The sensitivity to LPS toxicity differs considerably among various species of mammals. Lower vertebrates, such as frog and fish, show extreme resistance [35]. Nevertheless some observations suggest that at least some species of fish (Tilapia oreochnomis mossanbicus, Teleosti) react to LPS and shows an integumental and a cortisol response [36]. In contrast, the horseshoe crab (Limulus polyphemus) responds to LPS with fatal intravascular coagulation. This is due to the activation by endotoxin of a clottable protein of Limulus blood, which is produced by circulating amoebacytes [37-39].

It seems apparent that lipid A has no inherent toxicity, but rather, it is a highly conserved homologous epitope, which has been singled out during evolution by vertebrate and some invertebrate animals for defence purposes against Gram-negative bacterial infections [7,35,40]. Lipid-A, which is the specific homotope involved, is recognized by serum proteins and cell surface receptors, which are capable of activating the coagulation and complement systems and various members of the leukocyte series. This enables the animal to mount a rapid and effective immune defence reaction against all Gram-negative pathogens [7-12].

In the mouse approximately 7% of genes are mobilized during hepatic APR response to LPS. The extensive metabolic adjustments include suppression of pathways for cholesterol, fatty acid, and phospholipid synthesis. Increased expression of genes for innate defense was accompanied by coordinate induction of the major histocompatibility complex (MHC) class I antigen presentation machinery, illustrating an intersection between innate and adaptive immunity [41].

3.1.1 Cytokine response to endotoxin.

After systemic administration of LPS to mice, TNFα is significantly increased in the blood at 1-2 hours, which is followed by a decline, the TNF level returning to normal at around 4 hours. In ADX animals an exaggerated TNF response occurs, whereby TNF levels in the plasma rise approximately 60 times higher, and sensitivity to the lethal effects of TNF increases approximately 500 times over that is observed in normal control animals after LPS injection. This excessive response and increased mortality can be prevented if the animals are pre-treated with dexamethasone. The inhibition of cortisone synthesis in the adrenals by metyrapone also leads to an increased susceptibility (~15 times) to LPS [40-42]. Similar kinetics of TNF release was found in man after LPS infusion [44].

Interleukin-1 also rises in the blood of mice after endotoxin administration reaching the maximum at about 4 hours and elevated levels remain up to 24 hrs [43, 34]. Circulating IL-6 increases significantly after LPS administration. In man IL-6 peaked at 120 minutes after LPS administration, which was not inhibited by glucocorticoids or by repeated LPS administration [11, 45,46].

Leukemia inhibitory factor (LIF) rose moderately in mice after a sub-lethal injection of LPS and rose progressively during lethal septic shock induced by E-coli. LIF administration to mice induced catabolism and had a protective effect if given prior to the administration of bacteria [47].

Additional cytokines and mediators also play a role in endotoxin shock that include interleukin-8 [48], interleukin-10 [49], interferon-γ [50], TNF synthesis inhibitor [51], interleukin receptor antagonist [52], platelet activating factor, colony stimulating factor, prostaglandins and thromboxanes [11,12,53].

3.1.2 Neuroendocrine response to endotoxin.

Wexler et al [54] observed for the first time the stimulation of adrenocorticotropic hormone (ACTH) in rats by endotoxin as detected by the depletion of ascorbic acid and cholesterol in the adrenal glands. LPS was ineffective in causing these changes in the adrenal glands of hypophysectomized rats.

Endotoxin, infectious disease, and various forms of injury all elicit a neuroendocrine response via the stimulation of cytokines [11,12]. Profound changes occur in serum hormone levels. It is clear that dynamic and diurnal changes of hormones should be taken into consideration. Much remains to be elucidated about the significance of these hormonal alterations. Nevertheless, it is certain that the hypothalamus-pituitary-adrenal (HPA) axis exerts a powerful suppressive effect on the adaptive immune system and controls the level of inflammatory cytokines. Through the activation of this axis, specific immune reactions are profoundly suppressed, whereas the induction of acute phase proteins in the liver and natural antibody production are augmented by glucocorticoids and catecholamines, which are also elevated. Therefore, the conversion of the immune system from the adaptive mode of reactivity to the amplification of natural immunity is largely due to the activation of the HPA axis and of the sympathetic nervous system, e.g. sympathetic outflow [12,54-57]. Prolactin and growth hormone stimulate the adaptive immune system and usually rise within the first hour after endotoxin injection, which is followed by a decline and the level may become low normal to subnormal in serious cases of endotoxin shock. Luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogens, androgens, progesterone, and thyroid hormones all decline during infection and endotoxin shock, as a rule. Insulin,glucagon, α-melanocyte stimulating hormone (MSH), endorphin, leptin, corticotropin releasing hormone (CRH) and arginine vasopressin are increased during endotoxemia [11,12,40,58-62] (Table 1, Figure1) 

Legend: Figure 1. Immunoconversion during the acute phase response. The acute phase response (APR) is a systemic inflammatory reaction. Fever is a hallmark of the APR, of febrile illness. Immune –derived cytokines, primarily IL-1, IL-6 and TNF-alpha, are released by the immune system and act either on nerve terminals or on the brain. The cytokine signals eventually are registered in the hypothalamus and a powerful neuroendocrine and metabolic response to the infection/injury is initiated. From the hypothalamus corticotrpoin releasing hormone (CRH) and vasopressin (VP) are secreted during APR. Both of these peptides stimulate the hypothalamus-pituitay-adrenal axis (HPA). In addition VP has the capacity to stimulate prolactin (PRL) secretion. Initially CRH prevails and the activation of the HPA axis is dominant. This is coupled with “sympathetic outflow” from the adrenal gland. The increased level of glucocorticoids (GC) and cathecolamines (CAT), together with TNF alpha are of prime importance of inducing thymus involution (e.g. by inducing apoptosis of CD8+4+ thymocytes) and of inhibiting the T cell dependent adaptive immune system. Other factors that are likely to contribute to this suppression are the down-regulation of growth hormone and prolactin synthesis and zinc deficiency. During APR the synthesis of acute phase proteins (APP) is amplified in the liver by IL-6, GC and CAT. Serum C-reactive protein (CRP) will go up as much as 1000 times of the basal level within 24-48 hrs. CRP is capable of recognizing pathogenic organisms and to activate complement and leukocytes for phagocytosis and cytotoxicity. Other serum proteins with similar biology are lipopolysaccharide-binding protein (LBP) and mannan binging protein (MBP). Additional acute phase proteins (APP) are fibrinogen and a number of anti-inflammatory and enzyme inhibitory proteins, that also rise in the serum during APR. Natural antibodies, that are poly-specific similarly to CRP LBP and MBP, are also stimulated during APR and serve to identify pathogenic agents, which is followed by immune activation. Therefore the essence of febrile illness is to switch over the immune system from the adaptive (T cell dependent) mode of reactivity to the activation of innate/natural immune mechanisms. This process is coined as immunoconversion. During the chronic phase of inflammmatory disease CRH will subside and VP will take over the regulation of the HPA axis. Because VP also stimulates PRL secretion, it is hypothesized that VP alters the neuroendocrine milieu to favor the restoration of adaptive immunocompetence. This process is named immunoreversion, which will lead to recovery from acute illness.

Table 1. Major neuroendocrine changes induced by endotoxins

HPT and GLH Hormones Response The HPA Axis Response Gonatatropin & Sex Hormones Response

TRH ↓ CRF ↑ LH ↑↓ TSH ↓ VP/AVP ↑ FSH ↓ T4 ↓ ACTH ↑ E2 ↑↓ T3 ↓ GC ↑ TS ↑↓ PRL ↑↓ αMSH ↑ DHEA ↓ GH ↑↓ βEND ↑ PS ↑↓ IGF-I ↓ CAT ↑ IN  ↑ GLU ↑ LEP ↑ Please see the list of abbreviations at the end of this chapter. . 0 = no effect. This table is modified from reference [10]..

PRL receptor (PRLR) mRNA and protein levels were down-regulated in hepatic tissues after i.p. LPS injection. A suppressive effect on mRNA expression was also observed in prostate, seminal vesicle, kidney, heart, and lung tissues. PRLR mRNA levels were increased in the thymus, and did not change in the spleen. The proportion of transcripts for the different receptor isoforms (long, S1, S2, and S3) in liver and thymus was not altered by LPS injection [63].

Peripherally administered LPS induces anorexia, which is prevalent in females. Estradiol is capable of inducing this response. Estradiol affects meal frequency [64]. Hepatic dehydroepiandrosterone (DHEA) sulfotransferase (Sult2A1) activity and serum levels of DHEA-sulfate were significantly decreased in LPS-treated animals. TNF and IL-1 caused a significant decrease in the mRNA level of Sult2A1 in Hep3B human hepatoma cells [65]

3.1.3 Endotoxin shock.

Geller et al [66] observed that the administration of the glucocorticoid (GC), cortisone, to mice prior to, or simultaneously with, a lethal dose of LPS protected the majority of the animals from death, but there was no protection when cortisone was administered after LPS injection. The protective effect of GC against endotoxin shock has been since observed repeatedly in various species [67,68].

No correlation was found between TNF serum levels and the lethal effects induced by different types of LPS in rats [69]. The co-treatment of rats with low doses of TNF and LPS resulted in the rapid demise of the animals leading to 100% mortality within 4 hours [70].Pre-treatment of rats with a single low i.v. dose of TNF prevented subsequent death from a lethal dose of TNF or of LPS applied 24 hours later [71]. The lethal effect of endotoxin could be inhibited in various animal models by the opioid antagonist, naloxone, by indomethacin, by monoclonal antibodies to TNFα and by the pharmacological inhibition of platelet activating factor (PAF) [11]. Anti-inflammatory cytokines, such as the IL-1 receptor antagonist [71], the TNF synthesis inhibitor [72], IL-10 [73], leukemia inhibitory factor [74], all participate in the down regulation of the noxious effects of endotoxin. Interferon-γ antagonizes the development of endotoxin tolerance [75]. In vitro observations revealed that the direct exposure of macrophages to LPS also leads to decreased activation and cytokine production upon re-exposure to LPS [76,77].

Previte et al [78] discovered that the exposure of endotoxin to ionizing radiation resulted in significant loss of toxicity. Subsequently Bertok and coworkers [79] demonstrated over a number of years that radiodetoxified endotoxin is capable of boosting host resistance against infectious agents, radiation, septic shock, tourniquet shock, intestinal ischemic shock, hemorrhagic shock, X-irradiation and even against immunosuppression by anti-lymphocytic serum [80, 81]. Radiodetoxified endotoxin has been tested clinically for the treatment of infectious disease in man [81]. Monophosphoryl lipid A preparations of low toxicity are also studied in animals and man for boosting host resistance to infections and traumatic events [82,83].

3.2 The Pathomechanisms of APR.

Clinically, APR is characterized by fever, loss of appetite, inactivity and sleepiness. Changes in sleep are hallmarks of the acute phase response to infectious challenge. The regulation of these responses involves a cytokine cascade within brain, including IL-1 and TNF, and several other substances such as growth hormone releasing hormone, PRL, nitric oxide and NFkappaB. These substances are also involved in the regulation of normal spontaneous sleep.

3.2.1 Cytokines and hormones

Acute febrile illness (e.g. APR) is mediated by cytokines, GC and cathecolamines. Cytokines appear in the circulation and function as acute phase hormones affecting the central nervous system (CNS), the neuroendocrine system and virtually the function of every other tissue and organ in the body. IL-1, IL-6 and TNFα have been identified as major mediators of the endocrine and metabolic changes characteristic of APR. However, several other cytokines have been found to be inducers of acute phase proteins (APP) [7, 9-12,85]. ACTH and GC, leptin (LEP), epinephrin (EP), norepineprin (NEP), glucagon (GLN), vasopressin (VP), and aldosterone (ALD) are elevated during the APR, whereas GH, PRL, estrogens, androgens, insulin (IN), and thyroid hormones may be either elevated or suppressed, depending on the severity of the condition [7-12,85, 86].

A subset of marrow-derived brain macrophages, termed perivascular cells, synthesize prostanoids after systemic cytokine or endotoxin challenges. These brain macrophages are critically involved in the interleukin-1-induced hypothalamo-pituitary-adrenal axis activation. This suggests a two-way interaction between perivascular and endothelial cells in monitoring circulating cytokine signals [87].

In most burn patients bone density dropped significantly 6 and 12 months post-injury indicating bone resorption. Cortisol was elevated, both in blood and in urine (free cortisol). There was very low testosterone, dihydrotestosterone (DHT) and free testosterone levels in blood of males, but not of females. 17beta-estradiol was elevated in many burned males; but was generally normal in burned females. DHEA-S levels were generally low. Triiodothyronine (T3) and of the free thyroxine (FT4) was vey low. Increased, even very high, PTH values were occasionally present. hGH and IGF-1 were generally normal. Total and ionized calcium levels were low after burn, 25-0H vitamin D was usually low or low normal. Osteocalcin levels were initially low to low normal, to increase later to the normal levels. In most instances elevated levels of TNFalpha, IL-2, IL-6 and IL-8 levels were found. The use of anabolics, of vitamin D, of calcium, and eventually of calcitonin was suggested for treatment [88].

During APR (eg. sepsis) the serum level of leptin rises rapidly. Cytokines, especially TNFα, causes this elevation. LEP inhibits glucocorticoid and IL-6 production. The levels of LEP in serum correlate positively with the survival of patients with septicaemia. LEP stimulates the production of IL-1 receptor antagonist, which protects against LPS toxicity in mice. LEP stimulated the production of IL-1β in murine glial cells. Exogenous LEP upregulated both phagocytosis and the production of proinflammatory cytokines in animals. Leptin is also involved in wound healing and angiogenesis [89-98]. Chronic leptin deficiency in ob/ob mice interfered with adequate control of zymosan-induced arthritis [98].

3.2.2 Acute phase proteins

An alteration of protein synthesis by the liver is most characteristic for APR.The synthesis of acute phase proteins (APP) is initiated, whereas the synthesis of some normal serum constituents such as albumin and transferrin is decreased. The concentration of APP increase dramatically in the serum. For example, in man, C reactive protein (CRP) and serum amyloid A (SAA) may increase over 1,000-fold within 24-48 hours. Fibrinogen, "1-antitrypsin and certain complement and properdin components (factor B and C3) show a more moderate increase [7, 12]. In man IL-6 exerted a hyperglycemic effect, whereas IL-2 induced a decrease in blood glucose concentration [100].

CRP binds to C-type pneumococcal cell walls in the presence of Ca²⁺. CRP has been identified in multiple species, including mammals, chicken, fish and crab. In the serum CRP consists of 5 identical subunits, which form a ring-shaped molecule named pentraxin. This term now also stands for a protein family.  In man, serum amyloid P also belongs to this family.The mature subunits consist of 206 amino acids with 23 kDa molecular weight [101].

CRP recognizes specifically several homotopes, such as phosphocholine, with polysaccharides containing galactose, with some biologic polycations, such as protamine, poly-L-lysin and with myelin basic protein. CRP is in its pentameric form, if Ca²⁺ is present, and binds phosphocholine and galactans, whereas in the absence of Ca²⁺, it becomes monomeric and binds various polycations. These homotope determinants are frequently present on the surface of bacteria, fungi, parasites and damaged cells and tissues. After combination with the specific ligand, CRP activates complement by the classical pathway, induces chemotaxis and enhances phagocytosis by neutrophilic leukocytes and monocytes and elicits tumoricidal activity in macrophages, all of which are complement dependent. In addition, CRP stimulates the synthesis of IL-1, TNF", and potentiates the cytotoxic activity of T lymphocytes, natural killer (NK) cells and platelets. CRP localizes in vivo at sites of inflammation. It binds platelet activating factor (PAF) and blocks its activity. The clinical determination of CRP is diagnostic for the presence of infectious and inflammatory disease [101-104].

CRP influence PMN adhesion, migration and expression of CD11b/CD18 and fibronectin receptors, and can modulate the action of IL-8 on polymorphonuclear cell (PMN) attachment to endothelium and fibronectin, and on PMN traffic through the extracellular matrix during transendothelial migration [105].

Upon denaturation or after attachment onto polystyrol plates, free CRP subunits attain a third conformation referred to as neoCRP. NeoCRP is a membrane protein on NK cells and macrophages and functions as galactose-specific receptor.It also accumulates at injured sites of tissue.  Monocyte/macrophages express a specific CRP receptor.Proteolytic fragments of CRP activate macrophages and neutrophils [101]. Human CRP protected mice from an otherwise lethal S. pneumoniae infection [104].

The LBP shows a 100-fold increase (from 0.5-50 µg/ml) in the serum during an APR. LBP is capable of opsonizing LPS bearing particles, and thus may be required for the activation of complement by endotoxin through the alternate pathway.LBP-LPS complexes are also potent stimulators of cytokines from monocytes and macrophages after combining with CD14 on the surface of these cells [26].

In man high-dose LBP (hd-LBP) suppressed the binding of both R-type and S-type LPS to CD14 and inhibited LPS-induced nuclear translocation of NF-kappaB. This inhibitory effect of serum could be mimicked by purified high-density lipoprotein (HDL) in serum-free medium, indicating an LBP-mediated transfer of preferentially S-type LPS to plasma lipoproteins such as HDL [106].

Haptoglobin is an acute phase protein. It binds haemoglobin, thus preventing iron loss and renal damage. Haptoglobin is an antioxidant, has antibacterial activity and plays a role in modulating many aspects of the acute phase response [107]. Haptoglobin selectively antagonized LPS effects in vitro by suppressing monocyte production of TNF-alpha, IL-10 and IL-12, but failed to inhibit the production of IL-6, IL-8 and IL-1 receptor antagonist. Haptoglobin knockout mice were more sensitive to LPS effects then were their wild-type counterparts [108].

The acute phase proteins alpha(1)-acid glycoprotein and alpha(1)-antitrypsin exert antiapoptotic and anti-inflammatory effects and contribute to the delayed type of protection associated with ischemic preconditioning in the kidney and in other insults [109].

Mannose-binding lectin (MBL) is a serum protein characterized by both collagenous regions and lectin domains, which plays an important role in innate immune defence. It binds to the repeating sugar arrays on many microbial surfaces through multiple lectin domains. Following binding, MBL is able to activate the complement system via an associated serum protease, MASP-2. There is an increased incidence of infections in individuals with mutations of MBL and an association with the autoimmune disorders SLE and rheumatoid arthritis [110]. MBL activates the complement cascade and inflammation following binding to carbohydrate structures. The serum concentration of MBL is subject to large individual differences. Growth hormone influences MBL levels [111]. Mouse MBL-A and MBL-C were studied in various strains and in APR.  The MBL-A and MBL-C levels in 10 laboratory mice strains were found to vary between 4 µg/ml to 12 µg/ml, and 16 µg/ml to 118 µg/ml, respectively. After the induction of APR by the i.p. injection of casein or LPS, MBL-A was found to increase approximately two-fold, with a maximum after 32 h, while MBL-C did not increase significantly. Serum amyloid A peaked at 15 h with an approximate 100-fold increase [112].

Other APP are proteinase inhibitors, such as "2-macroglobulin, "1-acid glycoprotein, antithrombin III, "1-acute phase globulin, and "1-proteinase inhibitor, which are abundant in the rat. Kupffer cells stimulate "2-macroglobulin synthesis by hepatocytes in vitro in the presence of 10^-9 M DEX. Fibrinogen is also and APP with an important role in blood clotting and healing. Alpha-macrofetoprotein ("-MFP) is a strong inhibitor of inflammatory mediators, such as histamine, bradykinin, serotonin, PGE2 and inhibit polymorphonuclear chemotaxis [113].

3.2.2.1   Regulation of APP production.

Catecholamines and GC induce "-macrofetoprotein (MFP) in normal rats. The "-MFP level induced by cathecolamines (CAT) was very high, comparable to those observed in the post-injury phase, whereas the effect of GC was moderate. In ADX rats the effect of CAT on "-MFP synthesis was greatly diminished, whereas the moderate effect of GC remained. The combination of GC and CAT induced extremely high "-MFP levels in ADX animals. Some other APP, such as haptoglobin and "1-major acute phase protein, were affected differently by these hormones [114].

Glucocorticoids (GC) exert a stimulatory effect on a variety of inflammatory response components. This is usually observed at near basal GC concentrations. For example, such stimulation was observcrd for the hepatic APR, for cytokine secretion, expression of cytokine/chemokine receptors, and for the pro-inflammatory mediator, macrophage migration inhibition factor [115].

IL-1 and TNF induced a full range of APP in vivo, but only a limited number of APP were induced by these cytokines in cultured liver cells compared with crude cytokine preparations from macrophages. This led to the discovery of IL-6 as a major inducer of APP synthesis. Additional cytokines, namely IFN-(, leukemia inhibitory factor, TGF-$, and oncostatin M, were found to be active as direct inducers of APP from the liver. IL-6 activates the genes of APP through the DNA binding protein called NF-IL-6. NF-IL-6 is a pleiotropic mediator of many inducible genes involved in the acute-, immune- and inflammatory responses, similarly to NF6B. Both NF-IL-6 and NF6B binding sites are present in the inducible genes, such as IL-6, IL-8 and several acute phase genes [116].

Adrenalin evokes a high level of IL-6 in rats, which can be antagonized by propranolol. When IL-6 release is blocked, the fast reacting APP, 2-macroglobulin and cysteine protease inhibitor are strongly depressed.  Isoprenalin, a 2-adrenergic receptor agonist, also causes very high levels of IL-6, indicating that 2 receptors are involved [117].

During chronic liver injury induced by biweekly application of CCl₄, deletion of the gp130 receptor in nonparenchymal liver cells and not hepatocytes resulted in fibrosis progression. This indicates the involvement of IL-6 in the pathogenesis of liver diseases and suggests a protective role of IL-6/gp130-dependent pathways in nonparenchymal liver cells during fibrosis progression in chronic liver diseases [118].

The relationship between spontaneously occurring activation of the acute phase response and leptin levels were examined in 29 chronic hemodialysis patients. When CRP was elevated, leptin levels were significantly reduced, as were the negative acute phase proteins albumin and transferrin. Serum amyloid A, ceruloplasmin, alpha-1 acid glycoprotein, and IL-6 were all significantly increased at the maximum CRP level, compatible with general activation of the acute phase response. The change in leptin correlated negatively with the change in CRP, as did changes in albumin [119].Pro-inflammatory cytokines, especially TNF, induce inflammatory hyper-leptinemia. This is an integral part of APR and necessary for comprehensive immunocompetence. This indicates the existence of an integrated communication network to co-ordinate the energy status of the animal with the ability to fight pathogens [120].

The thymus is severely affected by stress and APR, which rapidly leads to the loss of thymocytes manifesting in profound involution. The thymus is a central primary lymphoid organ responsible for the generation of mature, functional thymus-derived (T) lymphocytes. In turn T lymphocytes govern adaptive immune reactions through their regulatory function. In addition, the thymus exerts endocrine functions, the significance of which is not yet fully appreciated. A large body of evidence attests for the existence of a complex neuroendocrine control of thymus physiology. Long standing observations indicate that the thymus becomes involuted during the stress response. During stress or more forcefully, during APR, the HPA axis is activated. This results in the suppression of the T-cell-dependent adaptive immune response, which is supported further by the suppression of the hormones that are essential for the maintenance of the thymus and of T lymphocytes (e.g., PRL, GH, IGF-I). Catecholamines and glucocorticoids, which are released in large quantities during APR, induce apoptosis in the thymus with a striking efficiency.The elevated level of TNF" and zinc deficiency, that develop during APR, also contribute to thymic involution and to the suppression of the adaptive immune system  (Figure 1) [9].

Cytokine-induced hyperlipoproteinemia, clinically termed the 'lipemia of sepsis', represents an innate, non-adaptive host immune response to infection. Triglyceride (TG)-rich lipoproteins (VLDL and chylomicrons, CM) bind and neutralize LPS. CM-bound LPS attenuates the hepatocellular response to pro-inflammatory cytokines. Primary rodent hepatocytes pretreated with CM-LPS complexes for 2 h demonstrated a near 70% reduction in cytokine-induced NO. The lipemia of sepsis likely represents a mechanism by which the host combats sporadic, non-life-threatening episodes of endotoxemia. Also, it may indicate a negative regulatory mechanism for the hepatic response to sepsis, serving to effectively down-regulate the acute phase response [121 ].

The multiple organ failure induced by critical illness was suggested to be a primarily functional, rather than structural, abnormality with a potentially protective mechanism. The decline in organ function is triggered by a decrease in mitochondrial activity and in oxidative phosphorylation, leading to reduced cellular metabolism. This might be the consequence of acute-phase changes in hormones and inflammatory mediators [122].