The inflammatory response in cardiac surgery An uptodate overview with the emphasis on the role of heat shock

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1 : The inflammatory response in cardiac surgery. An up-to-date overview with the emphasis on the role of heat shock proteins (HSPs) 60 and 70 Pavel Kunes, Jiri Mandak, Miroslav Brtko, Martina Kolackova1, Manuela Trojackova Kudlova1, Ctirad Andrys1, Jan Krejsek1 Charles University in Prague, School of Medicine and University Hospital in Hradec Kralove Department of Cardiac Surgery 1Department of Clinical Immunology and Allergy Hradec Kralove, Czech Republic
2 : In most cases, clinical manifestations of SIRS are restricted to: transient tachycardia arterial hypo- or hypertension due to dysregulated vasomotion body temperature rising temporarily into febrile ranges without any proof of a concomitant infection or conversely, dropping temporarily to hypothermic values of = 35°C slight abnormalities in most blood chemistry tests Cardiac surgical operations are accompanied by the development of the systemic inflammatory response syndrome (SIRS).
3 : This mild form of SIRS should be viewed as a basically beneficial biological response, which: helps the host to withstand the loss of whole-body homeostasis inflicted by surgical injury is a prerequisite for a timely and proper wound healing, as well as for complete regain of regulatory homeostatic mechanisms on all organ levels last but not least, tightly controled SIRS alerts („primes“) the host´s immune system on the danger of infection. A subclinical form of SIRS is expressed virtually in all cardiac surgical patients with minor inter-individual variations.
4 : Fig.1: An example of the process depicting the priming of polymorphonuclear leukocytes (granulocytes) Killing mechanisms of granulocytes are either O2-independent or O2-dependent. O2-independent mechanisms rely redominantly on the action of various antibacterial peptides, such as defensins. O2-dependent mechanisms of killing are mediated by various oxygen reactive species which are generated from superoxide anion O2 -.. This molecule is produced by NADPH oxidase. NADPH oxidase is a multimolecular enzymatic complex which is assembled from both cytoplasmic and membrane bound subunits after the activation of granulocytes.
5 : Resting granulocyte – NADPH oxidase complex is disassociated.
6 : Primed granulocyte – assembly of NADPH oxidase complex in the cytoplasm.
7 : Activated granulocyte – translocation of assembled NADPH oxidase components to the cellular membrane and formation of oxygen reactive species resulting in microbial killing.
8 : septic patients, in whom SIRS presents mostly a serious problem with an uncertain prognosis multiple trauma patients, in whom the development of SIRS is proportional to the extent and the gravity of underlying injuries. In sum, the danger of SIRS is a kind of a trade-off paid for the progress in critical care medicine, which is now able to support, at least temporarily, basal life functions even in the most gravely affected patients who, before the advent of the sofisticated techniques of modern health care system, would have helplessly died long before the development of SIRS. SIRS also strikes other patient groups, such as:
9 : These life-threatening variants of systemic inflammation are referred to as: the multiple organ dysfunction syndrome (MODS) the multiple organ failure syndrome (MOFS). This unfavorable trajectory of systemic inflammation follows basically the same developmental pattern, irrespective of whether it has been set off by an infectious (e.g., sepsis or septic shock) or a sterile (e.g., ischemia-reperfusion or cardiopulmonary bypass) stimulus. Due to genetically determined susceptibility of individual cardiac surgical patients and/or due to adverse external conditions, such as an unfavorable perioperative course, unrestrained forms of SIRS evade the complex network of regulatory mechanisms and subsequently develop into clinically overt forms, which may ultimately proceed to death.
10 : SIRS is marked by the predominance of pro-inflammatory mediators, such as tumor necrosis factor (TNF) -?, interleukin (IL) -1? and/or IL-6 in the circulation right from the onset of the adverse event, whether or not it is of septic origin. MARS represents a mixed anti-inflammatory response syndrome with systemic levels of the pro- and anti-inflammatory mediators attaining more or less an equilibrium, thus heralding an intermediate state between diminishing SIRS and ascending CARS. CARS is an acronymon for the compensatory anti-inflammatory response syndrome which is characterized by predomi- nance in the systemic circulation of anti-inflammatory mediators, the most important being IL-10, IL-1 receptor antagonist (IL-1Ra) and soluble receptors of TNF?. This latter classification, by contrast to the former one, implies a favorable outcome of the underlying disease or injury. An alternative classification of the critically ill is supposed to mirror more closely their individual inflammatory fluctuations during trauma, surgery or sepsis. Accordingly,
11 : Irrespective of whether increased or decreased, these changes occur: early (up to 6 hours after the onset of the inciting stimulus) simultaneously concerning both the pro- and the anti-inflammatory mediators and correlate to the early but not to the late mortality or to survival. According to recent studies, however, SIRS is characte- rized by a concomitant increase in plasma levels of both pro-inflammatory (most notably IL-6) and anti-inflammatory (most notably IL-1Ra) mediators or, less frequently but with a comparable clinical picture and prognostic value, by a concomitant decrease in plasma levels of both the pro- and the anti-inflammatory mediators.
12 : As it is, the final outcome of SIRS does not seem to be incorporated into the early response, whether it be hyper- or hypoinflammatory. This multifaceted inflammatory response, although it has been recognized in septic animals, has been proposed to be recapitulated in cases of sterile systemic inflammation, such as that started off by the cardiopulmonary bypass in cardiac surgical patients.
13 : Determination of the extent of SIRS which has not attained a clear-cut clinical picture may be assessed by the capacity of the host to respond to ex vivo stimuli, i.e. probing into the cellular branch of the innate immunity/inflammatory response. Furthermore, original SIRS/CARS paradigm has not been defined solely on the basis of circulating cytokine concentra- tions, but also – and maybe more importantly – by the expression of various immune/inflammatory cellular components. Cells harvested from septic patients have been shown to exhibit a markedly reduced capacity to elaborate pro-inflammatory cytokines following an ex vivo stimulation.
14 : Ex vivo stimulation of these cells results in suppressed production of pro-inflammatory mediators, such as: IL-2 interferon (IFN)-? IL- 6 while the synthesis of anti-inflammatory mediators, such as: IL- 4 IL-10 is enhanced after a challenge of these cells either with lipopolysaccharide (LPS) or concanavalin. Specifically, it has been shown that the capacity of mouse splenic/peritoneal macrophages to release inflammatory cytokines 24 hours after cecal ligation and puncture (CLP) -induced sepsis is substantially diminished.
15 : Moreover, a CARS-specific decrease of major histocompatibility complex (MHC) class II expression on circulating monocytes has been found to occur early, i.e. within 24 hours, in both murine and human sepsis. Prognostic impact conferred by circulating mediators may not necessarily rely on their biological functions, but rather on the simple fact that the host is actually capable of producing them. Once again, the same principle applies not only to septic inflammation, but also to sterile inflammation evoked by cardiopulmonary bypass.
16 : Therefore, the patients´ blood is exposed to altered biophysical forces, which lead to activation of plasma protein cascades, such as: the complement system, the kallikrein-kinin system, the coagulation system. Cardiac on-pump operations are performed with the use of cardiopulmonary bypass. The extra-anatomical environment of the heart-lung machine, namely the tubing system and the oxygenator, is completely devoid of the endothelial layer.
17 : ABBREVIATIONS: C3a, C5a: Small spliting fragments of complement components C3, C5 ICAM: InterCellular Adhesion Molecule IL: Interleukin iNOS: inducible form of NO-Synthase NF-?B: Transcription Factor NF-?B PECAM: Platelet Endothelial Cell Adhesion Molecule VCAM: Vascular Cellular Adhesion Molecule Fig.2: Proposed scenario induced by CPB leading to systemic inflammation (SIRS).
18 :
19 : At the same time, cellular elements within the blood stream are activated, or more properly termed, they are primed. Priming means that the respective cellular elements are prepared to execute na early-immediate, often exaggerated response after they have been challenged by an additional activatory stimulus (the „second challenge“ or the „two-hit“ model).
20 : The priming paradigm can be translated from individual blood cellular elements up to the patients´ body in that the operation (the first challenge) gives rise to a subclinical form of SIRS which, up to a certain degree, may be synonymous with the acute-phase response and which, whenever the patient experiences an adverse event, such as a shock state or an infection (the second challenge), proceeds to its clinically overt manifestation.
21 : „Two-hit“ model depicting the immunologic and clinical consequences of severe injury. The same principles apply to CPB-operated cardiac surgical patients. Early after injury/operation, SIRS of diverse extent develops, putting the patient at risk of succumbing to MODS / MOFS, if the inflammatory response is excessive. SIRS is the proinflammatory arm of the innate immunity response. Patients who survive initial SIRS usually develop the CARS syndrome which may either favor healing or, due to its antiinflammatory nature, put the patient at risk of infection leading to secondary SIRS. ABBREVIATIONS: CARS: Compensatory Antiinflammatory Response Syndrome MODS: Multiple Organ Dysfunction Syndrome MOFS: Multiple Organ Failure Syndrome SIRS: Systemic Inflammatory Response Syndrome Fig.3: „Two-hit“ model of severe injury.
22 :
23 : Depending on the quality of the patient´s regulatory network and/or the virulence of the external noxious stimuli, the full-blown form of SIRS: 1 ) may help to combat infection (the SIRS ? CARS transition) 2a) may inflict serious damage to the patient´s own tissues (the SIRS ? MODS progression) 2b) or may even lead to death (SIRS ? MOFS).
24 : Leukocyte-endothelial cell interactions make up the first step in a complex sequence of events which lead to the deployment of inflammatory reactions within the interstitial space of tissues subjected to trauma or infection. For reasons which will be explained further, the heart and the lungs are the first and the most gravely affected by SIRS target organs in cardiac surgical patients.
25 : Interaction of activated leukocytes with endothelial cells which have acquired a proinflammatory phenotype. (A) Net result of these interactions is transendothelial migration of activated leukocytes from the blood stream into the interstitial space. (B) ABBREVIATIONS: ICAM: InterCellular Adhesion Molecule IL: Interleukin I-?B: Inhibitory Factor ?B JAM: Junction Adhesion Molecules LPS: Lipopolysaccharide MMP: Matrix MetaloProteinases NF-?B: Transcription Factor NF-?B PECAM: Platelet Endothelial Cell Adhesion Molecule PKC: Protein Kinase C ROS: Reactive Oxygen Species TLR: Toll-Like Receptor TNF?: Tumor Necrosis Factor ? VCAM: Vascular Cellular Adhesion Molecule Fig.4: Leukocyte-endothelial cell interactions
26 : A
27 : B
28 : In parallel to activation or priming, cells of the patient´s body set off production of the „danger signal“ molecules. These endogenous immune stimulators are released during unfavorable life conditions in order to alert the immune system on the need of starting off an inflammatory or innate immunity response. One of the most prominent „danger signal“ is carried by heat shock protein 60 (HSP60). The cipher of sixty denominates its molecular weight of 60 kDa.
29 : ABBREVIATIONS: Gp96: Glycoprotein HSP: Heat Shock Protein LOX-1: Lectin-like Oxidized low-density lipoprotein receptor 1 SR-A: Scavenger Receptor-A TLR: Toll-Like Receptor D.C.: Dendritic Cells Fig.5: Receptors and effects of selected HSP molecules on the immune system
30 :
31 : ABBREVIATIONS: HSE: Heat Shock Elements HSF: Heat Shock Transcription Factor JAK: Janus Kinase NO: Nitrogen Oxide ox-LDL: oxidized-Low Density Lipoprotein Ras/Rac/ERK: Signaling Pathway kinases SR: Scavenger Receptor STAT: Signal Trandsducer and Activator transcription factor Fig.6: External stimuli and signaling pathways leading to HSP expression in vascular endothelial cells.
32 :
33 : „Chaperoning“ encompasses: proper folding of newly synthesized proteins safe transport of these proteins among various intracellular compartments or, when damaged, a timely repair of these proteins. „Chaperoning“ also implies that under normal conditions, HSP60 does not leave the intracellular milieu. Consequently, HSP60 remains hidden out of the reach of the immune system. Under normal life conditions, HSP60 is produced by all mammalian cells. This constitutive form of HSP60 is operative within the interior of its producing cell where it performs so-called „chaperone“ functions.
34 : The patient´s HSP60s increase predominantly in endothelial cells which must face diverse forms of cellular stress, such as: altered blood flow due to disturbed hemodynamic forces oxidative and/or osmotic stress within the cells unbridled activities of pro- and/or anti-inflammatory cytokines dissemination of micro-organisms which do not need to be pathogenic in humans with a properly tuned immune system. Whenever an individual cell or the whole body is submerged by adverse life conditions, an entirely different situation sets in. A cardiac surgical operation presents an illustrative example of a change in environmental conditions, no matter whether or not CPB has been employed.
35 : ABBREVIATIONS: TNF?: Tumor Necrosis Factor ? Fig.7: Adverse stimuli enhancing cardiovascular hsp-gene transcription and HSP synthesis in experimental models.
36 :
37 : HSP60 molecules that have been manufactured in excess are translocated from inside their maternal (e.g., endothelial) cell onto the cellular membrane. This is the first step bringing the HSP60s within the reach of the immune system. This message reads: „Beware of danger!“ The ensuing inflammatory/innate immunity response depends upon the fate of the HSP60´s maternal cell. Basically, this cell may be damaged but remain alive or die by apoptotic or necrotic death.
38 : Cells dying by necrotic death loose their HSP60s from the fragments of outer membranes. HSP60s are thereafter released into the extracellular space. Here, free HSP60s are engulfed by macrophages or dendritic cells, themselves producers in stressful conditions of large amounts of HSP60s. Thus, macrophages or dendritic cells accumulate HSP60s originating from dying or dead cells (e.g., endothelial cells), in addition to HSP60s that have been produced by themselves. Upon engulfement, newly acquired HSP60s contribute mightily to the activation of the entire immune system.
39 : HSP60 is not only an intracellular protein, but also an intercellular signaling molecule which induces cytokine secretion and adhesion molecules expression. ABBREVIATIONS: ICAM: InterCellular Adhesion Molecule IL: Interleukin sHSP60: soluble Heat Shock Protein TNF?: Tumor Necrosis Factor ? VCAM: Vascular Cellular Adhesion Molecule Fig.8: Extra-cellular HSP60.
40 :
41 : Specifically, HSP60 has been found to act: as an innate activator of T-lymphocytes, themselves executors of antigen-specific cellular immune responses, and/or as a mitogen of B-lymphocytes, themselves producers of antigen-specific antibodies. During endotoxemia, a HSP60/LPS (lipopolysaccharide) complexes are formed which are bestowed with the capacity to augment the overall activation of innate immunity system.
42 : In bacterial infection, HSP60 facilitates binding of LPS to the CD14/TLR4 complex. HSP60 enhances LPS-induced TLR4 signaling leading to enhanced production by APC of cytokines like IL-12 which is responsible for LPS-induced INF? production and T cells activation. Under sterile conditions, i.e., in the absence of LPS, HSP60 functions as a classical danger signal indicating tissue damage to the immune system. Here, HSP60 exploits yet unknown mechanisms that do not lead to the secretion of cytokines of IL-12 type by APC. Neverthless, LPS-free HSP60 enhances IFN? production in primary T cell activation. This effect is likely to be due to the induction by APC of type I IFNs, especially IFN?. Fig.9: HSP60 in bacterial and/or sterile inlammation
43 : ABBREVIATIONS: APC: Antigen Presenting Cell HSP60: Heat Shock Protein IFN: Interferon IL: Interleukin LPS: Lipopolysaccharide MHC: Major Histocompatibility Complex NF-?B: Transcription Factor NF-?B PAMP: Pathogen Associated Molecular Pattern TLR: Toll-Like Receptor Fig.9: HSP60 in bacterial and/or sterile inlammation
44 :
45 : Moreover, HSP60-influenced B-lymphocytes skew the T-lymphocyte response to a Th0 phenotype, which is characterized by concomitant production and secretion of both IFN-? and IL-10. Canonically, these two mediators are each other´s antagonists. By contrast, dendritic cells activated by the HSP60/LPS complex secrete high amounts of IFN-? and IL-12. Canonically, these two mediators are supporters of the antigen-specific Th1 lymphocyte response.
46 : Chlamydia pneumoniae Helicobacter pylori Porphyromonas gingivalis Borrelia burgdorferi Legionella pneumophila Histoplasma capsulatum Trypanosoma cruzi to name only a few examples with presumed or proven relation to cardiovascular diseases. In addition to endogenous HSP60s, there are also HSP60 molecules of exogenous origin. The latter HSP60 molecules are embedded in outer membranes of a vast array of micro-organisms, such as:
47 : From the evolutionary viewpoint, members of the HSP60 superfamily belong to the most conserved proteins in micro- and macro-organisms, displaying as much as 70% of sequence homology between prokaryotic (microbial) and eukaryotic (mammalian including humans) HSP60s. Microbial HSP60s represent typical „pathogen-associated molecular pattern“ molecules or PAMPs. These molecular structures are shared by different, often unrelated microbial species (vide supra).
48 : Another example of a PAMP is a lipopolysaccharide, the endotoxin of Gram-negative bacteria. Peptidoglycan, on the other hand, is the most important PAMP of Gram-positive bacteria. All relevant PAMPs, whose occurrence by far exceeds heat shock proteins, lipopolysaccharide or peptidoglycan, are incessantly screened for by the host´s cellular receptors which are collectively referred to as „pattern-recognition receptors“ or PRRs. Due to a complex interplay between the PAMP and PRR networks, it is not necessary for the host to discriminate on the individual antigenic level all pathogenic micro-organisms right from the moment they have pervaded his or her tissues.
49 : Instead, at the early stage of infection, inflammation or cellular damage, it is both sufficient and efficient for the host to take notice of pervading micro-organisms (foreign antigens or non-self) or altered/damaged own structures (self neoantigens or altered self) via the PRRs. The latter receptors reliably identify the conserved molecular patterns shared by bacterial and mammalian cells. Hence, defense reactions can begin with ample advance, well before any noxious particle has been definitely discerned at the antigenic level.
50 : Most cells which build up the human body are equipped with the CD14 receptor. The most important cells relating to the development of SIRS, are represented by: monocytes/macrophages endothelial cells smooth muscle cells of the vascular wall. Moreover, soluble CD14 receptor can be synthesized by hepatic cells as an acute phase protein. As often as not, LPS is attached to the the target cell in conjunction with extracellular HSP60. The latter´s origin – endogenous (human) versus exogenous (microbial) – is of no significance in the process of signaling to the host: „Beware of danger!“ LPS, the most prominent PAMP in the context of inflammatory/innate immunity reactions, is bound to the target cell by way of the membrane receptor CD14.
51 : The localization of the CD14 receptor is confined to the outer surface of the plasma membrane. Moreover, it is devoid of an intracellular domain which could transmit the HSP60/LPS complex-borne signal into the cellular interior. Therefore, an additional receptor or a set of receptors is needed that would compensate for this drawback by penetrating the entire width of the cellular membrane into the cellular milieu. This drop-out in signal transmission is repaired by the family of Toll-like receptors (TLRs), which themselves are archetypical pattern-recognition receptors.
52 : ABBREVIATIONS: CAD: Coronary Artery Disease IRAK: Interleukin-1 Receptor-Associated Kinase MyD88: Myeloid Differentiation factor 88 (an adaptor protein) SIRS: Systemic Inflammatory Response Syndrome TLR: Toll-Like Receptor Fig.10: Surgically relevant TLR pathways and their association with various diseases.
53 :
54 : Within the entire TLR family, which by now counts at least 10 members, TLR4 stands out as the most important LPS-binding PRR. The intracellular domain of TLR4 is closely reminiscent of corresponding domains of receptors for IL-1 and IL-18, respectively. Hence their collective denomination of TIR, which means „the Toll/interleukin-1 receptor.“
55 : The HSP60/LPS complex has been found to induce maturation of dendritic cells, which is demonstrable by up-regulation of costimulatory molecules: CD40, a costimulatory molecule indispensable for the very process of dendritic cell maturation, MHC II (major histocompatibility complex class II), B7-2, a costimulatory molecule important for Th2 responses, CD54 or ICAM-1 (intercellular adhesion molecule-1). In response to LPS, macrophages and dendritic cells use predominantly the TLR4 intracellular signaling pathway. Dendritic cells, in addition to TLR4, are also responsive to TLR2.
56 : In responding to HSP60, B-lymphocytes up-regulate their expression of: CD40 MHC II B7-1 , B7-2 costimulatory molecules CD69, an early activation marker B-lymphocytes, however, do not up-regulate the expression of ICAM-1 (CD54). T-lymphocytes respond to HSP60 via TLR2, B-lymphocytes, by contrast, respond to HSP60 via TLR4.
57 : The effects of: the HSP60/LPS complex on dendritic cells and of HSP60 on B-lymphocytes promote the capacity of the respective cell populations to act as antigen-presenting cells (APCs). This is an illustrative example of innate immunity passing over to acquired immunity.
58 : „Professional“ antigen presenting cells are represented by macrophages and dendritic cells. There are many other „non-professional“ APCs, endothelial cells being the most important ones from the surgical point of view. However, only the former ones, i.e., „professional“ APCs, can give rise to an antigen specific immune response. Fig. 11: HSP60/LPS complex-induced interaction between antigen presenting cells and T cells.
59 : ABBREVIATIONS: APC: Antigen Presenting Cell DC: Dendritic Cell GM-CSF: Colony Stimulating Factor for Granulocytes/Monocytes ICAM: InterCellular Adhesion Molecule IFN: Interferon IL: Interleukin LFA-1,-3: Leukocyte Function Associated adhesion molecules MHC: Major Histocompatibility Complex NF-?B: Transcription Factor NF-?B NOD: cytoplasmic receptor for danger patterns TcR: T-cell Receptor for antigen TNF?: Tumor Necrosis Factor ? TLR: Toll-Like Receptor Fig. 11: HSP60/LPS complex-induced interaction between antigen presenting cells and T cells.
60 :
61 : The external signal borne by the HSP60/LPS complex enters the intracellular space via the CD14/TIR receptor complex. Within the intracellular compartment, the signal is transmitted further by way of complex enzymatic cascades, all of which converge on the level of the transcription factor NF-?B.
62 : ABBREVIATIONS: sHSP60: soluble Heat Shock Protein ICAM: InterCellular Adhesion Molecule IL: Interleukin LPS: Lipopolysaccharide MyD88: Myeloid Differentiation factor 88 (an adaptor protein) NF-?B: Transcription Factor NF-?B TLR: Toll-Like Receptor VCAM: Vascular Cellular Adhesion Molecule Fig.12: sHSP60-initiated intracellular signal transduction pathways.
63 :
64 : In quiescent cells, e.g. those not affected by the HSP60/LPS?CD14?TIR signaling cascade, NF-?B remains in an inactive form within the cytoplasm. After the cell has been stimulated, an active form of NF-?B is made by assembling all of its individual components which, so far, were dispersed separately within the cell.
65 : After the assembly of all NF-?B components: the active form of the transcription factor is transferred from the cytoplasm to the nucleus here, transcription of the requisite genetic information from nuclear DNA to messenger RNA starts off.
66 : This process leads to de novo synthesis of: cytokines chemokines adhesion molecules growth factors. All of these newly formed proteins participate in the development of the inflammatory response. The syndrome of SIRS may be its resultant clinical manifestation. See also figure No.: 2
67 : Terminologically, SIRS may overlap with: the sepsis syndrome in cases of bacterial infection the „sepsis-like“ syndrome in cases of sterile inflammation.
68 : SIRS may be evoked both in septic in sterile inflammation. In the former case, microbial HSP60s, often in conjunction with the self HSP60s originating from the host´s damaged cells, serve to start off the inflammatory process. In the latter case, microbial HSP60s are absent and only the host´s own HSP60 molecules take part in the inflammation. In sterile conditions, HSP60 may be released from cells damaged by the cardiopulmonary bypass. ABBREVIATIONS: CpG: Bacterial DNA rich for Cytosine and Guanosine (Danger Pattern) HSP: Heat Shock Protein LPS: Lipopolysaccharide Fig. 13: HSP´s in sterile and septic inflammation.
69 :
70 : Basically, SIRS and SIRS-like reactions have been conserved during evolution in order to support the host so that he or she may survive in an unfriendly environment, such as: strenuous exercise of the „fight or flight“ nature multiple injuries or burns infections or, more recently, any major surgery, including cardiac surgery.
71 : ABBREVIATIONS: CPB: Cardiopulmonary Bypass HSP: Heat Shock Protein sHSP60: soluble Heat Shock Protein ox-LDL: oxidized-Low Density Lipoprotein SIRS: Systemic Inflammatory Response Syndrome TLR: Toll-Like Receptor Fig.14: Schematic view of a putative role of HSP60/70 in inflammatory steps converging in the development of SIRS.
72 :
73 : Whichever the underlying event, tight control of every step of the inflammatory processes must be exerted both on local and on systemic levels. It is here where activities of: the neuro-immune, the endocrine, the circulatory systems converge or overlap. If this control fails, morbidity and mortality increase dramatically. See also figure No.: 3
74 : Conceivably, the defense/inflammatory reactions leading to SIRS have not developed in response to cardiac surgery. Even less do they respond specifically to cardiopulmonary bypass. Nevertheless, the definitive extent of SIRS in cardiac surgical patients is fine-tuned by conditions relevant to the surgical procedure. These modifying conditions can be divided into three closely interrelated steps: contact activation of blood myocardial ischemia and reperfusion splanchnic hypoperfusion, with or without transient endotoxemia.
75 : In cardiac surgical operations performed with the use of CPB, the patients´ blood is temporarily diverted from the patient´s circulation into the tubing system of the heart-lung machine. This is a bona fide extra-anatomical vascular bed in which endothelial lining is totally absent.
76 : Contact of blood with the artificial material of the tubing system invariably results in: activation or priming of blood-borne cellular elements, notably the white blood cells and platelets activation of humoral immune/inflammatory cascades, such as: the complement system the kallikrein-kinin system the coagulation system.
77 : Plasma bactericidal capacity increases progressively even in the absence of any circulating bacteria due to activation of bactericidal enzymes such as: myeloperoxidase elastase phospholipase A2.
78 : Ongoing production of „proximal“ inflammatory cytokines such as: TNF-? IL-1? IL-6 elicits with some delay in time the synthesis of a huge wave of chemokines, such as: IL-8 MCP-1 RANTES which in turn support migration and accumulation of activated leukocytes in inflammatory foci. See also figure No.: 4A, 4B
79 : At the same time, synthesis of acute-phase reactants is set off, among which predominate: C-reactive protein (CRP) pentraxin-3 (PTX3) In the setting of clinically overt SIRS, both of these peptides lose their original protective activities at the expense of an overall damaging capacity.
80 : Both CRP and PTX3 activate the complement system via the alternative pathway. Ongoing production of anaphylatoxin C5a and the terminal complement complexes C5b-9(n) adds mightily to the damaging effects of cardiopulmonary bypass.
81 : During cardiac surgery, wheather or not performed with CPB, complement is invariably activated via the alternative pathway, which is set into motion via acute-phase reactants CRP and PTX3. Later on, a new wave of complement activaction occurs via the presence of heparine-protamine complexes. ABBREVIATIONS: B, D: factors of alternative pathway of complement activation C3: Component 3 of complement system MASP: Mannose Associated Serine Protease MBL: Mannose Binding Lectin Fig.15: Schematic review of complement activation.
82 :
83 : The phenotype of endothelial cells converts from the original non-adhesive anti-inflammatory quality into a pro-coagulant pro-inflammatory one. The latter phenotype is characterized by an unrestrained expression of: adhesion molecules coagulation factors. Inflammatory cytokines (TNF-?, IL-1?, IL-6) set off activation of the vascular endothelium.
84 : Concomitantly, synthesis of endogenous anti-inflammatory peptides such as: IL-10 TGF-? which in normal conditions would satisfactorily counter-balance the explosive activities of their pro-inflammatory counterparts, is substantially reduced.
85 : The blood-gas interface of the oxygenator, whose pro-inflammatory capacity is of extraordinary magnitude, supplies yet an activatory stimulus to all blood cellular elements and humoral cascades.
86 : The extent of the blood components activation is directly proportional to the duration of: aortic cross-clamping, which elicits global myocardial ischemia extracorporeal circulation of blood which, apart from contact activation of blood-borne elements, by-passes physiological oxygenation in the lungs. On the other hand, it must be emphasized that genetic background of each patient plays an important if not decisive role in these processes, including the outcome of SIRS (i.e. the SIRS?CARS or the SIRS?MODS/MOFS trajectory).
87 : In CPB patients, global myocardial ischemia is elicited by putting a cross-clamp on the ascending aorta. The heart itself has been arrested by a cardioplegic solution introduced into the aortic root (antegrade cardioplegia) and/or into the coronary venous sinus (retrograde cardioplegia). After the surgical procedure on the still-standing heart has been terminated, aortic cross-clamp is released and reperfusion of the myocardium sets in.
88 : Reoxygenation of any ischemic tissue unchains a complex array of inflammatory events. Many of the pro-inflammatory cascades have been alerted during the period of oxygen deprivation by way of transcription factors HIF-1? (hypoxia-inducible factor) Egr-1 (early growth response) Reintroduction of oxygen-rich blood overflows the heart with pro-inflammatory cytokines, mainly: TNF-? IL-6 Interleukin-6 is the predominant mediator elaborated by ischemic myocardial tissue.
89 : The „proximal“ cytokines (TNF-?, IL-1?, IL-6) incite up-regulation of endothelial adhesion molecules, such as: ICAM-1 (intercellular adhesion molecule) VCAM-1 (vascular cell adhesion molecule) E-selectin. Their presence on the endothelial surface sets the stage for leukocyte-endothelial interactions. Thereafter, activated neutrophils and monocytes transmigrate into the interstitial space of reperfused tissues. See also figure No.: 4A, 4B
90 : In CPB patients, all these events take place not only in the heart, but also in the lungs, whose blood perfusion has been interrupted during extracorporeal circulation with only bronchiolar arterioles remaining in a perfused state. The latter do not belong to the pulmonary circulation, but to the systemic circulation. As such, they do not take part in blood gas exchange.
91 : Activated neutrophils attack all cells, irrespective of: whether they are the host´s own cells that have been so far happy to survive the period of oxygen deprivation, whether they are pathogenic micro-organisms that must be disposed of as soon as possible. The cytotoxic armamentarium of activated neutrophils is made up by proteolytic enzymes and reactive oxygen species (ROS). Out of the latter, the superoxide radical O2?¯ is a relatively moderate weapon, whereas the hydroxyl radical ?OH is an extremely aggressive oxidant. Interstitial space of reperfused tissues is reminiscent of a virtual battle-field.
92 : Reintroduction of oxygen-rich blood into a previously ischemic tissue (e.g., the heart after the cross-clamp release) unchains an explosive free radical storm. The same occurs on the whole-body level by way of reactive oxygen species („free“ radicals) which have been generated via the effects of cardiopulmonary bypass. ABBREVIATIONS: BH4: Tetrahydrobiopterin EC: Endothelial Cells GC: Guanylate Cyclase iNOS: inducible form of NO-Synthase MAPK: Mitogen Activated Protein Kinase NADPH: NicotineAmide Adenin Dinucleotide-Phosphate NO: Nitrogen Oxide PKC: Protein Kinase C Fig.16: Oxidative stress in surgically reperfused heart.
93 :
94 : During reperfusion of globally ischemic myocardium, ROS generated by activated neutrophils attack cardiac muscle cells which are prone to die by necrotic or apoptotic death. Cardiac muscle cells that have so far withstood free radical attack are successful in their effort only at the cost of limiting their metabolic demands and biomechanical performance next to zero.
95 : By the time the reperfusion/reoxygenation of the globally ischemic myocardium in cardiac surgical patients has been achieved, many heart muscle cells are stunned or dead. Functional disorders which afflict the overall cardiac performance, are heralding the clinical picture of the early/immediate postoperative period. See also figure No.: 3
96 : The extent of the end-organ damage which has been inflicted by leukocyte-derived ROS depends for the most part upon the quality of myocardial protection achieved by cardioplegic solutions in the arrested heart. This exogenous protection is reinforced by endogenous protective mechanisms pertaining to the heart itself.
97 : Apart from anti-oxidant enzymes which, due to space limitations will not be discussed here, an important, if not yet entirely understood protection, is afforded by heat shock proteins. An outstanding member of the heat shock protein family which confers support to ischemic and reperfused myocardial cells is HSP70.
98 : The loose denomination of „HSP70“ encompasses at least two members of this protein family, namely: the constitutive form of HSP73, alternatively known as HSC70 the stress-inducible form of HSP70, also referred to as HSP72, which will be the leading topic of the following text A novel HSP70-like protein 1 (HSP70L1) has been discovered recently. HSP70L1 is a dendritic cell-derived protein, which skews immune responses in favor of the Th1 pathway. HSP70 is expressed in many organs including the heart.
99 : HSP70 is an intracellular protein, which under stress condition trans- locates from the cytosol into the nucleus. In case of cell death, HSP70 is set free just like HSP60. Once released into the extracellular space, HSP70 exerts activatory effects on the immune cells which are closely reminiscent of activities of HSP60. ABBREVIATIONS: HSP: Heat Shock Protein Fig.17: The HSP70 family.
100 :
101 : HSP70 plays a prominent role in manifold processes which retain both cellular metabolism and organ (i.e., cardiac) function in the course of ischemia/reperfusion- or hypoxia/reoxygenation-induced injury. Under conditions of cellular stress, HSP70 is translocated from the cytosol to the nucleus, where it enters into contact with the chromatin-binding enzyme called poly(ADP-ribose)polymerase.
102 : The quiescent form of this sensor enzyme is converted into an active one in the process of searching for nuclear DNA single-stranded breaks. Poly(ADP-ribose)polymerase also performs important activities in the repair of damaged DNA.
103 : Furthermore, this enzyme is expected to play a protective role in the process of reperfusion injury. After the oxygen supplies of ischemic cells have been replenished, continuing activation of poly(ADP-ribose)polyme- rase may exceed actual metabolic demands. Exaggerated activation of poly(ADP-ribose)polymerase during periods of reperfusion or reoxygenation will consume large amounts of cellular ATP and NAD+.
104 : Poly(ADP-ribose)polymerase is a branched polymer synthesized on acceptor proteins by PARPs using NAD+ as a donor of ADP-ribose units. Fig.18: Poly(ADP-ribose)polymerase synthesis.
105 :
106 : The cytoprotective and death-promoting effects of poly(ADP-ribosyla- tion) represent two opposing faces (the Yin and Yang paradigm). Oxidative stress-induced DNA breakage results in a high level of PARP activation, which leads to the depletion of intracellular NAD+ and ATP content, respectively, and consequently, to cellular death. On the other hand, poly(ADP-ribosylation) facilitates DNA repair in cells subjected to extensive free radical attack of e.g. ionizing radiation or ischemia/reperfusion. ABBREVIATIONS: ATP: Adenosine TriPhosphate NAD: Nicotin Amide Dinucleotide PARP: Poly(ADP-Ribose)Polymerase Fig.19: The Yin-Yang paradigm of PARP activation.
107 :
108 : The net result will be another form of lethal energy depletion. Conceivably, cells that have scarcely survived the stress of oxygen deprivation, will suffer another metabolic blow. Conversely, poly(ADP-ribose)polymerase inhibitors may ameliorate damage suffered by reperfused hearts.
109 : Exaggerated increase in the poly(ADP-ribose)polymerase activity in hypoxic/reoxygenated myocardial cells can be curtailed by heat shock treatment which results in HSP production. Out of various heat shock proteins which are thus generated, HSP70 represents the most abundant form. Hypoxia/reoxygenation-induced increase in poly(ADP-ribose)polymerase activity has been shown to relate inversely with post-hypoxic cell viability which, by contrast, is proportional to HSP70 content in the nuclei of affected cells.
110 : An alternative cytoprotective and prosurvival mechanism exerted by HSP70 under stress conditions resides in its capacity to inhibit lysosomal membrane permeabilization. Originally, lysosomes were considered „suicide bags“ which, through the release of unspecific enzymes, cause autolysis and tissue damage in uncontrolled or poorly controlled inflammatory/innate immunity reactions.
111 : Accumulating data show that lysosomes also function as death signal integrators. Cytotoxic lysosomal proteases and cathepsins translocate from the lysosomal lumen to the cytosol in response to a variety of cellular death stimuli, such as: TNF-? Fas/FasL p53 activation oxidative stress.
112 : Once released in the cytosol, cathepsins – especially cysteine cathepsins B and L and aspartyl cathepsin D – will incite cellular death featuring apoptosis- or necrosis-like morphology. HSP70 may effectively rescue cells form both apoptotic and necrotic death which is induced by: proinflammatory cytokines, notably TNF-? oxidative stress.
113 : The almost universal prosurvival capacity of HSP70 has got also its less friendly face, „the hidden side of the moon”. This protein has been found in many tumor cells which, due to its expression, can escape a timely removal from the tumorous tissue.
114 : If myocardial damage exceeeds the cytoprotective capacity of HSP70, this protein is released from the intracellular compartment into the extracellular space. As was the case with HSP60, HSP70 comes into the first contact with the surveillance mechanisms of the immune system.
115 : HSP70 activates immune cells by way of CD14 membrane receptor TLR-2 and/or TLR-4 membrane receptors. The cellular interior, in turn, is activated through the MyD88?IRAK?NF-?B cascade. As a result, the transcription factor NF-?B translocates from cellular plasma to the nucleus. See also figure No.: 12
116 : After cardiac surgery, extracellular presence of HSP70 is found almost regularly. This implies that some cardiomyocyte death has occurred despite all precautions taken. Peak circulation levels of HSP70 can be seen as soon as two hours after the beginning of surgery. These levels correlate closely with the duration of: the aortic cross-clamping the extracorporeal circulation.
117 : As has been found by Brit Dybdahl and coworkers, patients operated on without the use of CPB, i.e. the „off-pump“ patients, release as little as one fourth of the HSP70 amount released by „on-pump“ patients. Both groups of patients display some extracellular HSP70 even in wholly uncomplicated peri- and post-operative course.
118 : Peak postoperative levels of circulating HSP70 have been found to correlate with peak levels of troponin T MB isoenzyme of creatine kinase if some perioperative damage to the heart muscle has been inflicted.
119 : Moreover, a strong correlation between HSP70 and IL-10 levels has been proved. This correlation, however, is demonstrable only in „on-pump“ (i.e., CPB) patients. Their „off-pump“ counterparts release negligible amounts of extracellular IL-10. Futhermore, there is significant correlation between HSP70 and IL-6 levels, irrespective of the type of cardiac surgical operation.
120 : The target cells of extracellular HSP70 are predominantly monocytes, which are activated via the CD14?TLR4 pathway. The result of these serial events is an important support to the activation of inflammatory responses. In order for the surgical patient to act counter to the unwonted inflammation, monocyte receptors CD14, TLR-2 and TLR-4, respectively, are down-modulated immediately after surgery. In the following 24-48 hours, their density on monocyte surfaces is either normalized or up-regulated.
121 : Transient endotoxemia is the result of insufficient blood supply to the splanchnic vascular bed after a substantial amount of its volume has been excluded from the patient’s own vasculature into the tubing circuit of the heart-lung machine. Relative hypoperfusion of the splanchnic vascular bed underlies gut wall ischemia, manifested by: an increase of villous capillary permeability translocation of the endotoxin of Gram-negative bacteria or even the patient’s own intestinal flora into the systemic circulation.
122 : Scavenging capacity of the hepatic reticuloendothelial system, namely the Kupffer cells, which normally set up a reliable barrier against bacterial translocation from the portal vein blood into systemic circulation, is rapidly overwhelmed and broken down.
123 : Splanchnic hypoperfusion during cardiac surgery cannot be entirely prevented even by ample substitution of blood volume using blood transfusions, much less so by way of crystalloid and/or colloid fluid resuscitation. One reason of this therapeutic failure can be seen in laminar blood flow generated by roller pumps of the heart-lung machine instead of pulsatile flow generated with each contraction of the patient’s own heart.
124 : LPS concentrations in peripheral blood of cardiac surgical patients can increase up to levels which facilitate parallel entry into the blood stream of extracellular HSP60, even in the absence of bacterial infection. An intriguing question stands out whether the patient’s own commensal flora, which can supply large amounts of its HSP60, should be regarded as undergoing temporary conversion from symbiosis to infection.
125 : Whichever the case, it seems plausible to hypothesize that in cardiac surgical patients, whether or not surgery has been performed with the use of CPB, the ability of endogenous HSP60 to bind circulating LPS and to present the HSP60/LPS complex to the CD14?TLR2/TLR4?NF-?B signal transduction pathway is increased to an extent which can start off postoperative SIRS. See also figure No.: 12, 14
126 : Acknowledgments: The authors feel deeply indebted to Mrs. Hana Kotlandova for excellent technical assistance. Funding: This work was supported by Ministry of Education, Czech Republic, project No. MSM0021620812.


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