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RAGE and the innate immune response in infection and inflammation
van Zoelen, M.A.D.
Publication date 2009
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Citation for published version (APA):
van Zoelen, M. A. D. (2009). RAGE and the innate immune response in infection and inflammation.
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RAGE and the innate
immune response in infection
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M. A .D . v an Z oe le nRAGE
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the innate immune response in
infection and inflammation
RAGE and the the innate immune response in infection and inflammation Dissertation, University of Amsterdam, the Netherlands
Copyright © 2009, Marieke A.D. van Zoelen, Amsterdam, the Netherlands
All rights reserved. No part of this publicatn may be reproduced or transmitted in any form by any means, electronic or mechanical, including photocopy, recording or any information storage and retrieval system, without written permission of the author. Cover figures. RAGE staining of a murine lung.
Author: Marieke A.D. van Zoelen
Printed by: GVO drukkers en vormgevers B.V. | Ponsen & Looijen Financial support:
Tergooiziekenhuizen lokatie Hilversum, Hycult Biotechnology, J.E. Jurriaanse Stichting, Universiteit van Amsterdam, Beun-de Ronde
RAGE
and
the innate immune response in
infection and inflammation
Academisch proefschrift
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. D. C. van den Boom
ten overstaan van een door het college
voor promoties ingestelde commissie,
in het openbaar te verdedigen in de Agnietenkapel
op woensdag 1 juli 2009, te 14.00 uur
door
Marieke Antonia Dorothea van Zoelen
Promotiecommissie
Promotor: Prof. dr. T. van der Poll Overige leden: Prof. dr. D.J. Gouma
Prof. dr. E. de Jonge Prof. dr. M. M. Levi Prof. dr. P. Speelman Prof. dr. J. Roth Prof. dr. P.P. Tak Faculteit der Geneeskunde
121
141
ContEnts
1. General introduction and outline of the thesis 9
Part I: RAGE and infection
2. Receptor for advanced glycation end products facilitates host
defense during Escherichia coli induced abdominal sepsis in mice. 23 Journal of Infectious Diseases, 2009
3. The receptor for advanced glycation end products impairs host defense in
pneumococcal pneumonia. 43
Journal of Immunology, 2009
4. Receptor for advanced glycation end products protects against
Klebsiella pneumoniae induced pneumonia in mice. 67
Submitted
5. Receptor for advanced glycation end products is protective
during murine tuberculosis. 85
Submitted
6. Receptor for advanced glycation end products is detrimental during
influenza A virus pneumonia. 101
Virology, 2009
Part II: soluble RAGE, RAGE ligands and damage-
associated molecular patterns
7. Ligands of the receptor for advanced glycation end products, including high mobility group box (HMGB) 1, limit bacterial dissemination during Escherichia coli peritonitis.
Under revision
8. Systemic and local high mobility group box 1 concentrations during severe infection.
ventilation and ventilator-associated pneumonia. Shock, 2008
10. Role of Toll-like receptors 2 and 4, and the receptor for advanced glycation end products in high mobility group box 1 induced inflammation in vivo. Shock, 2008
11. Expression and role of myeloid-related protein-14 in clinical and experimental sepsis.
American Journal of Respiratory and Critical Care Medicine, 2009
Part III: soluble RAGE levels during inflammation
12. S100A12 and soluble RAGE levels during severe infection. Submitted
13. Neutrophil-derived S100A12 in acute lung injury and respiratory distress syndrome.
Critical Care Medicine, 2007
14. Urokinase plasminogen activator receptor deficient mice demonstrate reduced hyperoxia induced lung inury.
American Journal of Pathology, 2009
15. Endogenous monocyte chemoattractant protein-1 promotes lung inflammation induced by LPS and LTA.
Submitted
16. Summary, general discussion and future perspectives for research and therapy
Nederlands samenvatting voor niet-ingewijden Acknowledgements/Dankwoord List of publications List of contributors 159 173 185 207 219 235 253 271 281 287 290 292
1
General introduction and outline of
the thesis
Marieke A.D. van Zoelen1
1Center for Infection and Immunity Amsterdam (CINIMA), Center for Experimental and Molecular Medicine (CEMM), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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p
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Introduction
1. Receptor for advanced glycation end products (RAGE) and alarmins
1.1 Receptor for advanced glycation end products (RAGE)
The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily that is expressed at a high level in the lungs and at a low level in normal adult tissues, including on cells involved in the innate immune system, e.g. neutrophils, T and B lymphocytes, monocytes, macrophages, dendritic cells and endothelial cells (1-3). RAGE is composed of 3 immunoglobulin-like regions, a transmembrane domain, and a highly charged short cytosolic tail that is essential for intracellular signaling (Figure 1). The V domain in the extracellular part of the receptor is critical for ligand binding and interacts with a diverse class of ligands due to its ability to recognize 3-dimensional structures rather than specific amino acid sequences (4). Originally, RAGE was identified as a receptor for advanced glycation end products (AGEs), explaining the choice of its name. AGEs are products of non-enzymatic glycation and oxidation of proteins, lipids and other macromolecules that appear, in particular, under conditions of increased availability of reducing sugars and/or enhanced oxidative stress, especially when molecules turn over slowly and aldose levels are elevated (5, 6).
Figure 1. Schematic depiction of RAGE-ligand interaction showing domains of the receptor.
The V-type domain is critical for binding of RAGE ligands. Deletion of the cytosolic tail results in a modified form of RAGE that binds ligands, remains firmly embedded in the cell membrane, but does not transmit RAGE-mediated cellular activation (from (7)).
Ongoing studies revealed, however, that RAGE is able to recognize a wide range of endogenous molecules that are released during various conditions of inflammation and/or injury. The emerging concept of pattern recognition involves RAGE and Toll-like receptors (TLRs) in sensing not only “pathogen–associated molecular patterns” (PAMPs) but also (endogenous) damage-associated molecular patterns (DAMPs) (8, 9). This “novel” group of molecules has been introduced as important pro-inflammatory factors of innate immunity. Because of their release by activated or damaged cells under conditions of cell stress, the terms “endokines” or “alarmins” are used (10). Infection commonly is associated with the release of these endogenous proteins that serve to warn the host for eminent danger. Examples of alarmins and established ligands for RAGE are high mobility group box 1 (HMGB1) (11, 12) and some members of the S100 family (e.g. S100A12 (13)). Other examples of alarmins of the S100 family are S100A8 (myeloid-related protein 8, MRP8) and S100A9 (MRP14).
Ligand binding to RAGE leads to sustained receptor-dependent signaling and activation of nuclear factor-κB and mitogen-activated protein kinase pathways. Inhibition of RAGE signaling has been found to reduce inflammatory responses in an infection model of cecal ligation and puncture (CLP (14)) and several other (non-infectious) models of hepatic injury (15-17), diabetic atherosclerosis (18, 19), delayed type hypersensitivity (14, 20) and type II collagen induced arthritis (20). In chapter 2, we investigated the role of RAGE during abdominal sepsis induced by one of the major causative pathogens, Escherichia coli (E. coli), focusing on the outgrowth of bacteria at the primary site of infection and the subsequent dissemination, and the accompanying systemic inflammatory response syndrome. Given the ubiquitous expression of RAGE in the lungs, it is likely that this receptor plays a role in the regulation of lung inflammation. In chapter 3-6, we studied the role of RAGE during pneumonia caused by (myco)bacteria or influenza A.
1.2 High mobility group box 1 (HMGB1)
HMGB1 is a nuclear protein that for a long time was studied for its intranuclear functions only. During the last few years HMGB1 has been discovered to have multiple extracellular functions and to be a damage-associated molecular pattern (DAMP). This re-discovery of HMGB1 as an extracellular protein with cytokine-like activity (Figure 2) and its pivotal role as a late mediator of experimental sepsis have prompted clinical studies to characterize the release of HMGB1 in patients with sepsis. RAGE ligands in general, and HMGB1 in particular, have been implicated in the pathogenesis of abdominal sepsis. RAGE deficiency or administration of anti-HMGB1 antibodies improved survival in a model of abdominal polymicrobial sepsis induced by CLP (14, 21-23). Chapter 7 describes the expression and role of HMGB1 in experimental abdominal E. coli sepsis; furthermore, we showed the effects of soluble RAGE on the host defense during abdominal sepsis in that chapter. In chapter 8 we sought to determine the extent of systemic HMGB1 release in patients with
severe sepsis stratified to the three most common primary sources of infection (lungs, abdomen and urinary tract) and to examine local HMGB1 levels, at the site of infection, in patients with peritonitis and pneumonia. In chapter 9 we investigated the local release of HMGB1 in the pulmonary compartment during mechanical ventilation and ventilator-associated pneumonia (VAP). HMGB1 has been reported to transduce cellular signals in vitro by interacting with at least three receptors: TLR2, TLR4 and the RAGE (Figure 3). Chapter 10 describes a study in which we tried to determine the role of these pattern recognition receptors in the in vivo responses to HMGB1.
Figure 2. HMGB1 induces pro-inflammatory responses at various sites via activation of innate
1.3 S100 family/ Myeloid-related proteins
S100 proteins comprise a family with more than 20 members, from which at least three have been linked to innate immune functions by their expression by myeloid cells: S100A8 (also called calgranulin A or myeloid-related protein (MRP) 8), S100A9 (calgranulin B or MRP14) and S100A12 (calgranulin C or MRP6). S100A12 has been shown to exhibit its pro-inflammatory activities via interaction with RAGE (13). It has not been experimentally proven that all S100 proteins do so. MRP8 and -14 form heterodimers, which are the biologically relevant forms of these proteins (26-28). MRP8/14 complexes induce a variety of inflammatory reactions and the extent of MRP8/14 expression correlates with disease activity in several inflammatory disorders (29, 30). In chapter 11 we aimed to determine the extent of systemic MRP8/14 release in patients with severe sepsis and to examine local MRP8/14 levels at the site of infection in patients with peritonitis. In addition, we determined the role of MRP8/14 in specific host responses to murine E. coli abdominal sepsis in this chapter. S100A12 (or MRP6), a high-affinity RAGE ligand, has been described as a biomarker of neutrophil activation in inflammatory diseases, including pulmonary infections and sepsis (31, 32). In chapter 12, circulating S100A12 and sRAGE levels during sepsis and human endotoxemia are described. Because neutrophil activation is a hallmark of acute lung injury (ALI), we hypothesized that S100A12 may trigger innate immune processes during
Figure 3. Signaling pathways downstream of RAGE,
TLR4 and TLR2 that mediate the effects of HMGB1 (from (25)).
the acute respiratory distress syndrome (ARDS). We therefore analyzed pulmonary S100A12 and RAGE in patients with ARDS and in subjects instilled with endotoxin in chapter 13.
1.4 Soluble RAGE
Soluble RAGE (sRAGE), a truncated form of full length RAGE, is composed of only the extracellular ligand-binding domain (V-C-C’) lacking the cytosolic and transmembrane domains (i.e., the parts that transfer a signal into the cell, Figure 1) and therefore circulates in plasma. By competing with cell-surface RAGE for ligand binding, sRAGE may contribute to the removal/neutralization of circulating ligands, thus functioning as a decoy. For this reason, recombinant sRAGE has been extensively (and successfully) tested in several animal models for the treatment of RAGE-mediated diseases, including type II collagen induced arthritis (20), delayed-immune hypersensitivity (DTH) (14) and diabetic atherosclerosis (18, 19). Others suggested that sRAGE, besides being a decoy receptor, also has immunomodulating activity of its own. Clinical studies have recently shown that higher plasma levels of sRAGE are associated with a reduced risk of coronary artery disease, hypertension, the metabolic syndrome, arthritis and Alzheimer’s disease. In chapter 12 we described sRAGE levels in patients with sepsis and in healthy volunteers intravenously challenged with lipopolysaccharide (LPS). In addition, we showed local sRAGE levels in ARDS patients and in healthy volunteers after LPS inhalation in chapter 13.
sRAGE had been recently described as a marker of lung injury based on experimental studies in rats and in patients with acute lung injury (33). In chapter 14 and 15 respectively, we tried to establish whether lung injury induced by hyperoxia and LPS was associated with increased levels of sRAGE in the bronchoalveolar space. In chapter 14, we furthermore investigated the effects of urokinase plasminogen activator receptor deficiency during hyperoxia induced lung injury. Chapter 15 describes the role of endogenous monocyte chemoattractant protein-1 during LPS and lipoteichoic acid induced lung inflammation.
2. Infectious and inflammatory diseases
studied in this thesis
2.1 Sepsis
Severe sepsis remains a major challenge in the care of critically ill patients. The outcome is poor and mortality rates remain up to 30-40%. Recent insights into the molecular mechanisms responsible for the pathogenesis of the sepsis syndrome shows that sepsis
is associated with the activation of multiple inflammatory pathways, including the cytokine network and the coagulation system. Pro- and anti-inflammatory pathways are simultaneously activated. Subsequently, cellular activation involving leukocyte-endothelial cell interactions occurs leading to expression of membrane surface molecules such as TLRs, adhesion molecules and cytokine receptors. Furthermore, sepsis leads to a hemostatic imbalance which can result in disseminated intravascular coagulation. While any bacterial infection can theoretically progress and cause systemic inflammation, the respiratory tract and the abdominal cavity are the most frequent sources of sepsis (Figure 4) (34, 35). In chapter 8, 11 and 12 we determined the extent of systemic HMGB1, MRP8/14, S100A12 and sRAGE release in patients with severe sepsis and their subgroups peritonitis, pneumonia and urinary tract infection as primary infections. We also examined local levels of these parameters at the site of infection in patients with peritonitis in these chapters.
Figure 4.
Sites of primary infection in patients with sepsis. Adapted from (35).
2.2 Peritonitis
Peritonitis is most often caused by the presence of bacteria in the otherwise germ-free peritoneal cavity (Figure 5) and is caused predominantly by the perforation of intestines. Acute bacterial peritonitis is a potentially life-threatening disease with a mortality rate ranging between 30% and 50%. Furthermore, peritonitis is the second most common cause of sepsis and the mortality of peritonitis-induced sepsis can be as high as 80%. Therefore we investigated the role of RAGE and the DAMPs HMGB1 and MRP8/14 in the inflammatory response to abdominal sepsis (chapter 2, 7 and 11). The most common causative organisms are enteric gram-negative bacteria. Since Escherichia (E.) coli is found in 60% of the cases (36) (Table 1), we used E. coli to induce abdominal sepsis in mice.
2.3 Endotoxemia
Systemic inflammation and sepsis are leading causes of mortality worldwide. The systemic spread of endotoxin (LPS) derived from gram-negative bacteria is considered one of the most important correlates of sepsis. Intravenous injection of LPS results in activation of several inflammatory pathways, thereby mimicking – in a qualitative way – the systemic
Figure 5. Peritoneal cavity. Table 1. Most frequent gram-negative bacteria isolated from
intra-abdominal infections (from (37)).
host response seen in sepsis. As such, this model allows for a close examination of the dynamic change in mediators, receptors and cells during systemic inflammation in humans. Therefore, we used this human model in chapters 8, 11 and 12.
2.4 Respiratory tract infections
Infections of the respiratory tract are the 7th leading cause of mortality in the United States
and bacterial pneumonia is the most frequent source of sepsis (34, 38-40). According to the acquisition of pneumonia and the pathogens involved, community-acquired pneumonia (CAP) can be distinguished from hospital-acquired pneumonia (HAP, also called nosocomial pneumonia). While the gram-positive bacterium Streptococcus pneumoniae is the single most frequent pathogen causing CAP, responsible for up to 60% of cases, Klebsiella pneumoniae, Haemophilus influenzae and Staphylococcus aureus are isolated in about 10% each (41-43). Pneumococcal pneumonia causes more than 150,000 hospitalizations in the United States annually and Streptococcus pneumoniae is the most frequent cause of lethal CAP (44, 45). Due to this high clinical importance and worldwide significance, we investigated the role of RAGE and the local release of HMGB1 during this particular disease in chapter 3 and 8, respectively. Klebsiella pneumoniae is a gram-negative opportunistic bacterium, which is an important cause of HAP and sepsis. We used this bacterium to study the role of RAGE in gram-negative pneumonia in mice in chapter 4. One of the most dramatic manifestations of chronic lung inflammation is tuberculosis. Chapter 5 describes the impact of RAGE deficiency on the immune response to instillation of live virulent Mycobacterium tuberculosis in mice. Finally, we studied the role of RAGE in pneumonia caused by influenza A virus in chapter 6. Recent outbreaks of highly pathogenic influenza A virus infections have had important economic consequences and the notion that a new influenza pandemic might occur in
Peritonitis Pathogen % E. coli 60 Enterobacter/Klebsiella 26 Proteus 22 Pseudomonas 8
the near future has triggered renewed interest in influenza infection. Furthermore, we induced sterile lung inflammation by instilling LPS, a constituent of the gram-negative bacterial cell wall, or LTA, a component of the gram-positive cell wall in mice (chapter 4 and 15) and in humans (chapter 13).
2.5 Mechanical ventilation
The majority of patients at the intensive care unit are intubated and mechanically ventilated. Although mechanical ventilation certainly is a cornerstone of intensive care management, mechanical ventilation may also aggravate pre–existing lung injury or even initiate pulmonary damage in patients without lung injury at the start of mechanical ventilation (a phenomenon referred to as ventilator–associated lung injury (46, 47)). In addition, mechanical ventilation puts patients at risk for a nosocomial infection, so called ventilator-associated pneumonia (VAP) (48). Similar to ALI/ARDS and pneumonia, ventilator–associated lung injury is associated with local production of pro-inflammatory mediators. In chapter 9 we investigated the local release of HMGB1 in the pulmonary compartment during mechanical ventilation and VAP.
2.6 Hyperoxia induced lung injury
Patients with ALI or ARDS commonly receive supportive care consisting of low tidal volume protective mechanical ventilation (see above) and the administration of high concentrations of inspired oxygen (49, 50). Prolonged exposure to high oxygen concentrations, however, can worsen or induce lung damage in already injured or previously healthy lungs (51). Hyperoxia induced lung injury is characterized by infiltration of neutrophils in the lungs. In chapter 14 we determined the expression and role of urokinase plasminogen activator receptor (uPAR) during hyperoxia induced lung injury. In this study, we determined local sRAGE levels in bronchoalveolar lavage fluid.
Aim and outline of this thesis
The general aim of this thesis is to enhance our knowledge and insights into the specific role of RAGE and RAGE ligands, as well as of other damage-associated molecular patterns, in the innate immune response during severe infection in order to identify potential new treatment targets for these diseases.
In chapter 2 we investigated the role of RAGE in the inflammatory responses and host defense against E. coli peritonitis. We studied the role of RAGE in pneumonia caused by Streptococcus pneumoniae, Klebsiella pneumoniae, Mycoplasma tuberculosis and influenza A virus in chapters 3-6. Chapter 7 reports on the effects of treatment of sRAGE and anti-HMGB1 antibodies on the immune response during E. coli peritonitis. In chapter 8 we sought to determine the extent of systemic HMGB1 release in patients with severe sepsis
stratified to the three most common primary sources of infection (lungs, abdomen and urinary tract) and to examine local HMGB1 levels, at the site of infection, in patients with peritonitis and pneumonia. Chapter 9 focuses on the local release of HMGB1 in the pulmonary compartment during mechanical ventilation and VAP. Chapter 10 addresses the role of TLRs 2 and 4 and RAGE in the in vivo response to HMGB1. In chapter 11 we studied the role of MRP8/14 (or S100A8/A9) during E. coli induced abdominal sepsis. Chapter 12 characterizes systemic S100A12 and sRAGE concentrations during severe infection and human endotoxemia and in chapter 13 these parameters were analyzed in patients with ARDS and in healthy subjects after inhalation of LPS. Chapter 14 reports on sRAGE levels and the role of uPAR during hyperoxia induced lung injury. Lastly, chapter 15 addresses sRAGE levels and the role of endogenous MCP-1 during LPS and LTA induced lung inflammation.
References
1. Bierhaus, A., D. M. Stern, and P. P. Nawroth. 2006. RAGE in inflammation: a new therapeutic target? Curr. Opin. Investig. Drugs 7:985-991.
2. Chen, Y., S. S. Yan, J. Colgan, H. P. Zhang, J. Luban, A. M. Schmidt, D. Stern, and K. C. Herold. 2004. Blockade of late stages of autoimmune diabetes by inhibition of the receptor for advanced glycation end products. J. Immunol. 173:1399-1405.
3. Moser, B., D. D. Desai, M. P. Downie, Y. Chen, S. F. Yan, K. Herold, A. M. Schmidt, and R. Clynes. 2007. Receptor for advanced glycation end products expression on T cells contributes to antigen-specific cellular expansion in vivo. J. Immunol. 179:8051-8058. 4. Raucci, A., S. Cugusi, A. Antonelli, S. M. Barabino, L. Monti, A. Bierhaus, K. Reiss, P.
Saftig, and M. E. Bianchi. 2008. A soluble form of the receptor for advanced glycation endproducts (RAGE) is produced by proteolytic cleavage of the membrane-bound form by the sheddase a disintegrin and metalloprotease 10 (ADAM10). FASEB J. 22:3716-3727.
5. Schlueter, C., S. Hauke, A. M. Flohr, P. Rogalla, and J. Bullerdiek. 2003. Tissue-specific expression patterns of the RAGE receptor and its soluble forms--a result of regulated alternative splicing? Biochim. Biophys. Acta 1630:1-6.
6. Bierhaus, A., P. M. Humpert, M. Morcos, T. Wendt, T. Chavakis, B. Arnold, D. M. Stern, and P. P. Nawroth. 2005. Understanding RAGE, the receptor for advanced glycation end products. J. Mol. Med. 83:876-886.
7. Schmidt, A. M., S. D. Yan, S. F. Yan, and D. M. Stern. 2001. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J. Clin. Invest 108:949-955.
8. Bianchi, M. E. 2007. DAMPs, PAMPs and alarmins: all we need to know about danger. J. Leukoc. Biol. 81:1-5.
9. Hofmann, M. A., S. Drury, C. Fu, W. Qu, A. Taguchi, Y. Lu, C. Avila, N. Kambham, A. Bierhaus, P. Nawroth, M. F. Neurath, T. Slattery, D. Beach, J. McClary, M. Nagashima, J. Morser, D. Stern, and A. M. Schmidt. 1999. RAGE mediates a novel proinflammatory axis: a central cell surface receptor for S100/calgranulin polypeptides. Cell 97:889-901. 10. Oppenheim, J. J. and D. Yang. 2005. Alarmins: chemotactic activators of immune responses.
Curr. Opin. Immunol. 17:359-365.
11. Sorci, G., F. Riuzzi, C. Arcuri, I. Giambanco, and R. Donato. 2004. Amphoterin stimulates myogenesis and counteracts the antimyogenic factors basic fibroblast growth factor and S100B via RAGE binding. Mol. Cell Biol. 24:4880-4894.
12. Hori, O., J. Brett, T. Slattery, R. Cao, J. Zhang, J. X. Chen, M. Nagashima, E. R. Lundh, S. Vijay and D. Nitecki. 1995. The receptor for advanced glycation end products (RAGE) is a cellular binding site for amphoterin. Mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system. J. Biol. Chem. 270:25752-25761.
13. Moroz, O. V., A. A. Antson, E. J. Dodson, H. J. Burrell, S. J. Grist, R. M. Lloyd, N. J. Maitland, G. G. Dodson, K. S. Wilson, E. Lukanidin, and I. B. Bronstein. 2002. The structure of S100A12 in a hexameric form and its proposed role in receptor signalling. Acta Crystallogr. D. Biol. Crystallogr. 58:407-413.
14. Liliensiek, B., M. A. Weigand, A. Bierhaus, W. Nicklas, M. Kasper, S. Hofer, J. Plachky, H. J. Grone, F. C. Kurschus, A. M. Schmidt, S. D. Yan, E. Martin, E. Schleicher, D. M. Stern, G. G. Hammerling, P. P. Nawroth, and B. Arnold. 2004. Receptor for advanced glycation end products (RAGE) regulates sepsis but not the adaptive immune response. J. Clin. Invest 113:1641-1650.
15. Cataldegirmen, G., S. Zeng, N. Feirt, N. Ippagunta, H. Dun, W. Qu, Y. Lu, L. L. Rong, M. A. Hofmann, T. Kislinger, S. I. Pachydaki, D. G. Jenkins, A. Weinberg, J. Lefkowitch, X. Rogiers, S. F. Yan, A. M. Schmidt, and J. C. Emond. 2005. RAGE limits regeneration after massive liver injury by coordinated suppression of TNF-alpha and NF-kappaB. J. Exp. Med. 201:473-484.
16. Zeng, S., N. Feirt, M. Goldstein, J. Guarrera, N. Ippagunta, U. Ekong, H. Dun, Y. Lu, W. Qu, A. M. Schmidt, and J. C. Emond. 2004. Blockade of receptor for advanced glycation end product (RAGE) attenuates ischemia and reperfusion injury to the liver in mice. Hepatology 39:422-432.
17. Ekong, U., S. Zeng, H. Dun, N. Feirt, J. Guo, N. Ippagunta, J. V. Guarrera, Y. Lu, A. Weinberg, W. Qu, R. Ramasamy, A. M. Schmidt, and J. C. Emond. 2006. Blockade of the receptor for advanced glycation end products attenuates acetaminophen-induced hepatotoxicity in mice. J. Gastroenterol. Hepatol. 21:682-688.
18. Park, L., K. G. Raman, K. J. Lee, Y. Lu, L. J. Ferran, Jr., W. S. Chow, D. Stern, and A. M. Schmidt. 1998. Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts. Nat. Med. 4:1025-1031.
19. Goova, M. T., J. Li, T. Kislinger, W. Qu, Y. Lu, L. G. Bucciarelli, S. Nowygrod, B. M. Wolf, X. Caliste, S. F. Yan, D. M. Stern, and A. M. Schmidt. 2001. Blockade of receptor for advanced glycation end-products restores effective wound healing in diabetic mice. Am. J. Pathol. 159:513-525.
20. Hofmann, M. A., S. Drury, B. I. Hudson, M. R. Gleason, W. Qu, Y. Lu, E. Lalla, S. Chitnis, J. Monteiro, M. H. Stickland, L. G. Bucciarelli, B. Moser, G. Moxley, S. Itescu, P. J. Grant, P. K. Gregersen, D. M. Stern, and A. M. Schmidt. 2002. RAGE and arthritis: the G82S polymorphism amplifies the inflammatory response. Genes Immun. 3:123-135.
21. Lutterloh, E. C., S. M. Opal, D. D. Pittman, J. C. Keith, Jr., X. Y. Tan, B. M. Clancy, H. Palmer, K. Milarski, Y. Sun, J. E. Palardy, N. A. Parejo, and N. Kessimian. 2007. Inhibition of the RAGE products increases survival in experimental models of severe sepsis and systemic infection. Crit Care 11:R122.
22. Yang, H., M. Ochani, J. Li, X. Qiang, M. Tanovic, H. E. Harris, S. M. Susarla, L. Ulloa, H. Wang, R. DiRaimo, C. J. Czura, H. Wang, J. Roth, H. S. Warren, M. P. Fink, M. J.
Fenton, U. Andersson, and K. J. Tracey. 2004. Reversing established sepsis with antagonists of endogenous high-mobility group box 1. Proc. Natl. Acad. Sci. U. S. A 101:296-301. 23. Qin, S., H. Wang, R. Yuan, H. Li, M. Ochani, K. Ochani, M. Rosas-Ballina, C. J. Czura,
J. M. Huston, E. Miller, X. Lin, B. Sherry, A. Kumar, G. Larosa, W. Newman, K. J. Tracey, and H. Yang. 2006. Role of HMGB1 in apoptosis-mediated sepsis lethality. J. Exp. Med. 203:1637-1642.
24. Wang, H., H. Yang, and K. J. Tracey. 2004. Extracellular role of HMGB1 in inflammation and sepsis. J. Intern. Med. 255:320-331.
25. Lotze, M. T. and K. J. Tracey. 2005. High-mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat. Rev. Immunol. 5:331-342.
26. Vogl, T., J. Roth, C. Sorg, F. Hillenkamp, and K. Strupat. 1999. Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 detected by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry. J. Am. Soc. Mass Spectrom. 10:1124-1130.
27. Vogl, T., S. Ludwig, M. Goebeler, A. Strey, I. S. Thorey, R. Reichelt, D. Foell, V. Gerke, M. P. Manitz, W. Nacken, S. Werner, C. Sorg, and J. Roth. 2004. MRP8 and MRP14 control microtubule reorganization during transendothelial migration of phagocytes. Blood 104:4260-4268.
28. Vogl, T., K. Tenbrock, S. Ludwig, N. Leukert, C. Ehrhardt, M. A. van Zoelen, W. Nacken, D. Foell, T. van der Poll, C. Sorg, and J. Roth. 2007. Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat. Med. 13:1042-1049.
29. Foell, D., H. Wittkowski, T. Vogl, and J. Roth. 2007. S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J. Leukoc. Biol. 81:28-37. 30. Foell, D. and J. Roth. 2004. Proinflammatory S100 proteins in arthritis and autoimmune
disease. Arthritis Rheum. 50:3762-3771.
31. Foell, D., S. Seeliger, T. Vogl, H. G. Koch, H. Maschek, E. Harms, C. Sorg, and J. Roth. 2003. Expression of S100A12 (EN-RAGE) in cystic fibrosis. Thorax 58:613-617.
32. Foell, D., H. Wittkowski, I. Hammerschmidt, N. Wulffraat, H. Schmeling, M. Frosch, G. Horneff, W. Kuis, C. Sorg, and J. Roth. 2004. Monitoring neutrophil activation in juvenile rheumatoid arthritis by S100A12 serum concentrations. Arthritis Rheum. 50:1286-1295. 33. Uchida, T., M. Shirasawa, L. B. Ware, K. Kojima, Y. Hata, K. Makita, G. Mednick, Z. A.
Matthay, and M. A. Matthay. 2006. Receptor for advanced glycation end-products is a marker of type I cell injury in acute lung injury. Am. J. Respir. Crit Care Med. 173:1008-1015.
34. Bernard, G. R., A. P. Wheeler, J. A. Russell, R. Schein, W. R. Summer, K. P. Steinberg, W. J. Fulkerson, P. E. Wright, B. W. Christman, W. D. Dupont, S. B. Higgins, and B. B. Swindell. 1997. The effects of ibuprofen on the physiology and survival of patients with sepsis. The Ibuprofen in Sepsis Study Group. N. Engl. J. Med. 336:912-918.
35. Wheeler, A. P. and G. R. Bernard. 1999. Treating patients with severe sepsis. N. Engl. J. Med. 340:207-214.
36. McClean, K. L., G. J. Sheehan, and G. K. Harding. 1994. Intraabdominal infection: a review. Clin. Infect. Dis. 19:100-116.
37. Anaya, D. A. and A. B. Nathens. 2003. Risk factors for severe sepsis in secondary peritonitis. Surg. Infect. (Larchmt. ) 4:355-362.
38. 2005. Deaths: Leading Causes for 2002. National Vital Statistics Report. 53:1-90.
39. Dremsizov, T., G. Clermont, J. A. Kellum, K. G. Kalassian, M. J. Fine, and D. C. Angus. 2006. Severe sepsis in community-acquired pneumonia: when does it happen, and do systemic inflammatory response syndrome criteria help predict course? Chest 129:968-978.
40. Laterre, P. F., G. Garber, H. Levy, R. Wunderink, G. T. Kinasewitz, J. P. Sollet, D. G. Maki, B. Bates, S. C. Yan, and J. F. Dhainaut. 2005. Severe community-acquired pneumonia as a cause of severe sepsis: data from the PROWESS study. Crit Care Med. 33:952-961. 41. Bartlett, J. G., S. F. Dowell, L. A. Mandell, T. M. File Jr, D. M. Musher, and M. J. Fine. 2000.
Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society of America. Clin. Infect. Dis. 31:347-382.
42. Finch, R. G. 1990. Epidemiological features and chemotherapy of community-acquired respiratory tract infections. J. Antimicrob. Chemother. 26 Suppl E:53-61.
43. Bartlett, J. G. and L. M. Mundy. 1995. Community-acquired pneumonia. N. Engl. J. Med. 333:1618-1624.
44. Bartlett, J. G., S. F. Dowell, L. A. Mandell, T. M. File Jr, D. M. Musher, and M. J. Fine. 2000. Practice guidelines for the management of community-acquired pneumonia in adults. Infectious Diseases Society of America. Clin. Infect. Dis. 31:347-382.
45. 2002. Epidemiology and Prevention of Vaccine-Preventable Diseases, National Immunization Program. Centers for Disease Control and Prevention, U.S. Department of Health and Human Services.
46. Pinhu, L., T. Whitehead, T. Evans, and M. Griffiths. 2003. Ventilator-associated lung injury. Lancet 361:332-40.
47. dos Santos, C. C. and A. S. Slutsky. 2000. Mechanotransduction, ventilator-induced lung injury and multiple organ dysfunction syndrome. Intensive Care Med. 26:638-642. 48. Craven, D. E. and K. A. Steger. 1996. Nosocomial pneumonia in mechanically ventilated
adult patients: epidemiology and prevention in 1996. Semin. Respir. Infect. 11:32-53. 49. Wheeler, A. P. and G. R. Bernard. 2007. Acute lung injury and the acute respiratory distress
syndrome: a clinical review. Lancet 369:1553-1564.
50. Leaver, S. K. and T. W. Evans. 2007. Acute respiratory distress syndrome. BMJ 335:389-394.
51. Carvalho, C. R., S. G. Paula Pinto, B. Maranhao, and E. P. Bethlem. 1998. Hyperoxia and lung disease. Curr. Opin. Pulm. Med. 4:300-304.
2
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Receptor for advanced glycation
end products facilitates host defense
during
Escherichia coli induced
abdominal sepsis in mice
Journal of Infectious Diseases, 2009
Marieke A.D. van Zoelen,1,2 Ann-Marie Schmidt,3 Sandrine Florquin,4 Joost C. Meijers,5
Regina de Beer,1,2 Alex F. de Vos,1,2 Peter P. Nawroth,6 Angelika Bierhaus6 and Tom van
der Poll1,2
1Center for Infection and Immunity Amsterdam (CINIMA), 2Center for Experimental and Molecular Medicine (CEMM), 4Department of Pathology, 5Department of Vascular Medicine; Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
3Division of Surgical Science, Department of Surgery, Colombia University, New York, NY, USA
Abstract
Background
The receptor for advanced glycation end products (RAGE) mediates a variety of inflammatory responses.
Methods
To determine the role of RAGE in the innate immune response to abdominal sepsis caused
by Escherichia coli (E. coli), RAGE deficient (RAGE-/-) and normal wild-type (Wt) mice
were intraperitoneally injected with E. coli. In a separate experiment, Wt mice received either anti-RAGE IgG or control IgG.
Results
E. coli sepsis resulted in an upregulation of RAGE in the liver, but not in the lungs. RAGE deficient mice demonstrated an enhanced bacterial outgrowth in their peritoneal cavity and increased dissemination of the infection, accompanied by increased hepatocellular injury and exaggerated systemic cytokine release and coagulation activation, 20 h after intraperitoneal administration of E. coli. Wt mice treated with anti-RAGE IgG also displayed a diminished defense against the growth and/or dissemination of E. coli. RAGE was important for the initiation of the host response as reflected by a reduced immune and procoagulant response early after intraperitoneal injection of E. coli lipopolysaccharide.
Conclusion
These data are the first to suggest that intact RAGE signaling contributes to an effective antibacterial defense during E. coli sepsis, thereby limiting the accompanying inflammatory and procoagulant response.
Introduction
Sepsis is the most common cause of death in noncoronary critical care units in the United States with > 750,000 cases per year [1]. Peritonitis is the second most common cause of sepsis [2] with Escherichia coli (E. coli) as one of the major pathogens involved [3]. Since E. coli peritonitis is a life-threatening disease, an immediate and adequate host defense is necessary to contain and kill the pathogen.
The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily that is expressed in all tissues on a wide range of cell types, including cells involved in the innate and adaptive immune system [4-6]. RAGE is one of the major signal transduction receptors for advanced glycation end products (AGEs). Ongoing studies revealed, however, that RAGE is able to engage classes of unrelated (including non-AGE) molecules, recognizing their tertiary structures rather than amino-acid sequences [4]. Its known ligands include the damage-associated molecular patterns (DAMPs, i.e. endogenous molecules that signal tissue and cell damage [7]) high mobility group box 1 (HMGB1) [8, 9] S100A12 [10], S100B [11], and amyloid [12].
RAGE has been suggested to be involved in the inflammatory response in several ways. First of all, the cellular effects resulting from the activation of RAGE by above mentioned endogenous pro-inflammatory ligands are mediated by multiple intracellular signaling pathways, including NF-κB, leading to the transcription of pro-inflammatory factors [13, 14]. In addition to binding ligands that participate in inflammatory and immune responses, in vitro studies showed that RAGE on endothelial and epithelial cells [15, 16] can function as an adhesive receptor that interacts with leukocyte β2-integrins, thereby being directly involved in inflammatory cell recruitment [15, 16].
RAGE deficiency improved survival in a model of abdominal polymicrobial sepsis induced by cecal ligation and puncture (CLP) [17, 18]. These studies suggested that inhibition of RAGE during sepsis attenuates the systemic inflammatory response and ensuing organ damage. However, the CLP model is less suitable to study the influence of an intervention on bacterial growth and dissemination, considering that the infection is polymicrobial involving a large number of aerobic and anaerobic pathogens and considering that the antibacterial response is dependent on the extent of necrosis of the cecum and the formation of a local abcess [19]. Therefore, we here investigated the role of RAGE during abdominal sepsis induced by one of its major involved pathogens, E. coli, focusing on the outgrowth of bacteria at the primary site of infection and the subsequent dissemination, and the accompanying systemic inflammatory response syndrome.
Methods
Mice
8-10-week-old female RAGE-/- mice on a C57Bl/6 background (backcrossed ten times)
were generated as described [17]. Age and sex matched wild-type (Wt) C57Bl/6 mice were obtained from Harlan Sprague Dawley Inc. (Horst, The Netherlands). The Institutional Animal Care and Use Committee of the Academic Medical Center, University of Amsterdam, approved all experiments.
Experimental groups
E. coli peritonitis and LPS-induced inflammation were induced as described [20-22]. In some studies Wt mice were injected intraperitoneally (ip) with either rabbit anti-RAGE IgG polyclonal antibodies, produced as described before [23] (200 µg given 20 h before and 4 h after infection) or normal rabbit IgG (R & D Systems, Abingdon, United Kingdom). Mice were sacrificed 20 h after E. coli injection. Assays were performed as described [20, 21]. For measurements by ELISA liver and lung homogenates were lysed in lysis buffer and treated as described [20, 21].
Histological examination
Lungs and livers were harvested after 20 h, fixed in 4% formaldehyde, embedded in paraffin and cut in 4-µm thick sections for staining procedures. Immunostaining for RAGE was performed on paraffin slides after deparaffinization and rehydration using standard procedures. Primary antibodies used were goat anti-mouse RAGE polyclonal antibodies (Neuromics, Edina, MN) and secondary antibodies were biotinlylated rabbit anti-goat antibodies (DakoCytomation, Glostrup, Denmark). Hematoxylin-eosin stainings were performed as described [24]. Liver and lung injury were semi-quantitatively scored as described [21]. Fibrin(ogen) stainings were performed as earlier described [20, 25].
Statistical analysis
All data are expressed as means ± SEs. Differences between groups were analyzed by Mann-Whitney U test. Values of P< .05 were considered to represent a statistically significant difference.
Results
RAGE is upregulated in the liver but not in lungs during
E. coli
peritonitis
To determine whether RAGE expression changes during E. coli peritonitis, we performed immunhistochemical stainings of RAGE of liver and lung tissue from Wt mice after ip administration of E. coli. In accordance with the literature [26-28], we found that normal, healthy mice showed modest if any RAGE staining in their livers (figure 1A). Livers from mice injected with E. coli displayed clearly more diffuse hepatic RAGE expression (figure 1B). Moreover, the surface of sinusoidal cells (capillaries between the hepatocyes, figure 1B), leukocytes in the vessels and some smooth muscle cells were stained positively for RAGE (figure 1B). Remarkably, areas of necrosis showed decreased RAGE staining (figure 1B, arrows). In the lungs, RAGE was extensively present in both healthy mice and mice infected with E. coli (figure 1D-E). Pulmonary RAGE expression was not increased in
E. coli injected mice compared to healthy mice. Liver and lung tissue from RAGE-/- mice
were used as negative controls for the RAGE staining and displayed some background staining only (figure 1C and F, respectively).
RAGE
-/-mice have an enhanced bacterial outgrowth and dissemination to
distant organs during
E. coli peritonitis
To examine whether RAGE deficiency influences bacterial outgrowth during peritonitis, we established the number of E. coli cfu at 20 h after infection in PLF, blood, liver and lungs of Wt and RAGE-/- mice. RAGE-/- mice had significantly higher bacterial loads in
their PLF than Wt mice (P< .01; figure 1G). In addition, blood, liver and lungs from RAGE-/- mice contained more bacteria compared with Wt mice (all P< .01 with the
exception of lungs, P = .03; figure 1H-J). Hence, RAGE-/- mice showed a clearly increased
outgrowth of E. coli at the primary site of infection, which was associated with an enhanced dissemination of bacteria to distant organs.
Inflammatory cell influx, chemokine and cytokine levels
At 20 h after infection, Wt and RAGE-/- mice had similar numbers of total leukocytes,
neutrophils and macrophages in their peritoneal fluid (table 1). The mouse CXC chemokines KC and MIP-2 are important mediators in the attraction of neutrophils during inflammation [29, 30]. Therefore, we measured the concentrations of these chemokines in peritoneal fluid. RAGE-/- mice displayed higher MIP-2 levels than Wt mice (P< .05),
whereas KC levels in PLF were similar in both mouse strains (table 1). To determine whether RAGE influences the production of cytokines during septic peritonitis, local and systemic concentrations of pro- and anti-inflammatory cytokines were measured in Wt and RAGE-/- mice (table 2). TNF-α, IL-6 and IL-10 were significantly higher in PLF
Figure 1. Expression of RAGE during Escherichia coli (E. coli) peritonitis and increased local
bacterial outgrowth and dissemination in RAGE deficient (RAGE-/-) mice. Representative view of a liver and lung from a normal, uninfected wild-type (Wt) mouse (A and D, respectively) displaying modest if any staining in the liver and strong RAGE expression in the lung. Liver from a Wt mouse 20 h after intraperitoneal (ip) injection of 5 x 104 cfu of E. coli showed more diffuse hepatic RAGE staining (B). The surface of sinusoidal cells (capillaries between the hepatocyes), leukocytes in the vessels and some smooth muscle cells were stained RAGE positive (B). Arrows indicate areas of necrosis (B). Absence of RAGE positivity in the liver of a RAGE-/- mouse (C). Lungs from a Wt mouse 20 h after the inoculation of E. coli (E) and from a healthy Wt mouse (D). Absence of RAGE positivity in the lung of a RAGE-/- mouse (F). RAGE staining; original magnification x10. Bacterial loads in peritoneal lavage fluid (PLF, G), blood (H), liver homogenate (I) and lung homogenate (J) were determined in Wt (white bars) and RAGE-/- (black bars) mice 20 h after ip injection of 5 x 104 cfu E. coli (n = 6-16 mice/genotype). Data are means ± SEs. ** P< .01 vs. Wt mice, *** P<.005 vs. Wt mice.
Table 1. Leukocyte counts and chemokine levels in peritoneal lavage fluid (PLF) Wt RAGE-/- Cell count (x 104/mL) Total 227.1 ± 32.9 179.5 ± 17.3 Neutrophils 185.8 ± 28.5 150.7 ± 15.4 Macrophages 36.5 ± 6.6 28.3 ± 3.0 Chemokines (pg/mL) KC 3758 ± 1982 17859 ± 10400 MIP-2 422 ± 126 763 ± 101a
NOTE Data are means ± SEs of 14-16 mice/group at 20 h after intraperitoneal injection of
5 x 104 cfu Escherichia coli. Wt, wild type; RAGE-/-, receptor for advanced glycation end
products gene deficient; KC, cytokine-induced neutrophil chemoattractant; MIP-2, macrophage inflammatory protein-2.
a P <.05 vs. Wt mice.
from RAGE-/- mice (all P< .05 vs. Wt mice). Moreover, these three cytokines were higher
in liver homogenates from RAGE-/- mice, whereas in plasma IL-6 and IL-10 levels were
elevated in these mice (table 2). In addition, RAGE-/- mice displayed higher concentrations
of MCP-1 in PLF and liver, whereas plasma MCP-1 levels tended to be higher in this mouse strain. The concentrations of these mediators were similar in lung homogenates of Wt and RAGE-/- mice (data not shown). Hence, RAGE deficiency was associated with an
increased release of cytokines, in particular in the peritoneal cavity and liver.
RAGE
-/-mice display more severe liver damage whereas lung inflammation
is unaltered
Our model of E. coli peritonitis is associated with liver injury and focal hepatic necrosis [21]. Both Wt and RAGE-/- mice showed signs of inflammation in liver tissue as
characterized by the influx of leukocytes into the hepatic parenchyma (figure 2A-B). RAGE-/- mice clearly showed more signs of liver necrosis (figure 2B, asterisks; figure 2C;
P< .05 vs. Wt mice). In addition, RAGE-/- mice demonstrated more thrombus formation
(figure 2F, arrow; figure 2C, P<. 05 vs. Wt mice). Clinical chemistry confirmed the existence of more profound hepatocellular injury in RAGE-/- mice, i.e. RAGE-/- mice had
significantly higher plasma AST and ALT levels compared with Wt mice (figure 2D, both
not shown). Pulmonary inflammation did not differ between the two mouse strains, as reflected by similar lung histology scores, relative lung weights and MPO levels in lung homogenates (data not shown). In conclusion, RAGE deficiency was associated with more extensive hepatocellular injury and necrosis and thrombus formation in the liver, while lung inflammation was unchanged.
RAGE
-/-mice demonstrate enhanced coagulation activation during
E. coli
peritonitis
This model of abdominal sepsis is associated with thrombin generation and activation of the coagulation system [20, 21]. To determine the role of RAGE herein, we performed fibrin(ogen) stainings on liver tissue slides. RAGE-/- mice demonstrated increased
fibrin(ogen) depositions compared with Wt mice (figure 2F vs. figure 2E). Both Wt and RAGE-/- mice showed strongly elevated TATc (figure 2G-H) and D-dimer (figure
2I-J) concentrations in plasma (figure 2G and I) and PLF (figure 2H and 2I-J). Importantly,
NOTE Data are means ± SEs of 14-16 mice/group at 20 h after intraperitoneal
injection of 5 x 104 cfu Escherichia coli. Wt, wild type; RAGE-/-, receptor for
advanced glycation end products gene deficient; TNF, tumor necrosis factor; PLF, peritoneal lavage fluid; IL, interleukin; MCP-1, monocyte chemoattractant protein-1.
a P <.05 vs. Wt mice; b P <.01 vs. Wt mice.
Table 2. Cytokine and chemokine concentrations in peritoneal lavage fluid, plasma and liver 20 h after intraperitoneal injection of Escherichia coli
Wt RAGE-/- Cytokines (pg/mL) TNF-α PLF 166 ± 51 266 ± 35a plasma 366 ± 206 759 ± 250 liver 26 ± 11 34 ± 6a IL-6 PLF 3248 ± 1049 8269 ± 925b plasma 3218 ± 1093 7146 ± 1455a liver 221 ± 66 1251 ± 327b MCP-1 PLF 4674 ± 1128 10217 ± 793b plasma 4355 ± 1260 5797 ± 1638 liver 4429 ± 865 8838 ± 1019b IL-10 PLF 215 ± 144 454 ± 88b plasma 78 ± 67 730 ± 233a liver 501 ± 59 717 ± 88a
coagulation activation was more profound in RAGE-/- mice, as reflected by higher plasma
and PLF TATc and D-dimer levels (figure 2G-J, all P< .05 vs. Wt mice). Together, these data indicate that RAGE deficiency enhances the activation of coagulation both locally and systemically during E. coli peritonitis.
Figure 2. RAGE-/- mice display more extensive liver necrosis and liver thrombi formation and enhanced systemic and local activation of coagulation. Wild-type (Wt) and RAGE-/- mice were intraperitoneally injected with 5 x 104 cfu Escherichia
coli. Representative
hematoxylin-eosin stainings of liver tissue at 20 h after injection in Wt (A) and RAGE -/- (B) mice. Original magnification x200. Arrow points out thrombi and asterisks point out necrotic areas. Graphical representation of the degree of liver thrombi and necrosis (C) determined according to the scoring system described in the Methods section. Plasma concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (D). Representative fibrin(ogen) staining of liver tissue of Wt (E) and RAGE -/- (F) mice. Arrow points out thrombi. Original magnification x20. Thrombin-anti-thrombin complex (TATc, G-H) and D-dimer (I-J) concentrations were measured systemically (plasma, G and I) and locally (PLF, H and J) in Wt (white bars) and RAGE-/- (black bars) mice (n = 6-16 mice/genotype). Dotted lines represent the mean values from normal (uninfected) mice. Data are means ± SEs. * P< .05 vs. Wt mice; **
Ta ble 3. I m pact of R A G E de fic ie nc y o n Esc her ich ia col i l ip op ol ysacc haride ( L PS)-i nd uce d r es po nses t = 0 h t = 2 h t = 6 h Wt R A G E -/-Wt R A G E -/-Wt R A G E -/-P LF Cel ls (x 10 4/m L) T ot al c el ls 54.4 ± 3.5 37.5 ± 9.5 14.6 ± 2.5 11.5 ± 3.8 27.6 ± 7.6 17.3 ± 3.1 N eu tr op hils 0.0 ± 0.0 1.0 ± 0.9 3.3 ± 0.7 2.4 ± 0.5 27.5 ± 5.8 11.2 ± 2.7 a M ac ro ph ag es 50.9 ± 4.1 34.1 ± 3.5 10.0 ± 2.2 5.5 ± 2.7 7.0 ± 4.2 4.7 ± 1.4 C he mo ki nes ( pg/m L) K C N d N d 3881 ± 255 3175 ± 388 954 ± 320 1349 ± 307 M IP -2 N d N d 954 ± 129 763 ± 106 167 ± 68 217 ± 49 Plas m a T N F-α ( pg/m L) 7.4 ± 2.1 7.2 ± 0.8 5184 ± 836.5 2543 ± 370.9 b 39.2 ± 18.4 33.6 ± 7.4 TA Tc (µg/L) 5.8 ± 0.7 8.8 ± 1.6 29.5 ± 4.8 11.6 ± 1.4 a 13.6 ± 3.4 14.6 ± 1.9 N OT E D at a a re mea ns ± S Es of 4 ( t = 0 h ) t o 12 m ic e ( t = 2 a nd 6 h ) m ic e/g rou p a t 0, 2 o r 6 h aft er i nt ra pe rit onea l i nj ect io n of 250 µg lip op olysac ch ar ide (L PS) fr om Esc he ric hia c oli. W t, w ild t yp e; R A G E -/-, re ce pt or fo r ad va nc ed g lyc at io n e nd p ro duct s g ene deficie nt ; PL F, p er ito nea l l av ag e fl uid ; K C , c yt ok ine -ind uc ed neu tr op hil che mo at tr act an t; nd , not det er m ine d; M IP -2, m ac ro ph ag e i nfl am m at or y pr ot ei n-2; T N F, t umo r ne cr osis f act or ; T AT c, t hr omb in -a nt i-t hr omb in c om plex es . a P <.05 v s. W t m ic e; b P <.01 v s. W t m ic e.
RAGE
-/-mice demonstrate a diminished inflammatory response to
E. coli
LPS
We next investigated whether the exaggerated host response in RAGE-/- mice during E.
coli peritonitis was the consequence of the higher bacterial loads in these animals or an inhibitory effect of RAGE. For this we injected Wt and RAGE-/- mice with E. coli LPS
ip and harvested plasma and PLF 2 and 6 h later. RAGE-/- mice had two times lower
neutrophil counts in their peritoneal fluid than Wt mice at 6 h after injection (table 3,
P< .05); KC and MIP-2 levels did not differ in PLF of RAGE-/- and Wt mice (table 3).
Plasma TNF-α levels were clearly diminished in RAGE-/- mice 2 h post LPS (table 3,
P< .01), whereas MCP-1, IL-6 and IL-10 levels were similar in both genotypes (data not shown). Finally, at 2 h after LPS injection, the plasma concentrations of TATc were diminished in RAGE-/- mice (table 3, P< .05).
Anti-RAGE IgG enhances bacterial outgrowth and dissemination during
E. coli peritonitis
To exclude the possibility that the results with live E. coli bacteria obtained in RAGE
-/-mice were due to compensatory changes in these genetically modified animals unrelated to RAGE deficiency, we treated Wt mice with anti-RAGE IgG or control IgG antibodies and determined bacterial loads in various body compartments 20 h after induction of peritonitis. In strict accordance with the data generated in RAGE-/- mice, Wt mice with
anti-RAGE IgG antibodies had more bacteria in their PLF and in distant body sites (data not shown). Furthermore, the numbers of total leukocytes, neutrophils, and macrophages in their PLF were similar in anti-RAGE and control antibody treated mice, whereas the local concentrations (in PLF) of the CXC chemokines KC and MIP-2 were higher in mice that had received anti-RAGE antibodies (data not shown), results that are in line with the data obtained in RAGE-/- mice.
Anti-RAGE IgG increased fibrin deposition during
E. coli peritonitis
In line with the data obtained in RAGE-/- mice, livers of anti-RAGE IgG-treated miceshowed more thrombi formation 20 h after infection (figure 3B, arrow, vs. figure 3A; figure 3C; P< .05 vs. control mice). These data were supported by fibrin(ogen) stainings (figure 3F vs. figure 3E). However, in contrast to the RAGE-/- mice, anti-RAGE IgG
treatment was not associated with enhanced liver necrosis, hepatocellular injury (figure 3C-D) or TATc levels (figure 3G-H).
Figure 3. RAGE inhibition enhances hepatic fibrin(ogen) deposition during Escherichia coli
(E. coli) induced sepsis. Mice were intraperitoneally injected with 5 x 104 cfu E. coli and treated with either anti-RAGE IgG antibodies (α-RAGE) or control IgG antibodies (control). Representative hematoxylin-eosin stainings of liver tissue at 20 h after injection in IgG control (A) and anti-RAGE IgG (B) treated mice. Original magnification x200. Arrow points out thrombi. Graphical representation of the degree of the liver thrombi and necrosis (C) determined according to the scoring system described in the Methods section. Plasma concentrations of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (D) (n = 9-10 mice/group).
Representative fibrin(ogen) staining of liver tissue of control IgG (E) and anti-RAGE IgG (F) treated mice. Original magnification x20. Thrombin-anti-thrombin complex (TATc, G-H) concentrations were measured systemically (plasma, G) and locally (PLF, H). Data are means ± SEs. * P< .05 vs. control antibodies.
Discussion
Gram-negative peritonitis is a life-threatening condition frequently associated with systemic dissemination of bacteria and septic shock. Host defense in peritonitis is an established domain of the innate immune system as the rapid response to invading pathogens is essential for the host to survive. RAGE has the ability to activate signaling pathways leading to pro-inflammatory gene expression upon interaction with a range of distinct endogenous pro-inflammatory ligands. We here examined the in vivo role of RAGE during murine E. coli peritonitis, using RAGE-/- mice and anti-RAGE IgG. Our
key finding was that RAGE contributes to an effective antibacterial host response during E. coli infection. Indeed, RAGE deficiency caused an enhanced outgrowth of E. coli at the primary site of infection together with increased spreading of bacteria to other body compartments and more severe liver injury.
Knowledge of the role of RAGE in host defense against bacterial infection is highly limited. RAGE-/- mice displayed a reduced mortality after induction of polymicrobial
sepsis produced by CLP [17, 18]. Moreover, anti-RAGE therapy conferred a survival advantage even when administered 24 h after CLP in mice receiving antibiotic treatment [18]. In the latter investigation, RAGE deficiency or anti-RAGE therapy was reported not to influence bacterial loads in PLF, liver or spleen. It should be noted, however, that in this study all mice were treated with broad spectrum antibiotics and bacterial loads were only determined in mice that survived (i.e. not at predefined time points after CLP). Together with the fact that host defense against CLP at least in part relies on the extent of intestinal necrosis and the formation of a local abcess [19], the possible role of RAGE in antibacterial defense can not be easily determined from this earlier investigation [18]. Although our model does not resemble clinical abdominal sepsis as closely as CLP, it is a relevant tool to study the role of endogenous receptors and/or mediators in limiting the growth and dissemination of bacteria after a primary intraabdominal infection and to determine the contribution of these host proteins to specific immune responses [20, 21]. As such, we here provide evidence, using RAGE-/- mice and anti-RAGE IgG that RAGE
signaling contributes to an effective antibacterial response. Most likely, RAGE exerts this effect indirectly and not via direct interaction with E. coli, considering that RAGE
-/-leukocytes demonstrated an unaltered capacity to phagocytose and kill E. coli in vitro (data not shown). In addition, the observation that RAGE deficiency in general was associated with an exaggerated host response during E. coli sepsis and a reduced response to bolus E. coli LPS injection suggests that although RAGE is involved in the initation of an immune reaction to E. coli, this function can be compensated for by other receptors in the presence of a growing bacterial load. Previously, in vitro data have shown that interaction of RAGE with its ligand HMGB1 can induce activation of intracellular signaling pathways [8, 9,
31] and thereby inflammation. Since HMGB1 has been reported to transduce cellular signals in vitro and in vivo by interacting with at least two other receptors, i.e. TLR2 and TLR4 [32-35], one possible explanation for the enhanced inflammation in the RAGE
-/-mice could be that the absence of RAGE facilitates the interaction between HMGB1 and TLR2 and TLR4.
The current data do not necessarily contradict the previously described protective effect of RAGE deficiency with regard to CLP-induced mortality [17, 18]. The immune response to bacterial infection can act as a double-edged sword, on the one hand protecting the host against invading pathogens, on the other hand potentially damaging cells and tissues. It is conceivable that bacterial growth and dissemination do not impact significantly on the outcome of CLP-induced sepsis, in particular in the context of antibiotic therapy. Future research is warranted to investigate RAGE-mediated antibacterial activity against other pathogens associated with abdominal sepsis without the use of antibiotics. RAGE deficiency did not influence mortality in our E. coli model 14 out of 15 Wt mice and all 13 RAGE-/- mice died with the first lethalities occurring 25 hours after infection in both
groups; data not shown). In this respect it should be noted that the mortality curves after infection of previously healthy mice with this E. coli strain are very steep: whereas low doses do not cause lethality, doses that do cause lethality almost invariably do so. Hence, we consider this model less suitable to determine the impact on mortality and rather make use of it to study early host defense mechanisms.
Plasma IL-10 levels were 10-fold higher in the RAGE-/- mice, while other plasma
cytokine levels doubled at most. Earlier, it has been established that the recovery of E.
coli is diminished in IL-10-/- mice [36]. Similarily, treatment of mice with anti-IL-10
antibodies resulted in a lower bacterial outgrowth in a model of peritonitis induced by the intraperitoneal injection of the gram-negative bacterium Klebsiella pneumoniae [37]. In the latter study all mice received gentamicin. Together, these and our data suggest a (direct or indirect) association of high IL-10 levels with enhanced bacterial outgrowth during Gram-negative peritonitis. Of note, IL-10-/- mice demonstrated more
organ damage during E. coli peritonitis despite an accelerated bacterial clearance [36]. In that report, anti-TNF-α partially attenuated neutrophil recruitment and multiple organ damage in the IL-10-/- mice. These results imply that although endogenous IL-10
facilitates the bacterial outgrowth during E. coli peritonitis, it protects mice from organ damage by a mechanism that involves inhibition of TNF-α release. In contrast to these IL-10-/- mice, our RAGE-/- mice show more severe organ (liver) damage and elevated
TNF-α concentrations. Therefore, the role of elevated IL-10 in RAGE-/- mice during E.
RAGE deficiency resulted in enhanced organ injury with more necrosis of the liver in our model of E. coli induced sepsis. Sepsis is also associated with organ failure of the heart which contributes to hypotension, impaired perfusion and mortality [38]. The precise mechanism of this sepsis related myocardial dysfunction is unknown. Similar to some innate immune cells, cardiomyocytes are able to respond to “danger” signals with an innate immune inflammatory response [39, 40]. In addition, cardiomyocytes express multiple Toll-like receptors (TLRs) that signal predominantly through NF-κB when stimulated by pathogen-associated molecular patterns, leading to decreased cardiocyte contractility [41]. Boyd et al. demonstrated that RAGE coimmunoprecipitated with both S100A8 and S100A9 in hearts of mice injected with LPS [42]. Furthermore, it has been demonstrated that myocardial RAGE expression is upregulated in a model of ischemia/reperfusion in rats and that RAGE deficient mice are protected from ischemia/reperfusion injury of the heart [43, 44]. It remains to be established whether myocardial RAGE plays a role during E. coli sepsis. RAGE immunochemistry in mice has not been published before and unfortunately in our hands yielded unreliable results using multiple commercially antibodies.
In line with previous findings [20], E. coli peritonitis was associated with activation of the coagulation system. RAGE-/- mice displayed more activation of coagulation, as
reflected by increased TATc and D-dimer concentrations in PLF and plasma and more fibrin deposition in the liver. Knowledge of the involvement of RAGE in activation of coagulation is limited. In a model of chronic vascular inflammation in diabetic apolipoprotein E deficient mice, administration of soluble RAGE for 6 weeks suppressed aortic levels of tissue factor [45], the main initiator of coagulation in sepsis in general [46] and in our model of abdominal sepsis in particular [20]. In addition, anti-RAGE IgG has been reported to inhibit tissue factor expression by monocytes stimulated with serum amyloid A in vitro [47]. Clearly, these previous data cannot be readily extrapolated to our model of severe acute bacterial infection. Our finding that RAGE-/- mice demonstrated
lower plasma TATc levels upon bolus injection of LPS suggest that RAGE may play a role in acute activation of coagulation. The enhanced coagulation activation in mice with attenuated RAGE function during E. coli sepsis at least in part may have been caused by the higher bacterial loads in these animals. As such, our results provide evidence that although RAGE signaling may contribute to coagulation activation during acute infection, it is not essential for the procoagulant response during sepsis.
Although overall the effects of RAGE deficiency and anti-RAGE IgG on the host response
to E. coli peritonitis were largely similar, some differences were observed. In this respect
it should be noted that RAGE deficiency results in a complete absence of signaling via RAGE, whereas anti-RAGE IgG treatment most likely only partially prevents RAGE
signaling. Furthermore, these differences in effects could also be attributed to an additional deficiency of soluble RAGE in the RAGE deficient mice (in contrast to the anti-RAGE IgG treated mice that still can express soluble RAGE). In this respect it is important to note that most RAGE ligands (which can be bound by soluble RAGE) are promiscuous and are able to activate other cell-associated receptors besides RAGE [8-12, 48].
The present study is the first to document that intact RAGE signaling contributes to an effective antibacterial defense during abdominal sepsis caused by E. coli, thereby limiting the ensuing host systemic inflammatory and procoagulant response to infection. These data further illustrate the existence of a delicate balance between inflammation and anti-inflammation during severe bacterial infection where a certain degree of anti-inflammation is required to combat invading pathogens and exaggerated inflammation can result in tissue injury.
