Pathophysiological Mechanisms Of Septic Shockdoc

=== Pathophysiological mechanisms of septic shock ===

Ministry of Health of the Republic of Moldova

State University of Medicine and Pharmacy

"Nicolae Testemițanu"

GENERAL MEDICINE DEPARTMENT

Chair of Physiopathology and Clinical Physiopathology

THESIS

PATHOPHYSIOLOGICAL MECHANISMS OF SEPTIC SHOCK

Name of the student

year , group VI, gr. 16

Scientifical coordinator Feghiu Iuliana

assistant professor

Chișinău 2016

SUMMARY

INTRODUCTION………………………………………………………………..…

1.1. Actuality and medical importance of the studied problem…………………………

1.2. Aim and goals of the thesis………………………………………………………………..

1.3. Scientifical aspects of the results……………………………………………………….

1.4. Theoretical and applicative value of the thesis…………………………………………..

MECHANISMS OF SEPTIC-INDUCED MYOCARDIAL DYSFUNCTION

Mithocondrial dysfunction in septic shock……………..

Contractile dysfunction in septic shock …………………………….

Myofibril dysfunction in septic shock………………

ROLE OF INFLAMMATION AND INFLAMMATORY CYTOKINES IN DEVELOPMENT OF SEPTIC SHOCK……………………………………………

3.1. Role of inflammatory cytokines in development of septic shock…………

3.2. Role of TNF in development of septic shock…………………………….

3.3. Role of interleukins in septic shock ………………………………………..

3.3. Role of nitric oxide in pathogenesis of septic shock……………………………..

3.4. Role of other cytokines in septic shock…………………

ROLE OF ENDOTHELIAL CELL IN DEVELOPMENT OF SEPTICK SHOCK …

DISCUSSIONS……………………………………………………………………

CONCLUSSIONS……………………………………………………….

BIBLIOGRAPHY…………………………………………………………………..

INTRODUCTION

Actuality and medical importance of the studied problem

One of the most frequent and serious problems that clinicians face is the management of serious infections that trigger a systemic inflammatory response is septicemia. When sepsis results in hypotension and organ dysfunction, it is referred to as septic shock. Septic shock is the most common cause of death in intensive care units. In the USA alone it is estimated that more than 100 000 deaths occur each year due to septicemia and septic shock [4,12,22].

Sepsis and septic shock are common causes for admission to intensive care units (ICU). Despite aggressive treatment, the morbidity and mortality of these conditions remain unacceptably high. An epidemiological study in European ICU’s shows an incidence of 37% for sepsis and 30% for severe sepsis. Sepsis is one of the most common causes of death in European ICU. The mortality rate for sepsis is 27% and rises from 32% for severe sepsis up to 54% for septic shock. The most common infection leading to sepsis is pneumonia, followed by abdominal infections, primary bacteremia and urinary tract infections [24,31].

In the United States, sepsis and septic shock are the 10th most common cause of death with an incidence of sepsis-related deaths increasing by 1,5 % a year. Thus, sepsis and septic shock interfere significantly with national health care [22,33].

Despite continued advances in medicine and technology, the incidence of sepsis is increasing. From 2000 to 2008, hospitalizations for sepsis more than doubled from 326 000 to 727 000 according to the Center for Disease Control report

examining hospital admission data from the National Hospital Discharge Survey. With an in-hospital mortality rate ranging from 15 to 30%, sepsis represents the 11th

leading cause of death in the United States and is responsible for 7% of all childhood deaths. In addition to the many years of life which are lost, sepsis represents a significant economic burden with total hospital costs in 2007 for patients with severe sepsis estimated to be $24.3 billion nationally, a 57% increase from 2003 [5,8,9, 22,30,55].

The mortality rate in patients with septic shock ranges from 20 to 80%, and it can be related to both the severity of sepsis and the underlying disorder. Systemic inflammatory response can be triggered not only by infections, but also by noninfectious disorders such as trauma and pancreatitis. Sepsis associated with hypoperfusion that results in organ dysfunction syndromes, such as oliguria, lactic acidosis and altered mental function, and/or in hypotension can be referred to as septic shock, and has a poor prognosis. As the population ages, the chances are that physicians may have to manage more and more patients with septicemia and septic shock. In view of this, it is important that the underlying pathophysiologic mechanisms of this syndrome are well understood [4,7,36,54].

Septic shock is a multisystem response to infection and/or injury in which hypotension and insufficient perfusion of vital organs occurs that does not respond to fluid administration. It is believed that septic shock is due to inappropriate increase in innate immune response. Hence, to learn about septic shock, one has to understand how innate immunity functions and interacts with cell-mediated immune responses and the antibody recognition system [13,14].

The survival of humans and animals depends on their ability to recognize invading pathogenic organisms and respond to them rapidly and adequately. These defenses against microbial organisms are innate to the particular organism. The immune system is programmed to recognize the biochemical patterns displayed by these microbial organisms and to mount rapid responses to them. The innate immune system includes neutrophils, macrophages and natural killer cells, which can act directly against invading pathogens and eradicate them without involvement of the adaptive immune system. However, when necessary, these cells of the innate immune system release cytokines and express certain other stimulatory molecules that, in turn, can trigger adaptive immune responses by activating T and B cells. The adaptive immune system differs from the innate immune system in that it is highly

evolved, specific in its responses and ‘remembers’ the antigens presented to it. Thus, the innate immune response is a nonspecific one that attempts to keep the invading foe at bay until the adaptive immune system is ready with its more specific antibodies and T cells [1,10,23].

Myocardial depression is a well-recognized manifestation of organ dysfunction in sepsis. Importantly, the presence of cardiovascular impairment in sepsis is associated with a significantly increased mortality rate of 70 to 90 % compared with 20 % in septic patients without cardiovascular dysfunction. Animal experiments suggest left ventricular dilatation but this was not confirmed in a human study. A hypercirculatory state is associated with decreased systemic vascular resistance and a markedly increased cardiac index after adequate fluid resuscitation. Neither the pulmonary artery catheter, nor various regional and metabolic monitoring systems, have allowed progress in revealing the different hemodynamic patterns of septic shock. A comprehensive study suggested clearly both systolic and diastolic dysfunction, utilizing at that time two-dimensional and pulsed wave Doppler transoesophageal echocardiography [19,30,41].

Sepsis-related changes in the circulating volume and vessel tone inevitably affect cardiac performance. These are nevertheless not the only underlying mechanisms of myocardial depression seen in sepsis. For example, mitochondrial dysfunction endangers the energy supply of the cardiomyocytes. Furthermore, down-regulation of beta-adrenergic receptors, depressed post-receptor signalling pathways, reduced calcium liberation from the sarcoplasmic reticulum and impaired electromechanical coupling at the myofibrillar level also play an important role. Cardiac depression during sepsis is clearly multifactorial [19,25,30,32].

Most of these mechanisms are regulated by cytokines, mainly TNFα and IL-1β, and nitric oxide. Note that myocardial depression can also be seen as an adaptation to limited cellular energy reserve. Activation of signalling pathways that lead to cellular death is hereby prevented and complete recovery is still possible.

The inflammatory response in sepsis leads to hemodynamic and circulatory changes. The degree of these alterations depends on many factors, as immune status of the patient, cardiovascular status of the patient and concomitant organ dysfunction. Peripheral circulation failure arises in consequence of vasodilatation, an inadequate regional blood flow and an abnormal microcirculation. Initially, during septic shock, patients encounter an early hyperdynamic phase with a high cardiac output from which they either recover or decline into a late-stage hypodynamic phase. This hyperdynamic status may be very short owing the overwhelming immune response and associated developing organ dysfunction. When hypovolemia (causing a persistent low preloading condition) appears and the myocardium gets damaged by the inflammatory response, the cardiac output lowers and the peripheral resistance rises in an attempt to keep the blood pressure stable. Several studies have clearly established decreased contractility and impaired compliance (due to myocardial edema and vascular leakage) as major factors that cause myocardial dysfunction is sepsis. Reduced cardiac output, with or without reduced cardiac compliance, causes an oxygen supply-demand imbalance. The oxygen delivery to the cell does not meet the cellular need. Patients in severe sepsis and septic shock present a 30 % increase of oxygen consumption and baseline metabolism compared with normal basal values. However, during multiple organ failure, patients seem to endure lower values of oxygen supply. It has therefore been speculated whether cells utilize less oxygen rather than suffering from a defective oxygen delivery to tissues during severe sepsis [19,25,32,37].

The purpose of the present review is to outline the most commonly reported underlying mechanisms of septic shock.

Aim of the thesis.

Goals of the thesis are:

Scientifical aspects of the results:

Theoretical and applicative value of the thesis:

MECHANISMS OF SEPTIC-INDUCED MYOCARDIAL DYSFUNCTION

Myocardial depression is a well-recognized manifestation of organ dysfunction in sepsis. Importantly, the presence of cardiovascular impairment in sepsis is associated with a significantly increased mortality rate of 70 to 90 % compared with 20 % in septic patients without cardiovascular dysfunction. Animal experiments suggest left ventricular dilatation but this was not confirmed in a human study. A hypercirculatory state is associated with decreased systemic vascular resistance and a markedly increased cardiac index after adequate fluid resuscitation [11,19,37].

Sepsis-related changes in the circulating volume and vessel tone inevitably affect cardiac performance. But these are not the only underlying mechanisms of myocardial depression seen in sepsis. More mechanisms are responsible for myocardial dysfunction in septic patients [4,11,34].

From these the most plausible involved mechanisms are:

Mitochondrial dysfunction which endangers the energy supply of the cardiomyocytes;

Down-regulation of beta-adrenergic receptors;

Depressed post-receptor signalling pathways;

Reduced calcium liberation from the sarcoplasmic reticulum;

Impaired electromechanical coupling at the myofibrillar level [4,19,25,32,48].

Cardiac depression during sepsis is clearly multifactorial. Most of these mechanisms are regulated by cytokines, mainly TNFα and IL-1β, and nitric oxide. Myocardial depression can also be regarded as an adaptation of the heart to limited cellular energy reserve. Activation of signalling pathways that lead to cellular death is hereby prevented and complete recovery is still possible [6,7,49].

In severe sepsis or septic shock, two major hemodynamic effects must be considered: relative hypovolemia and cardiovascular depression. Hemodynamic optimization is attempted by manipulating preload, afterload and myocardial contractility in order to restore systemic oxygen supply [11].

The inflammatory response in sepsis leads to hemodynamic and circulatory changes. The degree of these alterations depends on many factors like immune status of the patient, cardiovascular status of the patient and concomitant organ dysfunction. Peripheral circulation failure arises in consequence of vasodilatation, an inadequate regional blood flow and an abnormal microcirculation. Initially during septic shock, patients encounter an early hyperdynamic phase with a high cardiac output from which they either recover or decline into a late-stage hypodynamic phase. This hyperdynamic status may be very short owing the overwhelming immune response and associated developing organ dysfunction. When hypovolemia (causing a persistent low preloading condition) appears and the myocardium gets damaged by the inflammatory response, the cardiac output lowers and the peripheral resistance rises in an attempt to keep the blood pressure stable [30,32,37].

Several studies have clearly established decreased contractility and impaired compliance (due to myocardial edema and vascular leakage) as major factors that

cause myocardial dysfunction is sepsis. Reduced cardiac output, with or without reduced cardiac compliance, causes an oxygen supply-demand imbalance. The oxygen delivery to the cell does not meet the cellular need. Patients in severe sepsis and septic shock present a 30 % increase of oxygen consumption and baseline metabolism compared with normal basal values. However, during multiple organ failure, patients seem to endure lower values of oxygen supply. It has therefore been speculated whether cells utilize less oxygen rather than suffering from a defective oxygen delivery to tissues during severe sepsis [11,37,41,48].

Mithocondrial dysfunction in septic shock

By generating adenosine triphosphate (ATP), a triphosphate that supplies energy for various metabolic key processes, mitochondria are by far the most important cellular energy providers. The mechanism of producing ATP by the mitochondria is called oxidative phosphorylation. NO, TNFα en IL-1β, which levels are increased in septic shock, have inhibitory effects on the oxidative phosphorylation [25,29,40].

Tissue injury during sepsis and multi-organ dysfunction has been related to mitochondrial derangement. Early damage of the double mitochondrial membranes independently appears to influence the permeability. Endotoxin administered to volunteers augments the activity of some mitochondrial enzymes in muscle cells, which appeared to be independent from AMP-activated protein kinase activity. During septic shock there is increased production of superoxide and NO. High level of NO in combination with depletion of intra-mitochondrial antioxidants during sepsis also reduces oxidative phosphorylation. This acquired defect has been termed “cytopathic hypoxia”. Hereby the ATP-production of the cardiomyocytes is endangered, which contributes to myocardial depression [11,25,40,48,56].

Furthermore, energy depletion increases the risk of cellular death. Post-mortem studies of patients in septic shock have revealed that myocardial cell death is rare, despite the presence of cellular hypoxia, and thus is not sufficient to contribute to the observed cardiac dysfunction. Survivors had a fully restored cardiac function within 7 to 10 days [25,34,49].

Contractile dysfunction in septic shock

Disorders in adrenergic stimulation in septic shock

Cardiomyocytes contain β1-adrenergic receptors. These receptors are coupled to Gs – proteins that stimulate signalling transmission. Binding of a ligand to the receptor will activate the adenylylcyclase enzyme, through the coupled Gs– protein. Catecholamines are ligands for the β1-adrenerge receptors and in consequence activate the adenylylcyclase. This enzyme will transform ATP to cyclic adenosine monophosphate (cAMP). cAMP, in his turn, activates protein kinase A (PKA), which phosphorylates key enzymes that will finally stimulate both cardiac contractility and heart rate [11,48].

By the presence of hypotension in sepsis and septic shock, the body first responds by releasing catecholamines, mainly adrenaline and noradrenaline. Noradrenaline is primarily released by noradrenergic neurons of the orthosympatic nerve system. Adrenaline on the other hand is produced by chromaffin cells of the adrenal medulla. Short-term stimulation of the β1-adrenergic receptors with these catecholamines increases cardiac contractility and heart rate. In contrast, long-term and excessive stimulation can lead to myocardial damage by calcium overload. However, certain adaptive mechanisms are identified at the cardiomyocyte level in response of these prolonged elevated catecholamine levels as happening in septic shock [19,30,37].

In septic rats, the myocardial density of β1-adrenergic receptors was found to be diminished. Additionally, in both septic animals as septic patients, inhibitory G-proteins (Gi–proteins) were increased post-mortem. These changes lead to a decreased activity of adenylyl cyclase and hence a reduced formation of cAMP and PKA [32,56].

Furthermore, the sepsis-induced excessive release of NO stimulates the production of cyclic guanosine monophosphate (cGMP), that activates protein kinase G (PKG). This enzyme has an inhibitory effect on the formation of PKA, with a negative cardiac inotropic and chronotropic effect as a consequence [25,48].

Changes in calcium ion house holding in septic shock

With the more recent insights on the molecular basis of cardiac failure, in which it is accepted that calcium sensitivity of the myofilaments is at least decreased, a significant reduction of serum calcium has been shown in patients with acute decompensated heart failure [11]. In septic shock however, the potential advantages on improved outcome have not yet been demonstrated. In a rabbit based septic shock model, levosimendan was tested and appeared at least beneficial on cardiac function, in conjunction with noradrenaline [48].

Calcium plays an important role in muscle contraction. The concentration of free calcium in the interstitial fluid is about 12000 times larger than the cytoplasmatic concentration. An inwards directed calcium current occurs as a result of this transmembrane concentration gradient. Depolarisation of the cardiomyocyte sarcolemma opens voltage gated L-type calcium channels. A limited amount of calcium flows into the cell and binds to the ryanodine receptors located on the sarcoplasmic reticulum . This evokes the release of a much larger quantity of calcium from the sarcoplasmic reticulum: “calcium induced calcium release”. Binding of calcium on troponine C reveals the binding area of myosine on actine. When binding of myosine to this area takes place, this induces a configuration change in myosine provoking muscle contraction. So, finally we can state that the intracellular calcium concentration determines the force of contraction [19,30]. Calcium can also bind to calmoduline, forming a calcium-calmoduline complex. This complex activates the endothelial NO synthase (e-NOS) resulting in NO-production. To permit diastolic relaxation, the released calcium needs to be removed again from the cytosol back into the sarcoplasmic reticulum. This is mostly regulated by the Sarcoplasmic Endoplasmic Reticulum Calcium Adenosine triphosphatase, or SERCA-pump. SERCA activity is regulated by the protein phospholamban. A small part of calcium is removed into the mitochondria and transferred extracellularly by a sodium-calcium pump. Hence mitochondrial integrity is also of significant importance [25,32].

Endotoxin and cytokines released in septic conditions suppress voltage-gated L-type calcium channels in isolated rat cardiomyocytes. Hereby the intracellular calcium concentration is decreased and cardiac contractility is reduced. Furthermore endotoxin opens ATP-dependent potassium channels, thereby shortening action potentials and diminishing calcium currents. Ryanodine receptor density is also decreased, resulting in impairment of calcium-induced calcium release and, again, reduced cardiac contractility [25,56]. Disturbed diastolic calcium uptake into the sarcoplasmatic reticulum is especially seen in the late phase of sepsis and is associated with a deficient phosphorylation of certain proteins, such as phospholamban, and an increase of cAMP, leading to changes in the β-adrenergic signalling as described earlier. The decrease of the sarcoplasmatic reticulum calcium content not only reduces the availability of releasable calcium but also decreases the driving force for calcium efflux from the sarcoplasmatic reticulum, leading to depressed cardiac function [11,19,37,49].

Myofibril dysfunction in septic shock

Postmortem immunohistochemical analysis of cardiomyocytes in septic shock patients shows a partial disruption of myofilaments. This is possibly due to increased matrix metalloproteinase activity, as these enzymes can demolish both the cytoskeleton as the cellular contractile device. Also a reduced calcium sensibility of myofibrillar proteins seems to contribute to myocardial depression seen in sepsis [19,49,56].

ROLE OF INFLAMMATION AND INFLAMMATORY CYTOKINES IN DEVELOPMENT OF SEPTIC SHOCK

3.1 Role of inflammatory cytokines in development of septic shock

The syndrome of sepsis is triggered when bacteria, usually within a nidus of infection (pneumonia, abscess), release exotoxins (more frequently involved in toxic shock syndrome is toxin 1 or streptococcal pyogenic toxin A) or bacterial constituents into the local or systemic environment of the host. Bacterial constituents may include cell wall components (endotoxin in Gram-negative bacteria; techoic acid and other antigens in Gram-positive bacteria) and perhaps bacterial DNA. These products stimulate the generation of pro-inflammatory cytokines both locally and systemically [2,15,43].

Pro-inflammatory cytokines have multiple effects including the stimulation of production and release of other pro-inflammatory mediators. TNF-α and interleukin-1β (IL-1β) are the major, known pro-inflammatory cytokines and have overlapping and synergistic effects in stimulating the inflammatory cascade. The next phase in the cytokine response to infection is the endogenous counter inflammatory cascade in response to the systemic activity of pro-inflammatory cytokines. Cytokine inhibitors (IL-1 receptor antagonist [IL-1ra], soluble TNF receptor) and anti-inflammatory cytokines (IL-4, IL-10 and IL-13) are involved in this phase of the response. Thus, the cytokine network in sepsis involves pro-inflammatory cytokines, anti-inflammatory cytokines and cytokine inhibitors. It is the balance between these cytokines at different time points that determine the clinical manifestations and outcome of sepsis [4,7,53].

Gram-negative sepsis. Gram-negative bacteria such as E. coli have an endotoxin or lipopolysaccharide (LPS) component to their outer membrane. LPS released into the circulation may be bound by a specific protein lipopolysaccharide binding protein (LBP). The LBP-LPS complex is recognized by macrophages, which causes it to secrete potent cytokines (including TNF-a and interleukin-1). In addition, LPS can stimulate lymphocytes to produce interferon gamma (IFN-y), which intensifies the macrophage output of TNF-α and interleukin-β1. The amount of the mediators produced by the macrophage presumably depends on the intensity of the stimulus. It is possible that, in extreme manifestations, the output of TNF-α and interleukin-1β is so great that it produces an overwhelming induction of iNOS in vascular endothelial and smooth-muscle cells; this would result in pressor refractory, long-lived, severe vasodilatation, which is a characteristic phenomenon in septic shock [14,25,41].

Gram-positive sepsis. Bacterial products, including superantigens, of gram-positive organisms may induce the massive activation of host lymphocytes, which then produce cytokines such as interleukin-2 and IFN-γ that, in turn, stimulate macrophages. It has been proposed that some products of the gram-positive cell wall interact with LBP in the same manner as endotoxin to produce effects similar to LPS-LBP. In animals, Staphylococcus aureus cell wall components peptido-glycan and lipoteichoic acid act together to release TNFa and IFNγ and cause shock with iNOS expression [2,30,31].

3.2. Role of TNF in development of septic shock

TNF-α, called cachectin, was the first major cytokine implicated in the pathogenesis of septic shock. TNF-α is a 17 kDa protein produced principally by macrophages. TNF- α has many effects. The most important are:

Activates monocytes, macrophages and neutrophils;

Stimulates neutrophil margination;

Induces acute phase proteins (via IL-6);

Produces fever (via IL-1β);

Increases gluconeogenesis and protein synthesis [2,7,17].

Two distinct cell surface receptors for TNF-α have been described. Type I with a molecular weight of 55 kDa and type II with a molecular weight of 75 kDa. Stimulation of type I TNF-α receptors reproduce TNF-α activities as cytotoxicity, activation of NF-κB and expression of adhesion molecules on endothelial cells. Both receptors also occur in soluble forms and conserve their affinity for TNF-α. The soluble receptors compete with cell surface receptors for free TNF-α binding. Since free TNF-α is inactivated when bound by soluble receptor, soluble receptor generation represents an anti-inflammatory or regulatory response element [6,14,29].

Studies have documented elevated levels of circulating TNF-α in clinical sepsis and septic shock. Administration of endotoxin to humans results in a marked increase in serum TNF-α concentration concomitant with cardiovascular and metabolic perturbations qualitatively similar to human sepsis/septic shock [10,53]. In animal models, administration of recombinant TNF-α causes fever, lactic acidosis, disseminated intravascular coagulation, non-cardiogenic pulmonary edema and death. The cardiovascular pattern of such animals is similar to that of spontaneous human septic shock and is characterised by hypotension, increased cardiac output and low systemic vascular resistance. Similarly, many physiological and laboratory manifestations of septic shock are reproduced in humans receiving TNF-α infusions. These include chills, rigors and metabolic acidosis with respiratory alkalosis, leukopenia, thrombocytopenia and increased international normalization ratio. In addition, a hyperdynamic circulatory state with tachycardia, hypotension, increased cardiac output and decreased peripheral vascular resistance is typical [15,23,25].

High TNF-α levels correlate with severity and poor outcome in patients with sepsis. When given prior to challenge, antibodies to TNF-α protect mice and rabbits from endotoxic shock as well as protecting baboons from a lethal parenteral infusion of Escherichia coli. With respect to sepsis, TNF-α knockout mice were shown to be resistant to endotoxin-induced hemodynamic collapse and death [32,38,39].

3.3. Role of interleukins in development of septic shock

IL-1β is a 17 kDa polypeptide produced by mononuclear phagocytes and neutrophils. IL-1α is a related cytokine with similar activity. IL-1β increases cellular antimicrobial activity and stimulates the production of IL-6, IL-8 and TNF-α. IL-1ra is a circulating soluble inhibitor of IL-1β activity that is produced by the same cells as IL-1β. IL-1ra binds to IL-1β cell surface receptors with the same affinity as IL-1β. Binding of IL-1ra to cell surface receptors blocks IL-1β biological activity [6,10].

Most of the biological effects of TNF-α are also seen with IL-1β. Release of IL-1β is increased in experimental human and animal models of sepsis and septic shock (including endotoxic shock) and in clinical sepsis and septic shock. When elevated in clinical septic shock, IL-1β levels correlate with increased mortality. As with endotoxin, administration of TNF-α to humans and animals results in increased circulating levels of IL-1β. Infusion of IL-1β into animals reproduces many of the cardiovascular and metabolic manifestations of septic shock including prolonged (≥ 1 week) depression of ventricular ejection fraction in some models. The infusion of IL-1β to rabbits causes hypotension and a leukocyte infiltration in the lungs. Infusion into humans results in fever, chills, rigors, thrombocytopenia and marked cardiovascular abnormalities including hypotension [1,7,23,40,44].

IL-6 is a 21 kDa protein produced by activated monocytes, macrophages, endothelial cells, fibroblasts and activated B and T lymphocytes. IL-6 induces acute phase hepatic protein production and activates the coagulation system. When infused experimentally into animals, IL-6 produces mild clinical symptoms (chills and fever) without the hemodynamic changes or toxicity seen with TNF-α and IL-1β [43]. The role of IL-6 as a pro-inflammatory cytokine is not fully understood. However, high levels of IL-6 consistently correlate with poor outcome in sepsis patients, suggesting it is an important marker of systemic inflammation [14,15,31].

3.4. Role of nitric oxide in pathogenesis of septic shock

NO is unstable and has a life span of a few seconds. Because of its short half-life, the effects of NO must occur over short distances, and biologically active NO must be synthesized either within the cell (autocrine) or by cells nearby (paracrine). In aqueous solutions, such as plasma, NO is oxidized mainly to nitrite, which, in the presence of hemoglobin, is quickly oxidized to nitrate. Some of the physiologic characteristics of NO are related to its ability to bind to heme. The binding of NO to heme in hemoglobin results in the accelerated degradation of NO to nitrate, a feature that may further limit the lifespan of NO in the bloodstream. The binding of NO to the heme of guanylate cyclase, a smooth muscle enzyme, accelerates the conversion of guanosine triphosphate to cyclic guanosine monophosphate. The resulting increased levels of cyclic guanosine monophosphate apparently mediate muscle cell relaxation in a manner that is not well characterized [7,20,43].

Physiologic NO is synthesized from arginine by an enzyme complex called NO- synthase or NOS. Three distinct NOS enzyme complexes have been described. Neuronal nitric oxide synthase (nNOS) is found in cells of the central nervous system cells and is thought to support a neurotransmitter function. A second- constitutive NOS (cNOS) – is in endothelial cells and is thought to play a role in the maintenance of normal vascular tone. The third is a high output, inducible enzyme called inducible NOS (iNOS); this complex is found in many cell types but particularly endothelial and vascular smooth-muscle cells. It is proposed that iNOS is

the enzyme that plays a major role in septic hypotension [1,10,29,53].

In order to evaluate the potential role of NO in septic shock it is important to recognize the physiological role of NO normally produced in the endothelium. Endothelium-derived NO is involved in the physiological regulation of vascular tone in different organs and thereby plays an important role in the regulation of blood pressure and blood flow distribution. Basal release of NO is more pronounced in arteries than in veins and more pronounced in small vessels than in conductance vessels. In addition, NO release can be stimulated by different receptor-dependent (acetylcholine, bradykinin, histamine, adenine nucleotides and serotonin) and receptor-independent agonists (free fatty acids) [6,14]. Endothelial cells also act as mechanoreceptors and thereby transduce physical forces applied by the blood into

biochemical signals. As a consequence, NO production is influenced by viscosity, pulsatility and flow (flow-mediated vasodilation). The second major role of NO is its effects on platelets and leucocytes. NO reduces platelet aggregation, adhesion and activation. Both prostacyclin and NO act synergistically to inhibit aggregation and

to mediate platelet disaggregation. In contrast to prostacyclin, NO also inhibits platelet adhesion [14,31,43].

Platelets also have the ability to synthesize NO, which may act as an intrinsic negative feedback mechanism to regulate platelet reactivity. NO is a modulator of the leucocyte-vessel wall interaction and inhibits aggregation of leucocytes and release of superoxide anions from leucocytes. Experimental data also indicate that endothelial expression of adhesion molecules is upregulated during inhibition of NO synthesis. Proliferation of vascular smooth muscle cells is inhibited by both exogenous NO donors and endogenous NO production. Based on these diverse properties of NO, defects in NO homeostasis are suggested to be involved in the pathogenesis of cardiovascular diseases, such as hypertension, arteriosclerosis, restenosis after angioplasty and ischemia/ reperfusion injury [20,41].

Several observations indicate that nitrate production increases in humans in inflammatory states. Another study showed that in patients with septic shock, plasma

nitrite and nitrate concentrations are increased and correlate directly with endotoxin concentration and cardiac output. The same concentrations correlate inversely with systemic blood pressure, consistent with the role of NO as a mediator of the hemodynamic disturbances in sepsis [10,20].

Cytokines that are prominent in mediating the sepsis syndrome, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), induce the production of iNOS. Endothelial cells constitutively generate low levels of NO from cNOS, but they will respond to cytokines with iNOS synthesis and increased NO production [43].

Vascular smooth-muscle cells ordinarily lack NOS activity; however, they can be induced by TNF-ac and interleukin-I to form large amounts of iNOS. A major distinction between iNOS and cNOS is the amount of NO that is produced. The production of NO from iNOS may be as much as 1000-fold greater than the usual levels that result from cNOS. The enzyme requires hours to appear, however, because iNOS induction requires new protein synthesis. Once induced, the iNOS enzyme is likely to persist for many hours to days. The high levels of NO formed by this enzyme result in smooth-muscle cell relaxation (vasodilatation) refractory to commonly used pressor agents. These features make iNOS induction an appealing prospect for mediating the pressor refractory hypotension that appears several hours into the development of the sepsis syndrome [14,41,53].

Sepsis leads to high-level NO production. During septic shock, NO dysregulation correlates with apoptosis, as demonstrated in a non-ischemic ileal injury model. NO induces relaxation in vascular smooth muscle cells by producing cyclic guanosine monophosphate (cGMP), leading to vasodilatation, and hereby contributing to sepsis-related hypotension. It also plays an important role in myocardial depression. High doses of NO have been shown to induce contractile dysfunction by depressing myocardial energy generation. NO can bind to complex IV of the respiratory chain and then compete with oxygen, inhibiting this complex and increasing production of reactive oxygen species [32,41].

The adverse effects of NO might also, in part, be related to interactions between NO and superoxide anions with subsequent production of peroxynitrite (ONOO-). The latter has been shown to impair cardiac muscle contraction during sepsis and, like NO, depress mitochondrial respiration during experimental sepsis [1,32,44].

3.5. Other cytokines involved in development of septic shock

IFN-γ is produced by activated natural killer cells, T helper cells and cytotoxic T cells. IFN-γ production is enhanced by several cytokines including TNF-α, IL-1, IL-12 and IL-8. IFN-γ main actions in inflammation are induction of class II major histocompatibility complex (MHC) antigen expression on different cell types and activation of macrophages. IFN-γ also plays a role in stimulating production of antibodies against bacterial wall polysaccharides. Although possessing minimal cytotoxic activity alone, IFN-γ appears to synergise in in vitro cytotoxicity assays with endotoxin and other cytokines (including TNF-α and IL-1β) thought to produce adverse effects in septic shock. Similarly, it exerts little cardiovascular effect by itself in in vivo animal models but does appear to augment the hemodynamic effects of endotoxin and TNF-α [2,15].

IFN-γ is not consistently elevated in human sepsis. However, IFN-γ may still serve to amplify the adverse hemodynamic effects of TNF-α, IL-1β and various other pro-inflammatory factors involved in septic shock [16].

Of the anti-inflammatory cytokines, IL-10 has been most thoroughly investigated. IL-10 is an 18 kDa polypeptide produced by lymphocytes, monocytes and macrophages. Synthesis is stimulated by pro-inflammatory cytokines including TNF-α, IL-1β, IL-6 and IL-12. IL-10 inhibits pro-inflammatory cytokine production by activated mononuclear cells and inhibits expression of class II MHC expression by monocytes and macrophages. IL-10 also decreases killing of intracellular bacteria by macrophages and inhibits activation of the coagulation cascade. Pro-inflammatory effects have also been described; these include stimulation of cytotoxic T cells and enhancement of B cell function [10,29,31].

Transforming growth factor-beta (TGF-β), a 25 kDa polypeptide, is another important anti-inflammatory cytokine. TGF-β antagonizes the actions of TNF-α and IL-1β in the acute phase of the inflammatory response. This anti-inflammatory activity is produced partly through inhibition of cytokine production from macrophages and stimulation of soluble receptor (sTNF-R and IL-1ra) production. However, unlike IL-10, TGF-β also antagonizes the pro-inflammatory responses of target cells to TNF-α and IL-1β [7,17,53].

Both IL-10 and TGF-β seem to play important roles in limiting the inflammatory response that ultimately leads to sepsis and septic shock.

Several other less well-understood cytokines may have important roles to play in the development of sepsis and septic shock. These include IL-4, IL-8, IL-9, IL-12, IL-13 and perhaps, most notably, macrophage migration inhibitory factor (MIF) and high mobility group 1 protein (HMG-1). Most of these cytokines are known to be elevated during septic shock [2,7,14,29,53].

MIF has been shown to be produced by monocytes and macrophages following exposure to bacterial toxins including endotoxin, toxic shock syndrome toxin 1, streptococcal pyrogenic toxin A and to pro-inflammatory cytokines including TNF-α and IFN-γ. MIF results in expression of other pro-inflammatory mediators by monocytes/macrophages and activation of T cells. Increased MIF concentrations have been shown in the blood of mice subjected to peritonitis and in humans with septic shock. Injection of MIF during experimental murine E. coli peritonitis increases mortality [17,29,43].

HMG-1 also appears to have a key role in the pathogenesis of gram-negative sepsis. Mice show increased levels of HMG-1 in serum 8–32 h after endotoxin administration. Patients succumbing to septic shock also demonstrate increased serum HMG-1 levels. Administration of HMG-1 to normal and endotoxin-resistant mice induces dose-dependent mortality with signs consistent with endotoxic shock. In addition, experimental evidence suggests that one or more important mediators of septic shock remain unidentified [25,31].

ROLE OF ENDOTHELIAL CELL IN DEVELOPMENT OF SEPTIC SHOCK

In sepsis, microvascular dysfunction with reduced perfusion and oxygen could result in tissue hypoxia and, ultimately, in the development of organ failure. The precise mechanisms underlying microvascular dysfunction remain unclear, but include altered vasomotor tone, reduced functional capillary density, reduced red blood cell deformability, increased numbers of activated neutrophils, and endothelial cell dysfunction with increased permeability and apoptosis. The endothelium is the key in initiating, perpetuating, and modulating sepsis pathophysiology. A deeper understanding of the regulation of survival and death pathways in endothelial cells may facilitate the development of novel therapies to avoid the pathological events associated with endothelial injury during sepsis [45,47,50,54].

Disturbed microvascular perfusion has been implicated in multiple organ dysfunction and failure associated with severe sepsis. Recent research has shown that systemic hemodynamics can be maintained at the expense of impaired microcirculatory perfusion in sepsis. Microcirculatory perfusion is regulated by an intricate interplay of many neuroendocrine, paracrine, and mechano-sensory pathways. These mechanisms adapt to the balance between locoregional tissue oxygen transport and metabolic needs to ensure that supply matches demand. In sepsis, such a regulatory system is severely compromised because of:

Decreased deformability of red blood cells with inherent increased viscosity;

An increased percentage of activated neutrophils with decreased deformability and increased aggregability due to upregulation of adhesion molecules;

Activation of the clotting cascade with fibrin deposition and the formation of microthrombi;

Dysfunction of vascular autoregulatory mechanisms;

Secondary enhanced perfusion of large arteriovenous shunts.

These heterogeneous processes result in tissue dysoxia, either from impaired microcirculatory oxygen delivery and/or from mitochondrial dysfunction. Alterations in microvascular blood flow and oxygenation have been demonstrated in various models of sepsis. In a normodynamic septic model using cecal ligation and puncture in rats, reduced perfused capillary density and increased heterogeneity have been observed in striated muscles and intestinal mucosa. Meanwhile, it has been shown that, for the same level of hypotension in mice, mucosal perfusion disorders are considerably larger in endotoxin-induced hypotension than in hemorrhagic hypotension [18,30].

Although cardiac output is frequently increased in sepsis, high lactate levels and increased pCO2 in tissue indicate at least regional tissue dysoxia. This has been termed oxygen extraction deficit in sepsis and has been well documented in different animal models of shock. The heterogeneity of microvascular blood flow may help explain some of the alterations in oxygen extraction capabilities that are seen in sepsis. Using a mathematical model, an increase in blood flow heterogeneity has been indicated to be associated with an increase in critical delivery of oxygen, and gut and muscle blood flow heterogeneity has been shown to increase together with impaired oxygen extraction after endotoxin administration or fecal peritonitis [19,30,49].

However, it is still a matter of debate whether oxygen extraction deficit can be explained only by pathologic flow heterogeneity due to dysfunctional autoregulatory mechanisms and microcirculatory dysfunction. Hallmark clinical findings during sepsis include peripheral vasodilation with low systemic vascular resistance and high cardiac output. The reduction in peripheral vascular resistance is thought to be a key factor responsible for the death of patients with septic shock. However, these septic patients experience adrenergic unresponsiveness despite elevated circulating levels of catecholamines [32,41].

Numerous experimental studies of sepsis have clearly documented the presence of both in vivo and in vitro vascular responsiveness to α-adrenergic stimulation. Umans et al. have confirmed and extended these observations by demonstrating impaired contractile responses to angiotensin II and serotonin. Thus, sepsis changes vascular contractile responses to many vasoactive agents. Impaired vascular reactivity with an abnormal balance between vasoconstrictor and vasodilator tone should result, then, in an inability to regulate blood flow distribution between and within tissues, which can alter blood flow to vital organs such that organ failure will eventually occur [25,31].

It has been shown that inhibition of NOS is beneficial to largely restoring the contractile responses to agonists. Furthermore, the restorative effect of NOS inhibition can be maintained even in vessels in which the endothelium had been removed, indicating that in vivo endotoxin administration may lead to high expression of iNOS within vascular smooth muscle cells, thereby impairing contractile responses. In this regard, in situ hybridization and immunohistochemistry analysis of rat aorta following endotoxin administration in vivo and in vitro has suggested that aortic adventitia, in addition to the endothelium, is a potential source of iNOS. These changes correspond to the pathophysiological features of clinical and experimental sepsis and may account for the profound vasodilation and the limited response to the normal endogenous stimuli that can regulate blood flow distribution among organs. Finally, iNOS deficient mice have been found to be resistant to vascular hypocontractility. Accordingly, iNOS, which generates large amounts of NO, is likely to be a critical mediator of the diminished vascular contractility in sepsis [7,31,51].

Some authors in experimental studies have found different expression levels of iNOS between the two types of vessels from LPS-induced septic rabbits. Thus, the sepsis-induced increase in relative protein expression levels of iNOS was more marked in mesenteric (9.4-fold) than pulmonary (2.1-fold) arteries. This difference could lead to hypocontractile response to histamine in mesenteric but not in pulmonary arteries. These findings may provide a basis for the results of past investigations that iNOS induction-associated vascular hypocontractility was not observed in the pulmonary circulation despite the presence of iNOS mRNA in pulmonary vessels in vivo LPS [18,31].

The ATP-sensitive potassium (KATP) channel has been identified as an important modulator of arterial vascular smooth muscle tone. Particular attention has been focused on the involvement of KATP channels in both hypotension and vascular hyporeactivity induced by endotoxemia. In vivo evidence for KATP channel involvement in sepsis comes mainly from anesthetized rat, dog and pig models of endotoxemia. Rapid restoration of blood pressure can be achieved with glibenclamide, resulting largely from increased systemic vascular resistance rather than improved cardiac output. Glibenclamide also increases vasopressor reactivity to α1-adrenergic agonists in both the early phase (3 h) and the delayed phase (24 h from LPS challenge) of septic shock in the rat. These findings strongly suggest that KATP channels preferentially open during sepsis and are important underlying cause of hypotension and vascular reactivity [30,51].

Abnormal opening of KATP channels and, to a lesser extent, large conductance Ca2+-activated K+ (BKCa) channels, appears to be responsible for an increase in membrane hyperpolarization reported in mesenteric arteries and aortas from rats with endotoxic shock. Likewise, relaxations to KATP channel openers are potentiated in both in vitro and ex-vivo models of LPS-induced hyporeactivity, indicative of up-regulation of KATP channel function in vascular smooth muscle by endotoxin. An intriguing possibility is that endotoxin may alter KATP channel pharmacology such that channel inhibition via sulfonylurea receptors would become dysfunctional [18,31].

Excessive activation of KATP channels is clearly implicated in a number of crucial mechanisms in sepsis, including vascular hyporeactivity. However, channel opening may afford a degree of cellular protection. Furthermore, whether up-regulation of vascular expression of KATP channels is beneficial or detrimental in sepsis remains to be determined. It should be kept in mind that the involvement of KATP channels in vascular disturbances associated with sepsis is questioned in a recent placebo-controlled study in septic shock patients where glibenclamide has no effect on blood pressure or norepinephrine requirements. However, targeting of the vasculature with specific pore rather than glibenclamide might appear more appropriate in sepsis. Clearly further research is needed to better delineate the protective and harmful roles of KATP channels in sepsis [18,51].

Impaired endothelium-dependent vascular relaxations

Endothelial cells produce vasoactive molecules that regulate arteriolar tone and contribute to blood pressure control. These include the vasodilators, including NO and prostacyclin, and the vasoconstrictors, including endothelin, thromboxane A2, and platelet-activating factor. Impaired endothelium-dependent relaxations have been shown in blood vessels from endotoxemic animals [30]. Apart from anatomical injuries, such impairment observed in endotoxemic blood vessels may result from several mechanisms:

Alteration in endothelial cell surface receptors;

Modified signal transduction pathways such as receptor-eNOS uncoupling;

Altered function and expression of eNOS;

Changes in the pathways that lead to release of NO;

Changes in mechanisms that participate in subsequent degradation of NO.

eNOS expression is obviously diminished in blood vessels from rabbits and in lung tissues from mice following induction of sepsis with LPS. In other works, furthermore, it has been demonstrated that sepsis causes a progressive and profound reduction in phosphorylation of eNOS in rabbit mesenteric arteries, suggesting less production of NO by eNOS in sepsis.

Vascular hyperpermeability

In the intact vasculature, the endothelium forms a continuous, semipermeable barrier that varies in integrity and control for different vascular beds. During septic shock the breakdown of endothelial barrier function occurs. Thus, a central feature of the endothelium in sepsis is an increased permeability or loss of barrier function. The loss of fluid into the extravascular space leads to life-threatening edema in the lungs, kidney, and brain of septic patients [30,51].

There is evidence that LPS directly contributes to endothelial barrier dysfunction through a caspase-mediated cleavage of junctional proteins involved in regulating transport of material between the vascular space and tissue. Furthermore, the contribution of structural damage to endothelial cells to skeletal muscle edema has been shown in a pig model of septic shock. More importantly, the increase in endothelial permeability can be induced by a number of sepsis related factors [31]. Indeed, the cytokine TNF-α induces an increase in endothelial cell permeability both in vivo and in vitro. Under in vitro conditions, thrombin also increases endothelial cell permeability, while TNF-α and thrombin act synergistically to induce barrier dysfunction in vitro.A number of molecules that could be actually generated by activation of the NF-κB signaling pathway are involved in the development of vascular hyperpermeability during sepsis [25,51].

Endothelial cell apoptosis

Endothelial cell apoptosis is a highly regulated process. Normally, only a small percentage (<0.1%) of endothelial cells are apoptotic. Under in vitro conditions, certain pathogens are capable of inducing endothelial cell apoptosis. In vitro incubation of cultured bovine and ovine endothelial cells with LPS has been reported to induce apoptosis. Evidence that LPS-induced endothelial cell death is apoptotic in nature has been confirmed by several criteria, including morphological changes, DNA laddering, and transferase-mediated dUTP nick-end labeling (TUNEL). However, LPS alone fails to induce apoptosis in cultured human endothelial cells. The ability of endotoxin to induce apoptosis may be cell specific. Alternatively, the apoptosis effect of endotoxin in in vitro system may be dependent on whether the transduction molecules for the LPS signal are indispensably present in the endothelial cell line [3,18].

In in vivo animal models, purified LPS has been reported to elicit endothelial cell injury and apoptosis. Endothelial cell injury and detachment from the vascular wall have been demonstrated when LPS was injected into mice, rats, rabbits and sheep. In a mouse model of endotoxemia, intraperitoneal delivery of LPS from Salmonella typhimurium could result in widespread apoptosis of the endothelium in a process mediated via ceramide generation. In other study, intravenous administration of LPS in mice has been shown to induce endothelial cell apoptosis in the lung, but not the liver, pointing to organ-specific differences in programmed cell death. However, liver sinusoidal endothelial cells obtained from LPS-treated rats display enhanced activation of caspase-3, a central apoptotic effector protease. Interestingly, evidence of endothelial cell injury has been provided in postmortem biopsies obtained from patients who had died of sepsis-related ARDS [3,26,27].

Moreover, an increase in circulating endothelial cells has been found in septic patients, and the magnitude of this increase correlates negatively with survival. It has been shown that injection of a broad spectrum caspase inhibitor decreases endothelial cell apoptosis in the lung after LPS administration and improves survival in a murine model of acute lung injury [18,28].

In addition, LPS and TNF-α each up-regulate expression of other antiapoptotic proteins, including A1 and A20 in endothelial cells. Conversely, recent studies demonstrate that several signaling molecules originally described as mediators of apoptosis can contribute to the regulation of NF-κB activation. For example, transient overexpression of Fas-associated death domain (FADD), FLICE-like inhibitor protein (FLIP), or caspase-8 augments basal levels of NF-κB activation. FADD is an adaptor protein which can recruit pro-caspase-8 to the “death-inducing signal complex,” thereby causing its activation, and FLIP is an antiapoptotic protein with significant homology to caspase-8. Moreover, evidence that apoptotic signaling molecules can affect the ability of LPS to induce NF-κB activation has been shown by the finding that overexpression of Bcl-2 and Bcl-xL, antiapoptotic members of the Bcl-2 family, inhibits LPS induced NF-κB activation and NF-κB-dependent gene expression in endothelial cells. This inhibition of NF-κB activation corresponds with Bcl-2-mediated inhibition of IκBα degradation. Further evidence for a role for apoptotic signaling molecules in promoting NF-κB activation has been provided in a recent report showing that FADD downregulates LPS-induced NF-κB activation. However, additional work will be needed to delineate pathophysiological significance of cross talk between apoptotic and NF-κB signaling molecules [26,28,51].

LPS up-regulates expression of iNOS and increases NO production in endothelial cells. It has been demonstrated that suppression of iNOS induction and inhibitors of NOS activity protect against LPS-elicited endothelial cell injury, suggesting that iNOS-derived NO promotes LPS-induced apoptosis of endothelial cells. The mechanism by which NO overproduction contributes to LPS-elicited endothelial cell apoptosis remains unknown, but the generation of large amounts of NO following iNOS induction by LPS may result in NO reaction with superoxide anion to form peroxynitrite, a potent oxidizer. Endothelial cell injury resulting from the generation of peroxynitrite may synergistically enhance endothelial cell apoptosis elicited by LPS. Alternatively, high concentrations of NO can inhibit protein synthesis. Because protein synthesis inhibition has been established to sensitize human endothelial cells to LPS-induced apoptosis by inhibiting expression of the antiapoptotic protein FLIP, one may speculate that high levels of NO may result in a decrease in expression of this cytoprotective protein. On the contrary, NO has been shown to inhibit LPS induced apoptosis in endothelial cells. Increased production of NO due to iNOS overexpression could block LPS-elicited endothelial cell apoptosis [7,29,43]. The differential effects of NO on mediating LPS-induced endothelial cell apoptosis may be dependent on its concentrations, since it has been reported that moderate concentrations of NO confer protection, whereas higher concentrations of NO enhance LPS-induced apoptosis in endothelial cells [14].

DISCUSSIONS

It is evident from the preceding discussion that there is a complex network of events that occur in sepsis and septic shock. Because there is strong evidence for the involvement of MIF in sepsis and septic shock from animal studies, it may be necessary to evaluate its role in humans. Donnelly et al showed that MIF is detectable in the serum and the lung fluids and in alveolar macrophages of patients with ARDS. Furthermore, MIF inhibited the suppressive effects of dexamethasone on IL-8 production by lung macrophages. There is a close relation between the presence of IL-8 (a neutrophil chemotactic factor) in early bronchoalveolar lavage fluid samples and the development of ARDS. This suggests that both IL-8 and MIF can be used as prognostic indicators for the development of ARDS, which is common in septicemia and septic shock, and reinforces the significance of macrophages in the pathobiology of both ARDS and sepsis.

It is possible that methods designed to suppress the production of TNF and interleukins may still prove to be useful in septicemia and septic shock, even though anti-TNF antibody and IL-1 receptor antagonist have failed to benefit these patients. In this context, it is interesting to note that providing adequate amounts of glucose and insulin has been shown to antagonize the harmful actions of TNF-a. It was also observed that treatment with insulin can almost completely reverse the nutritional and histopathologic toxicity of sublethal doses of TNF in rats. Furthermore, insulin may have a regulatory role in superoxide generation. In addition, the expression of MIF in adipocytes can be modulated by insulin and glucose.

This evidence suggests that, during systemic inflammatory processes, MIF is secreted from the pituitary gland accompanied by an increase in glucocorticoid secretion (and macrophages will also produce MIF and TNF). The increase in plasma glucose concentration that occurs as a result of this glucocorticoid production is probably controlled by MIF, which has a positive effect on insulin secretion. Thus, glucose homeostasis during septicemia and septic shock is maintained by glucocorticoids, insulin and TNF by inducing insulin resistance. Because there is a

feedback control between MIF, glucose and insulin, it is possible that infusion of insulin and glucose can inhibit MIF production and release, which is similar to their action on TNF. If this hypothesis is correct, it suggests that a glucose–insulin–potassium regimen (which is used in the management of diabetic ketoacidosis) may be useful in the management of septicemia and septic shock, in which excess production of TNF-a and MIF seem to play an important role.

However, this concept remains to be verified. It is important to measure plasma TNF and MIF concentrations both before and after the glucose–insulin–potassium regimen in these patients, and to determine whether there is any correlation between the progress and outcome of septicemia and septic shock and the concentrations of TNF and MIF.

Refractory hypotension with end-organ hypoperfusion is an ominous feature of inflammatory shock. In the past fifteen years, nitric oxide (a diffusible, short-lived product of arginine metabolism) has been found to be an important regulatory molecule in several areas of metabolism, including vascular tone control. Vascular endothelial cells constitutively produce low levels of nitric oxide that regulate blood pressure by mediating adjacent smooth-muscle relaxation. In an inflammatory shock state, cytokines, like interleukin-1 and tumor necrosis factor-a, induce a separate, high-output form of the enzyme that synthesizes nitric oxide in both endothelial and smooth-muscle cells. The ensuing high rates of nitric oxide formation result in extensive smooth-muscle relaxation, pressor refractory vasodilation, and-ultimately-shock. The concept of the pathogenesis of inflammatory shock explains many limitations of current therapies and may foster the development of new interventions to mitigate the effects of nitric oxide overproduction in this syndrome.

GENERAL CONCLUSIONS

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