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Prepublished online as a Blood First Edition Paper on January 23, 2003; DOI 10.1182/blood-2002-07-2303.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Immunology, The Scripps Research
Institute, La Jolla, CA; and the Molecular Medicine Unit, Beth Israel
Deaconess Medical Center, Boston, MA.
In septic shock, tissue factor (TF) activates blood coagulation,
and cytokines and chemokines orchestrate an inflammatory response. In
this study, the role of Egr-1 in lipopolysaccharide (LPS) induction of TF and inflammatory mediators in vivo was
evaluated using Egr-1+/+ and Egr-1 Septic shock is induced in humans by the
presence of pathogenic bacteria or fungi in the blood and is associated
with a high mortality.1 In Gram-negative sepsis,
lipopolysaccharide (LPS [endotoxin]) is released from Gram-negative
bacteria and activates monocytes, endothelial cells, and epithelial
cells. In sepsis, the procoagulant molecule tissue factor (TF) is
expressed by circulating monocytes, vascular endothelial cells in the
spleen, and epithelial cells in the lungs and kidneys.2-7
TF-dependent activation of coagulation and the resulting intravascular
fibrin deposition contributes to multiorgan failure.8
Administration of anti-TF antibodies reduced mortality in baboon and
mouse models of septic shock.9,10 In addition,
administration of other coagulation inhibitors, such as antithrombin
III, protein C, and tissue factor pathway inhibitor in various animal
models of sepsis, reduced mortality.11-13 Plasminogen
activator inhibitor type 1 (PAI-1) inhibits both types of plasminogen
activators and reduces fibrinolysis.14 PAI-1 activity is
increased in the plasma of patients with Gram-negative sepsis15 and is likely to contribute to intravascular
fibrin deposition. Indeed, inhibition of PAI-1 prevents renal fibrin deposition in endotoxemic rabbits.16 In a mouse model of
endotoxemia, maximal levels of TF mRNA are observed at 8 hours, and
induction is observed in bronchial and tubular epithelial cells in the
lungs and kidneys, respectively.2,5-7 In contrast, LPS
treatment of mice induces maximal PAI-1 mRNA expression at 3 hours, and PAI-1 is expressed by endothelial cells.17,18
In Gram-negative sepsis, LPS triggers an inflammatory response that is
orchestrated by monocytes.19 LPS-stimulated monocytes express a variety of inflammatory mediators, which include tumor necrosis factor alpha (TNF The early growth response (Egr) family of zinc finger
transcription factors includes Egr-1, Egr-2, Egr-3, and
Egr-4.25 Egr-1 is the prototypic member of this family and
is induced in various cell types in response to a variety of
stimuli.26,27 In addition, Egr-2 is induced in activated
peripheral blood monocytes.28 Functional Egr-1 binding
sites have been identified in many genes, including TF, TNF In this study, we used Egr-1 Mice
Model of endotoxemia
Northern blot analysis Total RNA from the kidneys and lungs of mice was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (10 µg) was separated by electrophoresis, transferred to a nylon membrane, and hybridized with cDNA probes specific for mouse Egr-1 (303-1956 bp), TF (226-1044 bp), PAI-1 (1-1085 bp), ICAM-1 (140-887 bp), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Clontech Laboratories, Palo Alto, CA). The cDNA fragments were labeled with [ 32P]-deoxycytidine triphosphate (ICN, Costa Mesa,
CA) using a Prime-It kit (Stratagene Cloning Systems, San Diego, CA).
Bands were visualized by autoradiography.
Enzyme-linked immunosorbent assay (ELISA) Levels of murine TNF, IL-6, and MCP-1 were measured using
commercial ELISA kits (R & D Systems, Minneapolis, MN).
RNAse protection assay (RPA) Total RNA (10 µg) from the kidney and lung was hybridized with mouse cytokine (mCK-3b) and chemokine (mCK-5 and mCK-5b) RNA probes using a Riboquant Multiprobe RPA System (Pharmingen, San Diego, CA), following the manufacturer's instructions. The samples were separated by electrophoresis on 6% Tris borate-EDTA (TBE) urea-containing gels (Invitrogen), dried, and bands visualized by autoradiography. G3PDH and ribosomal protein L32 (L32) were used to monitor loading.In situ hybridization Expression of Egr-1 and TF mRNAs in kidneys and lungs from untreated and LPS-treated C57BL/6 mice were examined by in situ hybridization5 using 35S-labeled sense and antisense RNA riboprobes generated from a plasmid containing mouse TF cDNA (226-1044 bp)5 or from pSKEgr-1 (303-1959 bp), which was kindly provided by Dr V. Lindner (Maine Medical Center Research Institute, Portland).Immunoprecipitation and Western blotting Total protein from the kidneys and lungs was isolated and quantitated as described.35 Total protein (1 mg) from the kidney was immunoprecipitated overnight at 4°C using 300 µL radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz Biotechnology, Santa Cruz, CA), 1:100 dilution of anti-Egr-1 (sc-110) antibody (Santa Cruz Biotechnology), and 40 µL protein A Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ). Immunoprecipitates were collected by centrifugation and washed twice in RIPA buffer with detergent and twice with RIPA buffer without detergent. The final pellet was resuspended in 50 µL 4 × sample buffer, boiled for 5 minutes, and centrifuged at 14 000 rpm for 5 minutes at 4°C. Supernatant (20 µL) was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis using a rabbit anti-Egr-1 antibody (1:1000).LacZ expression LacZ staining of kidneys was performed using a -Galactosidase
Staining Kit (Stratagene Cloning Systems, La Jolla, CA). LPS or
saline-treated Egr-1+/ (LacZ) mice were perfused
intracardially with 6 mL phosphate-buffered saline (PBS)
followed by 6 mL fixing solution (2% formaldehyde, 0.2%
glutaraldehyde in PBS). Kidneys and lungs were cut into pieces approximately 2- to 3-mm thick and fixed for another 30 minutes. After
washing with PBS, the tissue was placed in X-Gal staining solution and
incubated at 37°C for 30 minutes. After staining, the tissue was
washed 3 times with PBS and placed in 70% ethanol. For
microscopic analysis, the tissue was embedded in paraffin and 7- to 8- µm sections were cut and counterstained with eosin. The
cell-type distribution of LacZ expression was analyzed by microscopy.
Images were captured using a digital Nikon camera (Melville, NY).
Histology For the histologic studies, kidneys and lungs from control or LPS-treated mice were fixed with a 10% formaldehyde solution and embedded in paraffin. Sections (3 µm) were stained with hematoxylin and eosin. Leukocytes in the tissues were identified using the napthol AS-D chloroacetate esterase reaction (Sigma Chemical), which stains esterase-positive cells red.36 For quantification of the neutrophil infiltration in the lung, 10 random fields (grid 10 × 10 mm area, × 400 magnification) were counted in LPS-treated Egr-1+/+ (n = 2) and Egr-1/ (n = 2)
mice by an investigator blinded to the groups.
Data analysis Band intensities were quantified by densitometric analyses using a Personal Densitometer and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Statistical analysis was performed using either the 2-tailed unpaired student t test or the log-rank test and differences were determined to be statistically significant at a P value less than .05.
LPS induction of Egr-1, TF, and inflammatory mediators in the kidney We measured the steady-state levels of Egr-1 mRNA in the kidney at various times after administration of LPS to mice. LPS induced a transient increase in Egr-1 mRNA expression that was maximal at one hour (Figure 1A). LPS also induced Egr-1 protein expression (Figure 1B). These data indicate that LPS induces Egr-1 expression in the kidneys of endotoxemic mice.
Local fibrin deposition in glomeruli and in the renal interstitium of the kidney results from increased TF and PAI-1 expression. In addition, inflammation is driven, in part, by the binding of leukocytes to ICAM-1 expressed by endothelial cells. Previous studies have reported that Egr-1 directly or indirectly regulates TF, PAI-1, and ICAM-1.29,31,32,37 We found that LPS induction of Egr-1 mRNA expression preceded the induction of PAI-1, ICAM-1, and TF mRNA expression in the kidneys of mice (Figure 1A). Maximal levels of PAI-1 and ICAM-1 mRNA were observed between 2 and 4 hours, whereas maximal levels of TF mRNA were observed at 8 hours (Figure 1A). Role of Egr-1 in LPS induction of TF, PAI-1, and ICAM-1 expression in the kidney The role of Egr-1 in LPS induction of TF, PAI-1, and ICAM-1 mRNA expression in the kidneys of mice was determined by comparing the levels of expression of these genes in Egr-1/ mice with
those observed in Egr-1+/+ mice. At 3 hours, there was no
difference in the level of LPS-induced TF mRNA expression in the
kidneys of Egr-1+/+ and Egr-1/ mice (Figure
2A). In contrast, at 8 hours, TF mRNA
levels were significantly reduced in Egr-1/ mice
compared with Egr-1+/+ mice (Figure 2B). LPS induced TF
mRNA levels 23.9-fold in the kidneys of Egr-1+/+ mice and
only 2.4-fold in the kidneys of Egr-1/ mice, which
represents a decrease of 86% (P = .008). Egr-1 deficiency did not affect LPS induction of PAI-1 and ICAM-1 mRNA expression at 3 hours (Figure 2C,E) but resulted in a 52% decrease in PAI-1 mRNA
expression and a 52% (P = .047) decrease in ICAM-1 mRNA
expression at 8 hours (Figure 2D,F). These results indicated that Egr-1
does not contribute to LPS induction of TF, PAI-1, and ICAM-1 mRNA expression at 3 hours but contributes to their expression at 8 hours in
the kidneys of endotoxemic mice.
Role of Egr-1 in LPS induction of IL-6 and MCP-1 in the kidney A previous study indicated that Egr-1 regulates the expression of various inflammatory mediators, such as MCP-1, in a murine model of lung ischemia-reperfusion injury.32 We determined whether Egr-1 regulates LPS induction of various cytokine and chemokine mRNAs in the kidneys of endotoxemic mice using RNAse protection assays. LPS induced IL-6 mRNA expression at 3 and 8 hours in the kidneys of Egr-1+/+ mice (Figure 3A). The level of IL-6 mRNA expression was decreased by 57% (P = .047) and 49% (P = .0013) at 3 and 8 hours, respectively, in the kidneys of Egr-1/ mice
injected with LPS compared with the kidneys of Egr-1+/+
mice (Figure 3A). Low levels of transforming growth factor 1 (TGF1) mRNA were detected in the kidneys of endotoxemic
mice, and these were not changed in Egr-1/ mice at 3 hours but reduced at 8 hours (Figure 3A). At 3 hours, LPS
induced only very low levels of TNF mRNA, which was detected in longer exposures, and this expression was not reduced in
Egr-1/ mice (data not shown). We did not detect
expression of TNF, lymphotoxin (LT),
interferon  (IFN), IFN, TGF-2, and
TGF-3 mRNAs.
MCP-1 mRNA expression was strongly induced in the kidneys of
endotoxemic mice (Figure 3B). At 3 hours, LPS induction of MCP-1 mRNA
expression in the kidney was similar in Egr-1+/+ and
Egr-1 Role of Egr-1 in LPS induction of TF and chemokine mRNAs in the lung We next determined whether Egr-1 was required for LPS induction of TF and chemokine mRNAs in the lung by comparing the induction of these genes in Egr-1+/+ and Egr-1/ mice. Previous studies have shown that maximal
levels of TF mRNA are expressed at 8 hours in the lungs of endotoxemic
mice.5,38 Administration of LPS increased the steady-state
levels of Egr-1 mRNA in the lungs with maximal levels at one hour
(Figure 4A). LPS also induced Egr-1
protein expression (Figure 4A). LPS did not induce a significant
increase in the steady-state levels of TF mRNA at 3 hours (data not
shown) but induced a 2.2-fold increase in TF mRNA levels at 8 hours in
the lungs of Egr-1+/+ mice (Figure 4B). Egr-1 deficiency
resulted in a 51% (P = .035) decrease in TF mRNA
expression in the lungs at 8 hours (Figure 4B). In addition, Egr-1
deficiency did not affect the level of MCP-1 mRNA at 3 hours, but its
expression was reduced by 54% (P = .012) at 8 hours
(Figure 4C). The expression of MIP-2, MIP-1, and MIP-1 mRNAs was
also reduced at 8 hours. LPS induction of RANTES mRNA was not affected
by loss of Egr-1 (Figure 4C). These results indicate that Egr-1
contributes to LPS induction of TF and MCP-1 mRNAs at 8 hours but does
not contribute to the induction of RANTES mRNA in the lungs of
endotoxemic mice.
Cell type-specific expression of Egr-1 and TF in the kidneys and lungs of untreated and LPS-treated mice Cell type-specific expression of Egr-1 mRNA in the kidneys and lungs of untreated mice was analyzed by in situ hybridization. In the kidney, Egr-1 mRNA was expressed in the urinary epithelium and by epithelial cells of the distal tubules and Bowman capsule (Figure 5A-B; data not shown). Proximal tubules, glomeruli, and endothelial cells did not express detectable levels of Egr-1 mRNA (Figure 5A-B). In the lung, Egr-1 mRNA was expressed in the bronchial epithelium (Figure 5C). LacZ expression was analyzed in tissues from untreated and LPS-treated Egr-1+/(LacZ)
mice. LacZ was expressed in the urinary epithelium and in epithelial cells of distal tubules and the Bowman capsule in kidneys of untreated and LPS-treated Egr-1+/(LacZ) mice (Figure 5D-E; data not
shown) in the same pattern as Egr-1 mRNA. LacZ expression in epithelial
cells in the kidneys and lungs of mice treated with LPS for 2 hours was
increased compared with untreated mice and no additional cell types
were found to express LacZ (data not shown). LacZ was also expressed in
bronchial epithelium in the lung (Figure 5F). These results indicate
that epithelial cells in the kidney and lung express Egr-1 in untreated
and LPS-treated mice.
Previous studies have shown TF expression in glomeruli but not in tubules in kidneys of humans, baboons, and rabbits.4,7,39 In contrast, TF is predominantly expressed in tubules and not glomeruli in mouse kidneys.2,5 In endotoxemic mice, TF mRNA expression is induced in tubular epithelium.2 In this study, we observed TF mRNA expression in distal tubules and urinary epithelium in kidneys and bronchial epithelium in lungs of untreated and LPS-treated mice (Figure 5G-I; data not shown). TF mRNA expression in epithelial cells was increased in kidneys and lungs from LPS-treated mice compared with untreated mice (data not shown). These data indicate that Egr-1 and TF are coexpressed in epithelial cells in the kidneys and lungs of untreated and LPS-treated mice. Does Egr-1 play a role in the recruitment of leukocytes to the lungs and kidneys in endotoxemic mice? Inflammation in the lungs and kidneys of endotoxemic Egr-1+/+ and Egr-1/ mice was evaluated to
determine whether the absence of Egr-1 resulted in a decrease in the
recruitment of leukocytes. LPS induced a large increase in the number
of leukocytes in the lungs at 8 hours, and similar numbers were
observed in Egr-1+/+ and Egr-1/ mice (3 per
group) (Figure 6A-F). In the kidney, we
observed fewer inflammatory cells compared with the lung, but, again,
similar numbers of leukocytes were observed adhering to the endothelium in endotoxemic Egr-1+/+ and Egr-1/ mice
(Figure 6G-L). Leukocytes in the lung were identified by esterase
staining, which permitted more accurate quantification of numbers
(Figure 7A-C). Similar numbers of
leukocytes were observed in the lungs from endotoxemic
Egr-1+/+ and Egr-1/ mice
(Egr-1+/+ mice, 80 ± 12 leukocytes/10 fields compared
with Egr-1/ mice, 94 ± 7 leukocytes/10 fields).
These data suggest that Egr-1 does not play a significant role in
leukocyte recruitment into the lungs and kidneys at 8 hours.
Effect of Egr-1 deficiency on the expression of inflammatory mediators in the systemic circulation and on the survival of mice in a model of endotoxemia We have shown that Egr-1 regulates the expression of TF and inflammatory mediators in the kidneys and lungs of endotoxemic mice. However, it is not known whether an absence of Egr-1 will affect the levels of circulating inflammatory mediators during endotoxemia. The expression of cytokines and a chemokine in the systemic circulation was determined in Egr-1+/+ and Egr-1/ mice
injected intraperitoneally with a high dose of LPS. Maximal levels of
TNF in plasma were observed at one hour in endotoxemic mice, and
levels were not different in Egr-1+/+ and
Egr-1/ mice (Figure 8).
Similar levels of IL-6 and MCP-1 were observed in endotoxemic
Egr-1+/+ and Egr -1/ mice between 0 and 6 hours, but MCP-1 levels were reduced in Egr-1/ mice at
8 hours by 42% (Figure 8). These data indicate that Egr-1 does not
contribute to the early expression of these inflammatory mediators in
the systemic circulation but contributes to MCP-1 expression at 8 hours
in a model of endotoxemia.
Finally, we determined the survival of Egr-1+/+ and
Egr-1
In this study, we showed that Egr-1 mRNA expression is rapidly and
transiently induced in the kidneys and lungs of endotoxemic mice with
maximal levels at one hour. LPS induction of Egr-1 mRNA in these organs
preceded the induction of mRNAs for the inflammatory mediators IL-6,
MCP-1, and ICAM-1, which are maximal at 3 hours, and TF mRNA, which is
maximal at 8 hours. We observed several different patterns of
Egr-1-dependent and Egr-1-independent gene expression. First, LPS
induction of IL-6 mRNA expression in the kidney was reduced at both 3 and 8 hours in Egr-1 Our data indicate that Egr-1 does not contribute to the expression of
the majority of inflammatory mediators in the early inflammatory
response in endotoxemic mice. IL-6 expression at 3 hours is an
exception because it is mediated, in part, by Egr-1. A recent study
reported that LPS induction of IL-6 expression in epithelial cells was
mediated by a nuclear factor- This study shows that Egr-1 is required for the sustained expression of
inflammatory mediators in endotoxemic mice. Unlike NF- Analysis of the cell type-specific expression of Egr-1 demonstrated
that Egr-1 was expressed by tubular epithelial cells in the kidneys and
bronchial epithelial cells in the lungs of untreated and LPS-treated
mice. Importantly, this study and previous studies showed that TF was
also expressed in tubular epithelial cells and bronchial epithelial
cells but not in endothelial cells in the kidneys and lungs,
respectively, of untreated and LPS-treated mice.2,5 Other
studies have shown TF expression in epithelial cells in the kidneys and
lungs of endotoxemic baboons and rabbits.4,7 Attempts to
show reduced TF mRNA expression in epithelial cells in the kidneys and
lungs of LPS-treated Egr-1 Epithelial cells express both CD14 and the LPS receptor toll-like receptor 4 (TLR4),48,49 which would permit the cells to respond directly to LPS. Indeed, one study using biotin-labeled LPS showed that LPS directly binds to epithelial cells.50 TLR4 expression by epithelial cells in the kidney is functionally important because C3H/HeJ mice, which express a dominant-negative form of TLR4, are more susceptible to infection by E coli than wild-type C3H/HeN mice.51 Our studies showing LPS induction of Egr-1 in epithelial cells suggest that Egr-1 may play a role in the innate immune response to microbial pathogens invading the body through epithelial surfaces.
We would like to acknowledge excellent technical assistance from M. Szeto, M. Tencati, and T. Holscher; preparation of the manuscript by C. Johnson; and critical reading of the manuscript by E. Adamson.
Submitted July 31, 2002; accepted December 30, 2002.
Prepublished online as Blood First Edition Paper, January 23, 2003; DOI 10.1182/blood- 2002-07-2303.
Supported by grants HL48872 and HL65226 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Nigel Mackman, The Scripps Research Institute, Departments of Immunology and Cell Biology, 10550 N Torrey Pines Rd, C-204, La Jolla, CA 92037; e-mail: nmackman{at}scripps.edu.
1.
Parrillo JE.
Pathogenetic mechanisms of septic shock.
N Engl J Med.
1993;328:1471-1477 2. Yamamoto K, Loskutoff DJ. Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator. J Clin Invest. 1996;97:2440-2451[Medline] [Order article via Infotrieve]. 3. Semeraro N, Triggiani R, Montemurro P, Cavallo LG, Colucci M. Enhanced endothelial tissue factor but normal thrombomodulin in endotoxin-treated rabbits. Thromb Res. 1993;71:479-486[CrossRef][Medline] [Order article via Infotrieve]. 4. Drake TA, Cheng J, Chang A, Taylor FB Jr. Expression of tissue factor, thrombomodulin, and E-selectin in baboons with lethal Escherichia coli sepsis. Am J Pathol. 1993;142:1-13. 5. Mackman N, Sawdey MS, Keeton MR, Loskutoff DJ. Murine tissue factor gene expression in vivo: tissue and cell specificity and regulation by lipopolysaccharide. Am J Pathol. 1993;143:76-84[Abstract]. 6. Mackman N. Regulation of the tissue factor gene. FASEB J. 1995;9:883-889[Abstract].
7.
Erlich JH, Fearns C, Mathison J, Ulevitch RJ, Mackman N.
Lipopolysaccharide induction of tissue factor expression in rabbits.
Infect Immun.
1999;67:2540-2546
8.
Levi M, ten Cate H.
Disseminated intravascular coagulation.
N Engl J Med.
1999;341:586-592 9. Taylor FB Jr, Chang A, Ruf W, et al. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock. 1991;33:127-134[Medline] [Order article via Infotrieve]. 10. Dackiw APB, McGilvray ID, Woodsie M, Nathens AB, Marshall JC, Rotstein OD. Prevention of endotoxin-induced mortality by antitissue factor immunization. Arch Surg. 1996;131:1273-1279[Abstract]. 11. Thijs LG. Coagulation inhibitor replacement in sepsis is a potentially useful clinical approach. Crit Care Med. 2000;28:S68-S73[CrossRef][Medline] [Order article via Infotrieve]. 12. Hack CE. Tissue factor pathway of coagulation in sepsis. Crit Care Med. 2000;28:S25-S30[CrossRef][Medline] [Order article via Infotrieve]. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
) and Egr-1
) mice. Data
are shown as mean ± SE. The asterisk indicates that the change in
MCP-1 is statistically significant (P < .05).
B (NF-