). The Dox-inducible I-Bmt mRNA abundance varied among organs in a pattern consistent with the enrichment of ECs in these organs (Fig. 1 B).
We performed immunofluorescence staining of lung section from mouse line 2/36 using anti–human I-B antibody, which has a low level of cross-reactivity with mouse I-B. Immunofluorescence staining showed Dox-induced I-Bmt protein expression on lung sections from mice fed with Dox (Fig. 1, D–F). To ascertain if Dox-induced I-Bmt protein expression is restricted to ECs, we isolated vascular ECs and fibroblasts from the lungs and macrophages from the peritoneal cavity of line 2/36 mice (TG) and their transgene-negative littermates (WT). These cells exhibited morphological characteristics of ECs, fibroblasts, and macrophages, respectively. The EC phenotype was further confirmed by staining for endothelial-specific markers, platelet/EC adhesion molecule 1, and intercellular adhesion molecule (ICAM) 1. Macrophages and fibroblasts were confirmed by staining with anti-Mac3 (macrophage marker) and anti-procollagen (fibroblast marker) antibodies. Western blotting using an antibody reactive to human and mouse I-B detected a single I-B band (endogenous mouse I-B) in the cytoplasmic protein of WT ECs, but two I-B bands in the cytoplasmic protein of TG ECs (Fig. 2 A, TG0).
The top band (Fig. 2 A, HI-Bmt) represents transgene product, and the bottom band (Fig. 2 A, MI-B) represents endogenous mouse I-B. The HI-Bmt band (top) was weak in control ECs (Fig. 2 A, TG0) but increased in Dox-treated ECs (Fig. 2 A, TG0.5 and TG1). The endogenous MI-B band (bottom) diminished after TNF- treatment (Fig. 2 A, WT-T), indicating degradation of endogenous I-B, whereas the Dox-induced HI-Bmt band (top) was not affected by TNF- treatment (Fig. 2 A, TG0.5 and TG1), indicating degradation resistance of the TG HI-Bmt protein. Western blotting detected a single I-B band in cytoplasmic proteins of both WT and TG fibroblasts from the same lungs where ECs were isolated (Fig. 2 B). The I-B band intensity was not affected by treatment with the same concentrations of Dox (Fig. 2 B) but was diminished by TNF- treatment (Fig. 2 B), representing endogenous MI-B. Similarly, both WT and TG macrophages expressed a single mouse I-B protein, which was significantly degraded by TNF- treatment (Fig. 2 C), confirming that TG macrophages do not express TG I-Bmt.
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Bmt transgene in our TG EC mice by performing RT-PCR analysis of I-Bmt mRNA expression in whole blood cells and lungs (as a positive control) from line 2/36 TG mice. As illustrated in Fig. 3, I-Bmt mRNA was not detected in either lungs or blood cells of the two TG mice that were not fed with Dox. The I-Bmt band was detected in lungs but not in whole blood cells from the three TG mice that were fed with Dox for 4 d (Fig. 3).
Thus, hematopoietic cells of our TG mice are unlikely to express I-Bmt mRNA. Collectively, our results demonstrate that our EC-rtTA/I-Bmt mice overexpress Dox-inducible I-Bmt selectively on endothelium.
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Bmt mice selectively block NF-B activation in ECsBmt protein and to confirm that NF-B blockade is endothelial restricted, TNF-– or LPS-induced nuclear translocation of p65 was compared between WT and TG lung ECs and fibroblasts, and peritoneal macrophages. To avoid the effects of Dox, all cells were treated with Dox. Compared with control cells, TNF- or LPS stimulation markedly increased p65 nuclear accumulation in all cell types (Fig. 4, A–C, WT-T and WT-L), which was blocked in TG ECs (Fig. 4 A, TG-T) but not in fibroblasts (Fig. 4 B, TG-T) and macrophages (Fig. 4 C, TG-L).
Thus, our TG mice selectively blocked TNF-– or LPS-induced NF-B activation in vascular ECs.
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Bmt miceB activation, we examined tissue levels of E-selectin, ICAM-1, and vascular adhesion molecule (VCAM) 1 expressions in the lungs, heart, and liver of WT and TG mice subjected to endotoxemia. Fig. 5 (A–C) depicts Western blot photographs showing tissue levels of E-selectin, ICAM-1, and VCAM-1 protein expression in three organs of WT-Con, WT-LPS, TG-Con, and TG-LPS mice.
Western blot bands were quantified using densitometry and summarized in Fig. 5 (D–F). Compared with WT-con and TG-con, tissue E-selectin, ICAM-1, and VCAM-1 protein levels increased significantly in all three organs of WT-LPS mice (Fig. 5). The LPS-induced expression of the three adhesion molecules was significantly reduced in the three organs of TG-LPS mice (Fig. 5).
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Bmt miceB blockade reduces neutrophil infiltration into multiple organs.
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B inhibition but not a result of other changes in TG mice, we also examined the protective effect of endothelial-selective I-Bmt overexpresion on LPS-induced multiple organ injury in mouse line 1/36, our second-best EC TG line. Likewise, the LPS-induced increases in EBD leakage index and tissue wet/dry ratio were reversed in all five organs of line 1/36 TG-LPS mice (Fig. 7, C and D).
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B blockade effectively prevents endotoxemic multiple organ injury.
Alleviated multiple organ injury and improved survival in septic EC-rtTA/I-Bmt mice
WT-sham and TG-sham mice showed similar endothelial EBD leakage index and tissue wet/dry ratio in the lungs, heart, liver, kidney, and intestine (Fig. 8, A and B).
Compared with WT-sham and TG-sham mice, WT-CLP mice exhibited significantly increased endothelial EBD leakage index and tissue wet/dry ratio (Fig. 8, A and B). The increases in EBD leakage index and tissue wet/dry ratio were prevented in all five organs of TG-CLP mice (Fig. 8, A and B), indicating that endothelial-selective NF-B inhibition prevents septic multiple organ injury. Ameliorated organ injury has resulted in a reduced mortality. As illustrated in Fig. 8 C, the 2-wk survival rate was 26% in WT-CLP mice and 67% in TG-CLP mice. Thus, we have demonstrated that endothelial-selective blockade of NF-B activation inhibits multiple organ inflammation, prevents multiple organ injury, and improves survival in mice subjected to endotoxemia or sepsis.
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Bmt miceB inhibition on systemic hypotension and coagulation in endotoxemic mice. Baseline systemic mean arterial blood pressure (MBP) was identical among WT-Con, TG-Con, WT-LPS, and TG-LPS groups of mice. At 4 h after LPS challenge, MBP dropped by 
50% in WT-LPS mice, which was virtually reversed in TG-LPS groups of mice (Fig. 9 A).
In parallel with a drop in MBP, WT-LPS mice showed a marked elevation in the plasma level of nitrite/nitrate, the stable end products of nitric oxide, which was significantly lower in that of TG-LPS mice (Fig. 9 B). Compared with WT-Con and TG-Con mice, WT-LPS mice had a significantly elevated plasma level of thrombin–antithrombin (TAT) complex, an indicator of intravascular coagulation (Fig. 9 C). Compared with WT-LPS mice, TG-LPS mice showed a significantly lower plasma level of TAT (Fig. 9 C).
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B had no effects on bacterial clearance capacityB in host defense response to eliminate bacterial pathogens, mice were infected with the intracellular bacterium Listeria monocytogenes, the extracellular gram-positive bacterium Streptococcus pneumoniae, and the intracellular gram-negative bacterium Salmonella enterica. Bacterial colonies that formed in blood and tissue homogenate cultures were counted and compared between WT and TG mice. Little bacteria grew in blood and tissue homogenates of WT-Con and TG-Con mice (Fig. 10, A–C).
Consistent with these organs being the main sites of Listeria infection, L. monocytogenes grew in liver and spleen homogenates of infected mice. The numbers of Listeria colonies formed were identical or similar between WT and TG mice (Fig. 10 A). S. pneumoniae and S. enterica grew in the lungs, liver, kidney, and spleen homogenates of infected mice (Fig. 10, B and C). The number of colonies formed varied among organs but were identical between WT and TG mice in all organs examined (Fig. 10, B and C). These results illustrate that WT and EC-rtTA/I-Bmt mice have identical capacities to eliminate invading bacteria, suggesting that endothelial NF-B does not play an important role in mediating host defense response against bacterial pathogens.
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DISCUSSION |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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B plays distinct roles in the inflammatory/injurious and host defense responses. Endothelial NF-B activity is critically required for the inflammatory and injurious responses that lead to septic multiple organ inflammation and injury. Compared with WT mice, TG mice that have the endothelial NF-B pathway blocked showed decreased expression of multiple adhesion molecules, reduced neutrophil infiltration into multiple organs, reduced systemic hypotension, alleviated endothelial injury, and ameliorated multiple organ injury when subjected to endotoxemia. Those TG mice also exhibited alleviated multiple organ injury and improved survival in the CLP model of sepsis. Thus, selective blockade of the endothelial NF-B pathway is sufficient to reduce multiple organ inflammation, prevent multiple organ injury, and improve survival, indicating that endothelial NF-B plays a critical role in septic multiple organ inflammation and injury. In supporting this contention, Henke et al. have recently reported that endothelial-specific NF-B suppression attenuates hypertension-induced renal damage (19).
In contrast, endothelial NF-B does not appear to play an important role in host defense response to eradicate invading bacterial pathogens. When infected with three different classes of pathogenic bacteria, S. pneumoniae, L. monocytogenes, and S. enterica, WT and TG mice with endothelial-selective NF-B inhibition exhibited identical bacterial clearance capability. Because clearance of the three pathogenic bacteria involves a different host defense mechanism (23), the lack of effect of blocking endothelial NF-B on bacterial clearance indicates that endothelial NF-B does not play an important role in host defense response to eliminate those pathogenic bacteria.
NF-B activation is a key component of host immune response (4). NF-B p50 knockout mice showed severely defective clearance of S. pneumoniae and L. monocytogenes (15). The lack of effect of endothelial-selective NF-B inhibition on bacterial clearance is somewhat surprising. It would be interesting to know which cellular NF-B system plays a major role in the host defense response against bacterial pathogens. TG mice whose hepatocyte NF-B pathway is selectively inhibited displayed a severely impaired capacity to clear L. monocytogenes (16), indicating a requirement of hepatocyte NF-B activity for the host immune response against L. monocytogenes infection. However, the same immune response does not appear to involve the endothelial NF-B system, because blocking endothelial NF-B had no effect on the clearance of this bacterium, as we demonstrated in this study. These results suggest that the involvement of a particular cellular NF-B system in the host defense response may be pathogen dependent. This question warrants further investigation.
Investigation into the role of NF-B activation in sepsis and other inflammatory conditions has been hampered by the fact that NF-B knockout mice have multifocal defects in immune responses and are embryonically lethal (15, 24–27). Conventional TG mice overexpressing I-Bmt selectively on endothelium have structural and functional defects in endothelium, as indicated by loss of endothelial tight junction, increased sensitivity to LPS-induced endothelial permeability, and enhanced susceptibility to tumor metastasis (18). To overcome those problems, we took a conditional TG approach. We generated TG mice that conditionally overexpress I-Bmt selectively on endothelium using a tetracycline-regulated gene expression system (20), which has been widely used to define various gene functions in TG animals (12, 16, 28–31). Our TG mice do not express I-Bmt until induced by feeding adult mice with Dox, and have normal NF-B activity that is critically required for embryonic development, avoiding embryonic side effects seen in NF-B knockout or conventional TG mice. NF-B inhibition in our TG mice is transient and restricted to endothelium, which would have minimal effects on immune cell differentiation, development, and function, allowing us to study septic or other inflammatory responses under a physiological setting.
I-Bmt bears two mutations at serine 32 and 36 residues, which prevent its phosphorylation and subsequent degradation (32), a key step leading to the activation of the classical NF-B pathway. As a specific and effective NF-B inhibitor, I-Bmt has been widely used to define the role NF-B in various pathological processes (12, 16, 33, 34). The overexpressed I-Bmt protein is degradation resistant and constantly binds to any form of NF-B dimer, resulting in a more effective blockade of the NF-B pathway and resolving the problem of functional redundancy between the NF-B family of proteins seen in single NF-B knockout mice.
Endothelial dysfunction, inflammation, and injury are increasingly recognized as an important pathogenic mechanism of many pathological conditions, including sepsis (35), hypertension (36), and atherosclerosis (37). Endothelial phenotypic changes are dictated by alteration in the expression of endothelial-specific genes. The ability to selectively manipulate endothelial gene expression in vivo will allow investigators to clearly define the role of individual endothelial-specific gene in the alteration of endothelial phenotypes. Therefore, there has been great interest in endothelial-selective gene knockout or TG animal models. Conventional gene knockout and TG animals have limitations such as developmental defects and embryonic lethality (18, 24–27), which limit their application and relevance to the adult disease state. In this context, the conditional TG approach is clearly advantageous. Although the Tet-regulated gene expression system (20) has been successfully applied to various fields (28–31, 33, 34), its application to endothelial-selective gene expression has been less successful. Coupling the tie2 promoter to the Tet-off system, Sarao and Dumont were able to direct the reporter gene (LacZ) expression only in embryonic endothelium (38). Using an extended Tie promoter coupled to the Tet-on system, Teng et al. were able to drive LacZ expression on the endothelium of adult mice, although those mice have leakiness (39). In the current study, we took a different approach by using the VE–cadherin-5 promoter (21) to drive endothelial-selective rtTA expression and by generating multiple independent rtTA and TreI-Bmt mouse lines. Intercrossing between those independent rtTA and TreI-Bmt lines produced 42 double TG mouse lines, which allowed us to select the best dual TG lines. Immunofluorescence staining and cell-culture experiments confirmed that our TG mice express Dox-inducible I-Bmt protein in ECs but not in fibroblasts and macrophages. The time-consuming and labor-intensive screening and selection processes were proven to be necessary, as 73.8% of the 42 double TG lines had little Dox-inducible I-Bmt expression, and 19% of them had a leaky problem, indicating the technical difficulty of applying the Tet-on system to endothelial-specific gene expression. To our knowledge, this is the first study that has successfully achieved endothelial-specific expression of a real mammalian gene in TG mice using the Tet-on system. This mouse model allows us to study endotoxemic or septic responses under a physiological setting and provides a useful tool for studying in vivo endothelial biology in any disease model in which endothelial dysfunction, inflammation, or injury plays a role. Our EC-rtTA mice can be intercrossed with any mouse strain carrying a Tre responder gene and would be a valuable tool for conditional gene manipulation in endothelium.
In summary, by using the Tet-on system, we have created double TG mice that conditionally express I-Bmt selectively on vascular endothelium. Our EC-rtTA/I-Bmt mice express no basal but a relatively high level of Dox-inducible I-Bmt on ECs, but not on fibroblasts, macrophages, and whole blood cells. When subjected to endotoxemia or sepsis, EC-rtTA/I-Bmt mice showed endothelial-selective blockade of NF-B activation, repressed expression of multiple endothelial adhesion molecules, reduced neutrophil infiltration into multiple organs, decreased endothelial permeability, ameliorated multiple organ injury, and improved survival. EC-rtTA/I-Bmt mice exhibited significantly reduced systemic hypotension under the condition of endotoxemia. Compared with WT mice, EC-rtTA/I-Bmt mice had an identical capacity to clear three major pathogenic bacteria. These results demonstrate that endothelial NF-B plays divergent roles in the inflammatory/injurious and host defense responses against bacterial infection. Endothelial NF-B activity is critically required for the inflammatory and injurious responses that lead to septic multiple organ inflammation and injury, but it plays little role in the host defense response to eradicate invading pathogenic bacteria.
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MATERIALS AND METHODS |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Bmt mice.
VeCadrtTA and TreI-Bmt transgene constructs are illustrated in Fig. 1 A. The 2.5-kb mouse VE–cadherin-5 promoter was generated by PCR amplification and inserted into a Tet-on plasmid DNA (Clontech Laboratories, Inc.), replacing the CMV promoter. TreI-Bmt was constructed by inserting the full-length of human I-Bmt cDNA (supplied by U. Siebenlist, National Institute of Allergy and Infectious Diseases, Bethesda, MD) into the TRE plasmid DNA (Clontech Laboratories, Inc.). Authenticities of PCR products and DNA constructs were confirmed by DNA sequencing.
The transgenes were linealized, purified, and injected into fertilized FVB mouse eggs at the Albert Einstein College of Medicine Transgenic Core Facility. Potential founders of VE-rtTA and TreI-Bmt mice were identified by Southern blot analysis of genomic DNA from tail biopsies. All founders were bred to homozygous and developed to mouse lines. RT-PCR analysis was performed using RNAs from the organs of VE-rtTA mice to verify rtTA mRNA expression. Dual TG EC-rtTA/I-Bmt mice that carry both the VE-rtTA and TreI-Bmt transgenes were created by intercrossing the VE-rtTA and TreI-Bmt mouse lines.
EC-rtTA/I-Bmt mice were fed without (for control) or with 1.5 mg/ml Dox in the drinking water for 4 d. The dose and duration of Dox were determined in separate time-course and dose–response studies. Total RNAs were extracted from the brain, heart, lungs, aorta, liver, spleen, kidney, stomach, intestine, thyroid, tongue, skeletal muscle, and whole blood cells; digested with RNase-free DNase; and reverse transcribed into cDNAs. The cDNAs (50 ng) were PCR amplified using transgene-specific primers and conditions as follow: 1 cycle of 95°C for 4 min; 35 cycles of 95°C for 45 s, 60°C for 40 s, and 72°C for 45 s; and 1 cycle of 72°C for 10 min.
Animal groups.
We studied four groups of mice: transgene-negative control (WT-Con), transgene-negative LPS (WT-LPS), EC-rtTA/I-Bmt control (TG-Con) and EC-rtTA/I-Bmt LPS (TG-LPS) mice.
Western blotting.
Cytoplasmic, nuclear, and membrane proteins were extracted from cultured cells or various organs of the mice. Equal amounts of protein were separated on 7.5–10% SDS-PAGE, electroblotted onto a polyvinylidene fluoride membrane. Western blotting was performed as we previously described (40), using antibodies against I-B, p65, E-selectin, ICAM-1, VCAM-1, and actin.
Histology and immunofluorescence staining.
For histological examination, 5-µm paraffin-embedded sections were prepared from the lungs, kidney, and liver of WT and TG mice fed with Dox, stained with hematoxylin and eosin, and examined under a light microscope (Optithot; Nikon) with a digital camera (DXM1200; Nikon). For immunofluorescence staining, 8-µm cryosections were prepared from lungs, fixed with paraformaldehyde, permeabilized with Triton X-100 in PBS, and blocked with blocking reagents. Cellular localization of I-Bmt protein was detected using anti–human I-B antibody, which a has low level of cross-reactivity with mouse I-B (Santa Cruz Biotechnology, Inc.), and FITC-conjugated secondary antibody.
Cell isolation and culture.
Vascular ECs and fibroblasts were isolated from the lungs of WT and TG mice according to the protocols previously described (41, 42). Peritoneal macrophages were isolated by peritoneal lavage of the four groups of mice 4 d after intraperitoneal injection of 0.5 ml of thioglycolate medium. ECs were cultured in EC culture medium containing 20% FCS, DMEM, 2 mM L-glutamine, 2 mM sodium pyruvate, 20 mM Hepes, 1% nonessential amino acids, 100 µg/ml streptomycin, 100 U/ml penicillin, 100 µg/ml heparin, and 100 µg/ml EC growth supplement. Fibroblasts were cultured in DME containing 10% FCS. Macrophages were cultured in DMEM containing 10% FCS. EC phenotype was confirmed by positive staining of the endothelial-specific markers platelet/EC adhesion molecule 1 and ICAM-1. Macrophages and fibroblasts were confirmed by staining with Mac3 (macrophages) and procollagen (fibroblasts) antibodies. Subconfluent cells were plated out and incubated with Dox for 48 h. Some ECs and fibroblasts were stimulated with 100 ng/ml TNF- for 15 min, and macrophages were stimulated with 100 ng/ml LPS for 30 min before protein extraction.
Measurement of tissue MPO activity.
We used tissue MPO activity as an indicator of tissue neutrophil infiltration. The lungs, heart, kidney, liver, and intestine were collected from WT-Con, WT-LPS, TG-Con, and TG-LPS mice at 4 h after LPS. Tissue MPO activity was determined, as we previously described (13), and was normalized to tissue weight.
Bronchoaleolar lavage (BAL) and neutrophil counting.
BAL was performed by three intratracheal instillations of 0.5 ml of warmed PBS, followed by gentle aspiration. Total fluids from the three collections were pooled and centrifuged to pellet cells. Cells were resuspended in PBS and centrifuged to cytospin slides, which were stained. Cells were differentially counted.
Assessment of endothelial permeability and organ injury.
Microvascular endothelial permeability was assessed using EBD leakage index as a marker (43, 44). Mice in the WT-Con and TG-Con or in WT-LPS and TG-LPS groups were injected with saline or 10 mg/kg LPS i.p. At 3.5 h after LPS, 20 mg/kg EBD was injected i.v. At 5 h after LPS, mice were injected with 200 U heparin i.v. After blood withdrawal, the organ vasculature was flushed free of blood by gentle infusion of 10 ml of prewarmed PBS through the left ventricle. The lungs, heart, liver, kidney, and intestine were then excised, and weighed before and after being dried at 70°C for 16 h. Dry tissues were homogenized in formamide, incubated at 60°C for 16 h, and centrifuged, and supernatant absorbances at 620 and 740 nm were recorded. Tissue heme pigment contamination was corrected using A740 readings. Tissue EBD content (mg EBD/g fresh tissue) was calculated by comparing tissue supernatant A620 readings with an EBD standard curve. Organ edema was assessed by determining organ wet/dry ratio, which was calculated by dividing the wet weight of each organ by its dry weight.
CLP model of sepsis.
Mice in the WT-sham and TG-sham groups were subjected to sham, and mice in the WT-CLP and TG-CLP groups were subjected to CLP operation, as previously described (45) using an 18-gauge needle. Mice were injected with 20 mg/kg EBD i.v. at 22 h and heparinized at 24 h after operation. Microvascular endothelial permeability in the lungs, heart, liver, kidney, and intestine were assessed as described in the previous section. In separate experiments, WT and TG mice were subjected to CLP, and survival was monitored for up to 14 d.
Monitoring systemic arterial blood pressure.
Mice in each group were anesthetized with 300 mg/kg tribromoethanol i.p., intubated, and ventilated with a mouse ventilator, as we previously described (12). The carotid artery was cannulated with a mouse arterial catheter, which was connected to a pressure transducer connected to a chart recorder. Systemic arterial blood pressure was recorded before and at 4 h after LPS challenge, and compared. Plasma levels of nitrite/nitrate and thrombin/antithrombin were measured using commercial kits.
Bacterial clearance.
Mice in control and bacteria groups were injected with saline or one of the following bacteria: intracellular bacterium (L. monocytogenes), extracellular gram-positive bacterium (S. pneumoniae), and intracellular gram-negative bacterium (S. enterica). To eliminate the effects of Dox, which was used to induce I-Bmt expression in TG mice, all four groups of mice were fed with an identical dose of Dox for 4 d before i.v. injection of bacteria, which was prepared in Dox-containing media.
12 h after S. enterica (108 CFU/mouse) or S. pneumoniae (107 CFU/mouse) injection, or 24 h after L. monocytogenes (108 CFU/mouse) injection, mice were killed by exsanguination. The blood, lungs, liver, spleen, and kidney were collected. Organ homogenates were prepared in a series of 10-fold dilutions aseptically, spread onto culture plates, and incubated in culture media and conditions, as recommended by the American Type Culture Collection. Bacterial colonies formed were counted and expressed as colony-forming units per gram of tissue or per milliliter of blood.
Statistical analysis.
Data were expressed as mean ± SEM. Multiple group comparisons were made using analysis of variance or the Kruskal-Wallis rank test. Comparisons between two groups were analyzed using a Bonferroni-corrected t test or a Mann-Whitney U test. The null hypothesis was rejected at the 5% level.
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Acknowledgments |
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Bmt cDNA, and Drs. S.M. Goyert and P. Wang for helpful discussions. This study was supported by the National Institute of General Medical Science (grant 063907 to S.F. Liu) and in part by the Faculty Award Program of the Feinstein Institute for Medical Research.
The authors have no conflicting financial interests.
Submitted: 9 July 2007
Accepted: 27 February 2008
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REFERENCES |
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TopABSTRACTRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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