Lóránd Bertók, “Fodor József” National Center of Public Health and “Frédéric Joliot-Curie” National Research Institute for Radiobiology and Radiohygiene, H-1221 Anna u. 5, Budapest, Hungary.
 

Published by: Elsevier Science
ISBN:0-444-51755-3
Neuroimmune Biology: Vol.5: Natural Immunity

Description:  Showcases the significant expansion in the understanding of the scope of natural immunity in order to strengthen the basis for fundamental and applied research. Topics covered include host defense mechanisms, the natural immune system, and regulation.

Audience:

Neurologists, psychologists, psychiatrists, immunologists, endocrinologists, physiologists, practising clinicians, veterinarians, animal scientists.

Contents:

NATURAL  IMMUNITY

Foreword: Istvan Berczi, Lóránd Bertók and, Donna A. Chow

Preface: Donna A. Chow

SECTION  I.  Host Defense Mechanisms

Host Defense: An Interaction of Neuroendocrine, Metabolic and Immune Mechanisms in the Interest of Survival. 
Istvan Berczi, Lóránd Bertók and, Donna A. Chow

SECTION  II.  Epithelial, Secretory and Endogenous Host Defense

Antimicrobial Peptides - The Defense Never Rests.
Kenneth M. Huttner 

Endogenous Cytoprotective Mechanisms. 
Hector R. Wong 

The Role of Bile Acids in Natural Resistance: Physico-Chemical Host Defense 
Lóránd Bertók

SECTION  III. The Natural Immune System.

A Historical Introduction of Natural Killer (NK) Cells and Current Status of Their Role in Host Defenses
Ronald B. Herberman

 The Role of the Reticuloendothelial System in Natural Immunity
 George Lázár, Elizabeth Husztik and George Lázár Jr. 

Effector Mechanisms of Natural Immunity: an Invertebrate Perspective
 Edwin L. Cooper 

Natural Immune Activation: Stimulators/Receptors. 
Donna A. Chow

Signalling in Natural Immunity: Natural Killer Cells
Laura N. Arneson and Paul J. Leibson

Pathogen recognition by Toll-like Receptors
Trude H. Flo and Alan Aderem

SECTION  1V: Regulation of Natural Immunity

Molecular Control of leukocyte Trafficking - Internal Regulatory Circuits of the Immune System: Leukocyte Circulation and Homing. 
Steven E. Bosinger, Karoline A. Hoisawa, Cheryl M. Cameron, Mark E. Devries, Jeff C. Coombs, Mark J. Cameron and David J. Kelvin.

Neuroendocrine Regulation of Natural Immunity. 
Istvan Berczi

Natural Immunity -Effect of Exercise. 
Bente K. Pedersen

New Prospect for the Enhancement of Natural Immunity. 
Lóránd Bertók

SECTION  V: Physiological,  Pathological and Behavioral Significance.

Physiological Regulation by the Natural Immune System 
Donna A. Chow

Pathological Relevance of the Natural Immune System 
Stefano Salvioli, Miriam Capri, Cristiana Fumelli, Francesco Lescal, Daniela Monti and Claudio Franceschi.

Behavioral Mechanisms for Defense Against Pathogens 
Susan J. Larson and Adrian Dunn
 

Article reprint used with permission, NIB 2005;(Vol:5: 215-262)

NEUROENDOCRINE REGULATION OF NATURAL IMMUNITY

ISTVAN BERCZI, DVM and PhD

Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, R3E 0W3, Canada

ABSTRACT

Natural killer (NK) cells, γδ T lymphocytes and CD5+ B lymphocytes are key effector cells in the natural immune system. These cells utilize germ-line coded receptors that recognize highly conserved, homologous epitopes (homotopes). Cytokines, hormones and neurotransmitters regulate natural immunity. Under physiological conditions the natural immune system is regulated similarly to the adaptive immune system: growth and lactogenic hormones (GLH), insulin-like growth factor-I (IGF-I), insulin, leptin, some steroid (glucocorticoid at physiological concentrations, dehydroepiandrosterone and some of its derivatives) and thyroid hormones are stimulatory. The peptides of the hypothalamus-pituitary-adrenal axis (CRF, AVP, ACTH, αMSH, βEND) exert an immunosuppressive, anti-inflammatory and anti-pyretic effect. Opioid peptides and estradiol are immunomodulators that promote some immune activities while inhibiting others. High (pathophysiological) levels of glucocorticoids, progesterone and testosterone act as immunosuppressive hormones. Beta-adrenergic agents are immunosuppressive and anti-inflammatory, whereas cholinergic agents promote immunity and inflammation. Substance P and calcitonin-gene related peptide are pro-inflammatory and promote immunity, whereas somatostatin is an antagonoist of these neuropeptides.

        Mild infection or a sublethal dose of endotoxin elicits a brief elevation of GH and PRL in the serum. Severe trauma, sepsis and shock results in the elevation of TNF-alpha, IL-1 and IL-6 in the blood stream, the GLH-IGF-I axis is suppressed, whereas the hypothalamus-pituitary-adrenal axis is activated.. LH, FSH, estrogens, androgens, progesterone, and thyroid hormones all decline during infection and endotoxin shock, as a rule. Leptin, insulin, glucagon, α-MSH, endorphin, and arginine vasopressin are increased during endotoxemia. A “sympathetic outflow” leads to elevated blood levels of catecholamines. Fever and catabolism prevails, whereas acute phase proteins in the liver, cell proliferation in the bone marrow, and protein synthesis by leukocytes are increased. This is an acute emergency reaction to save the organism after the adaptive immune system has failed to contain and eliminate the pathogenic agent. During sepsis and endotoxin shock, glucocorticoids potentiate the production of acute phase proteins and regulate pro-inflammatory cytokine production. Catecholamines also inhibit inflammatory responses and promote, even initiate, the acute phase response. Leptin regulates energy metabolism and it is a major stimulator of the immune system. If the acute phase reaction fails to protect the host, shock will develop and death will follow.

        The acute phase response leads to immunoconversion, which involves the suppression of the T-cell regulated adaptive immune system and the amplification of natural immunity. Natural antibodies, C-reactive -, endotoxin binding- and mannose binding proteins are boosted and serve as polyspecific recognition molecules for leukocytes. The natural immune system provides the first and the last line of host defence and its functional integrity and massive activation is largely dependent on the neuroendocrine system.

1.     INTRODUCTION

Natural immunity provides the first and last line of host defence against infectious disease, tissue injury and against a variety of noxious agents. Innate resistance may be divided into non-immune mechanisms and natural immune defence [1-6]. The natural immune defence system is comprised of highly specialized cells, such as natural killer (NK) cells, γδ T lymphocytes, and CD5+ B cells that secrete natural antibodies (NAb). However, the adaptive αβ T cells and B lymphocytes may also be activated by “superantigens” and other microbial mitogens, by the alternate complement pathway and cytokines during natural immune reactions. Neutrophilic, eosinophilic and basophilic leukocytes and mast cells also are integral to the natural immune system. Indeed, the entire immune system may be activated by natural immune mechanisms. The effector mechanisms of natural immune reactions are identical with those of adaptive immune reactions and include phagocytosis, cytotoxicity by the membrane attack pathway and by the induction of apoptosis, and most frequently, inflammation [5,7-10]. Non-immune mechanisms are diverse and it is beyond the scope of this chapter to discuss them in detail. Here we present the neuroendocrine regulation of the primary lymphoid organs (e.g., the bone marrow and thymus), of the cells involved in the natural immune system, and of the effector mechanisms, including inflammation, phagocytosis and cytotoxicity.

2.    GROWTH AND LACTOGENIC HORMONES, INSULIN-LIKE GROWTH FACTOR AND INSULIN

2.1. Embryonic development of the immune system

In foetuses that lack the pituitary gland the immune system develops normally [11]. It is likely that placental GLH support the development of the foetal haemolymphopoietic system. Bone marrow function, thymus cellularity and various immune reactions can be restored in hypophysectomized (Hypox) rats by human placental lactogen (PL) [12,13]. Human PL is mitogenic for Nb2 rat thymic lymphoma cells [14]. Prolactin (PRL) and pituitary grafts placed onto the chorioallantoic membrane of decapitated chicken embryos stimulated the early maturation of thymocytes [15]. In neonatal rats anti-growth hormone (GH) serum significantly decreased thymus and spleen weights, cellularities and the antibody response, all of which were corrected by treatment with bovine GH [16].

2.2. Bone marrow

Hormones have long been known to regulate bone marrow function [17]. Jepson and Lowenstein [18] discovered the erythropoietic effect of PRL. The anaemia, impaired bone marrow DNA and RNA synthesis, leukocytopenia and thrombocytopenia of Hypox rats were restored by syngeneic pituitary grafts (SPG) or by PRL, GH or human PL [12, 19-22]. PRL stimulated the phosphorylation of PRL-receptor-associated Janus tyrosine kinase (JAK)-2 in rat bone marrow and spleen cells, which led to the activation of signal transducer and activator of transcription (STAT) 5b protein, the interferon regulatory factor-1 (IRF-1) gamma activation sequence (GAS) and the IGF-I gene [23, 24]. Recombinant human PRL enhanced bone marrow function, accelerated lymphoid and myeloid reconstitution and promoted immune function in animals [25]. >

      Human haemopoietic progenitor cells formed granulocyte and erythroid colonies if stimulated with PRL in the presence of interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF) and erythropoietin (EPO) [26]. Human GH increased the total number of macrophage precursors in bone marrow cultures [27]. IGF-I mediated the action of GH on the bone marrow, including B lymphocyte growth [28]. PRL regulated immunity and the function of the bursa of Fabricius in birds [29-30].

2.3. The thymus

The stimulatory effect of GH on thymus has long been established [31-33]. GH induced thymus growth and increased immunocompetence in hormone deprived and old animals. GH was mitogenic for thymocytes and stimulated the production of thymic hormones [21,34-36]. Many effects of GH on the thymus are mediated by IGF-I [37-39]. In mice with severe combined immunodeficiency (SCID), human GH promoted the engraftment of human thymocytes [40]. In Hypox rats SPG, or treatment with GH or PRL restored DNA synthesis, cell proliferation and weight of the thymus and reversed immunodeficiency [21]. Pituitary grafts increased thymus weight and the number of thymocytes in Ames dwarf mice [41]. In thymocytes, PRL stimulated the expression of the Thy-1, LT-34 (CD4), and TL antigens [36, 42, 43]. PL selectively increased thymus growth in Snell-Bagg pituitary dwarf mice and PRL stimulated thymic hormones [38, 44, 46].

2.4. The antibody response

The immunization of rats with sheep red blood cells (SRBC, a T cell-dependent antigen) increased hypothalamic thyrotropin releasing hormone (TRH) mRNA, pituitary TRH receptor mRNA and plasma PRL levels with no change in TSH or GH. The hypothalamus-pituitary-adrenal (HPA) suppressive response appeared 5-7 days after SRBC treatment. In contrast, after treatment with lipopolysaccharide (LPS, a T-independent antigen), TRH mRNA decreased and an early corticosterone peak was induced [45].

2.5. Cell mediated immunity

The T cell dependent induction of macrophage tumouricidal activity was prevented by bromocriptine (BRC) and reversed by PRL [46]. Physiological concentrations of PRL stimulated B, T and NK cell responses to mitogens, and 5 to 10-fold higher levels inhibited the T cell response to IL-2 [47, 48]. PRL significantly increased interferon (IFN)γ secretion by human NK cells, which stimulated NK and lymphokine activated killer cell (LAK) cytotoxic activity [49]. PRL stimulated the growth and cytotoxic activity of purified NK cells, but there was no effect on NK activity in mixed populations of peripheral blood lymphocytes (PRL). This was due to the activation of suppressor cells in PBL. PRL did not induce novel cytotoxic NK cells, but stimulated novel LAK cytotoxicity. Both the NK and T cells participated in LAK induction. PRL had a diphasic (i.e. stimulatory and inhibitory) effect on NK cells with peaks either at 25 or 200 ng/ml, whereas LAK activation occurred only at 200 ng/ml. Physiological concentrations of PRL stimulated the generation of NK and LAK activities when combined with low doses of IL-2. Pathologically high concentrations of PRL reversibly inhibited the generation of LAK cells, whereas IL-2 activated NK cells were stimulated [50, 51]. PRL enhanced the cytotoxicity of mouse tumour-associated macrophage, which correlated with elevated NO2(-) and O2(-) release and was enhanced by IF-G [52]. GH increased NK cells and cytotoxicity in normal and GH-deficient humans [53-56].

2.6. The effect of GLH on phagocytic cells

GH, PRL and GH releasing hormone (GHRH), at very high concentrations, enhanced H₂O₂ production in human monocytes stimulated with phorbol myristate acetate (PMA). IGF-I had no effect [57]. In neutrophils, GH stimulated lysosomal enzyme production, oxidative metabolism, adhesiveness, modulated chemotaxis, and priming for superoxide production [58,59]. GH treatment (100 ng/ml) of human polymorphonuclear cells (PMC) inhibited apoptosis and up-regulated the production of reactive oxygen intermediates and had no effect on apoptosis of monocytes and lymphocytes [60]

       Neutrophilic leukocytes of aged rats show reduced superoxide anion secretion and bactericidal activity. This deficiency was corrected by treatment with IFN-γ or GH. Neutrophils from aged rats grafted with a syngeneic GH secreting pituitary tumour, GH3, responded normally to priming by IFN-γ for superoxide secretion [61]. The phagocytic activity of monocytes and PMC was increased significantly in children treated with GH for 6 months and in those on long term GH replacement therapy [62]. Neutrophils from patients with acromegaly and hyperprolactinemia showed a decrease in chemotactic activity [63].

2.7.  The effect of GLH on cytokine production

In bovine foetal thymocytes at mid-gestation, GH down-regulated c-jun and c-fos mRNA and increased the transcript levels for IL-1α, -β  , IL-6, and GM-CSF [64]. PRL enhanced IFN-γ production by murine spleen cells and by human peripheral blood mononuclear cells (PBMC) [46,65,and 66]. GH increased the release of IFN-γ and inhibited the enterotoxin A induced release of IL-1α from murine splenocytes. PRL also decreased IL-1α, but had no effect on IFN-γ  release under these conditions [67]. Placental and pituitary GH reduced IL-5 production slightly and stimulated IFN-γ  production in cultures of human peripheral blood lymphocytes (PBL). PRL also enhanced IFN-γ  [68]. IRF-I gene expression and IFN-γ  production were induced by PRL in Nb2 rat lymphoma cells and in T lymphocytes. GH stimulated the production of IL-2 by human lymphocytes and IL-1, TNF-α and superoxide anion production by monocytes [69-71]. GH activated monocytes for superoxide but not for TNF production, and for cell adherence or killing of M. tuberculosis [57].

        Murine splenocytes were stimulated with protein A (PA), toxic shock syndrome toxin-1 (TSST-1) and streptolysin S (SLS). In splenocytes stimulated with PA, GH induced a 40% and 50% drop in IL-1α and IFN-γ  release respectively, compared to controls, while no change was seen in IL-4 release. The release of IFN-γ  by TSST-1-stimulated splenocytes fell by 30%, but no changes were shown in IL-1α and IL-4 release after GH treatment. The release of IL-1α by SLS-stimulated splenocytes increased by 50% in the presence of GH, and no changes were shown in IFN- and IL-4 release [72]. High dose GH (13 IU/m²/day) did not affect TNF-α and IL-6 in patients undergoing laparoscopic surgery, nor did it in healthy individuals. The incubation of PBL with the GH antagonist, B2036, had no effect on the production of these cytokines [73].

        Enzymatically cleaved (16K)-PRL, but not full-length PRL, stimulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) by pulmonary fibroblasts and alveolar type II cells. The potency of 16K-PRL was comparable to that of IL-1β , IFN-γ , and TNF-α and occurred through a distinct receptor. Pulmonary fibroblasts endogenously produce 16K-PRL [74].

2.8.   Experiments in genetically altered mice

PRL gene and PRL receptor deficient mice are immunocompetent [75, 172]. In such animals GH must maintain immunocompetence. The over expression of IGF-II in FVB/N mice stimulated only T cell development. IGF-II transgenic Snell-dwarf mice are deficient in PRL, GH, TSH, and have low serum IGF-I. In these mice T cell development was stimulated to the same extent as in FVB/N mice. IGF-II also increased the number of nucleated bone marrow cells, including immature B lymphocytes. Mature B cells were not affected in the spleen [76,77].

2.9.   Insulin (INS)

Insulin induced an anaphylactic inflammatory promoting factor, potentiated anaphylaxis and enhanced fibrinolysis and phagocytosis. Fc receptors of guinea pig macrophages were downregulated and antibody-dependent cytotoxicity (ADCC) was inhibited by INS. Insulin suppressed the production of IL-1 and IFN-γ induced in murine spleen cells by Staphylococcal entertoxin-A. Glucagon and somatostatin antagonized INS action in lymphoid tissue [36, 68, 78].

3.      THE HYPOTHALAMUS-PITUITARY-ADRENAL AXIS AND OPIOID PEPTIDES

3.1.   Corticotropin releasing factor (CRF)

CRF produced in the hypothalamus releases ACTH from the pituitary gland and also mediates, in part, the cytokine induced ACTH release. CRF integrates the stress response in the central nervous system (CNS) and also acts centrally as an immunosuppressive agent. This is mediated by the stimulation of sympathetic outflow. Immunocytes have CRF receptors and produce CRF [79,80]. Immune derived-CRF production increases during inflammatory responses and it is an important anti-inflammatory hormone, although some forms of inflammation may be enhanced by CRF [81]. T lymphocyte proliferation, IL-1, -2, and -6 secretion and NK cell activity are all influenced by CRF [82].

         CRF type 1 receptor-deficient (CRFR1-0) mice show marked impairment of the HPA axis. Plasma ACTH concentrations of unstressed mutant mice are normal. Arginine vasopressin (AVP) is a major ACTH secretagogue in resting CRFR1-0 mice. Such mice are still able to mount an HPA response via mechanisms that do not depend critically on either CRF or AVP action [82, 83]. In mice CRF overexpression leads to a profound impairment of lymphocyte development and function mediated by corticosteroids [84].

         During immune/inflammatory reactions, cytokines, such as IL-1, -2, -6, -10, IFN-α, GM-CSF, leukaemia inhibitory factor (LIF) and oncostatin M [85-90] provide feedback signals for the activation/regulation of the HPA axis. IL-6 receptors are present on pituitary corticotrophs and on adrenocortical cells, which explains the ability of IL-6 to bypass CRF in the augmentation of adrenal function [91]. Radio-detoxification blunts the capacity of LPS to stimulate the HPA axis [92].

         A single intracerebroventricular (icv) injection of IL-1β increased CRF, AVP, ACTH and β -endorphin in the spleens of both sham-operated and adrenalectomised (ADX) rats. IL-1β  increased the thymic contents of CRF and ACTH in sham-operated but not in ADX rats [93]. Locally expressed CRF seems to release opioid peptides from immune cells in inflamed tissue, which inhibits pain sensation by peripheral nerves [94].

         Urocortin (UCN) is a new mammalian member of the CRF family and is a candidate endogenous ligand for type 2 CRF receptors. UCN mRNA expression increases within the thymus after immune activation in a corticosterone-dependent manner, which is the consequence of HPA axis activation [95]. UCN given icv (1 ng) to rats produced a marked decrease in the proliferative response of splenocytes, which was mediated by the sympathetic nervous system [96].

3.2.    Adrenotorticotropic hormone (ACTH)

ACTH has an anti-inflammatory effect and influences leukocyte recirculation in various species of mammals and birds, both due largely to the stimulation of glucocorticoids in the adrenal gland. ACTH inhibited antibody formation, antibody mediated reactions (anaphylaxis, Arthus type hypersensitivity) and cell mediated immunity (graft rejection, tuberculin response) [36]. ACTH suppressed both the Ca⁺⁺-dependent and -independent phagocytosis of murine peritoneal macrophages [97]. ACTH exerts an anti-pyretic effect by acting on the CNS [98]. Acute stress and ACTH stimulated IL-18 mRNA in glucocorticoid-producing cells of the adrenal cortex of adult male Sprague-Dawley rats, which was not inhibited by cortisone [99].

3.3.    Beta-endorphin (βEND) and other opioid peptides (OP)

Opioid receptors of κ-, δ- and μ-types are present on lymphocytes, monocyte/macrophages and on polymorphonuclear leukocytes [100-104]. Beta-END is derived in the pituitary gland and in other tissues from the proopiomelanocortin (POMC) peptide by enzymatic cleavage. CRF and cytokines, such as IL-1β and TNFα, regulate the production and secretion of βEND in the pituitary [105, 106]. Lymphoid cells also produce βEND, especially in inflamed tissues. Opioids in general and βEND in particular exert an anti-inflammatory effect and downregulate the immune response [107,108]. 

          Opioid peptides have a diverse effect on immune function. Antibody production, NK and LAK cell activity, cytotoxic T lymphocytes, the production of IL-1, -2, -4, -6, IFNγ and prostaglandins, mast cell degranulation and the activity of neutrophilic leukocytes were all affected by OP. Opioids modulate the immune system by acting through the CNS [109-118]. Opioid peptide-containing immune cells migrate to inflamed sites, where they release βEND which inhibits pain [119]. iNOS was expressed and cAMP levels were raised in human peripheral blood monocytes after incubation with βEND [120]. Met-enkephalin (MENK) and βEND enhanced chemiluminescence, induced a chemotactic response and up-regulated the expression of CD11b and CD18 by human neutrophils [121].

          Endomorphine-1 (EMO-1) and EMO-2 are present in the nervous system, in spleen and thymus, and have a very high specificity for the : receptor. EMO-1 and EMO-2 inhibited the production of super oxide anions by stimulated neutrophils [122,123]. Leucine-enkephalin (LENK) potentiates the immune response through the OP-1 ( δ) receptor and suppresses it through the OP-2 (κ) receptor. The OP-3 receptor has a permissive effect for centeral immunomodulation of endogenous opioid peptides and LENK. MENK enhances immune function by OP-1 receptors independent of OP-3 [124].

3.4.     Alpha-melanocyte stimulating hormone (α-MSH)

The POMC derived α-MSH is a major regulator of fever and inflammation. α-MSH is a cytokine antagonist and inhibits the pyrogenic and proinflammatory effects of IL-1, -6, TNF and IFNγ and promotes the secretion of IL-10. It acts within the brain to inhibit fever and peripheral inflammation. However, on the periphery α-MSH clearly exerts an anti-inflammatory effect [125-127]. Alpha-MSH inhibits inflammation by three general mechanisms: (I) inhibition of the production of inflammatory mediators; (ii) inhibition of inflammatory action of mediators; and (iii) inhibition of peripheral hosts cells [128]. Exogenous "-MSH antagonizes the stimulatory effects of IL-1 on the HPA axis. The ACTH response of rats to IL-1β was enhanced by icv infused α-MSH antiserum. [126, 129-131].

          α-MSH is produced by immune cells, by keratinocytes and is also present in the aqueous humour of the eye [132-135]. α-MSH inhibits thymocyte proliferation, induces neutrophilia and suppresses the production of acute phase proteins by the liver, TNF and IFN-γ production, the induction of prostaglandin E in fibroblasts and contact sensitivity reactions. The immunosuppressive effect of IL-1b, given icv, could be blocked by the simultaneous infusion of α-MSH [136-143]. A tripeptide from α-MSH strongly induced IL-10 in purified monocytes. T lymphocytes did not produce IL-10 in response to α-MSH [144, 146]. The α-MSH derived peptide (1-13) and its carboxy-terminal tripeptide α-MSH (1-13) have exerted a potent antiinflammatory effect in all major models of inflammation [129].

4.1.    Thyrotropin releasing hormone (TRH)

B and T lymphocytes have receptors for TRH [145]. TRH treatment of rats significantly increased the proliferative response of spleen cells to Con-A [147]. TRH stimulated T cell development in the gut and not in the spleen [148]. In man TRH elevated serum IFN-γ levels [150]. Repeated TRH administration in critical illness resulted in a repetitive increase of TSH, PRL, GH, thyroxin (T4), and triiodothyronin (T3), without increasing reverse T3 [151].

4.2.    Thyroid stimulating hormone (TSH)

TSH receptors (TSHR) are present in B and T cells, NK cells, monocytes and at high levels in dendritic cells (DC). TSHR are not detectable on foetal and neonatal immune cells. TSH significantly stimulated IL-2 and IL-12, IL-1β responses and enhanced the phagocytic activity of DCs from adult animals, enhanced the proliferative response of murine spleen cells to IL-2, significantly increased IL-2 induced NK cell cytotoxicity and enhanced the expression of MHC-II by human thyroid epithelial cells [149, 152-156]. In bone marrow cells IL-6, IFNβ, TNFα, TNFβ, TGFβ2, and lymphotoxin-$β responses were reproducibly induced by TSH [158]. Lymphocytes and monocytes synthesize TSH [149, 158].

4.3.    Thyroxin (T4) and Triiodothyronin (T3)

          T4 inhibited the development of TCRα,β CD8 in intestinal intraepithelial lymphocytes (IEL) in 6-8 week old euthymic mice [167]. The NK cell number and/or cytolytic activity of healthy subjects > 90 years old correlated positively with serum levels of vitamin D, while T3, FT4, i-PTH hormones and lean body mass were correlated only with NK cell number [168].

          Dendritic cells inhibited the proliferation of rat thyroid follicles, which was mediated by IL-1β [169]. IL-1 in moderate to high concentrations inhibited thyroid cell (TEC) function, which was supported by TNF and IFNγ. IL-1 induced the release of NO and cGMP, inhibited the adenylate cyclase mediated pathways and stimulated the guanylate cyclase mediated pathways in TEC. IL-1 receptor antagonist counteracted these IL-1 effects [170]. The binding of thyroid hormone receptors to the DR4 thyroid hormone responsive element was markedly decreased in the spleens of rats with adjuvant arthritis (AA) or with AA + adrenalectomy [171]. Thyroid hormone deficient mouse strains showed a defective primary B cell development. Other haematopoietic cell lineages and mature B lymphocytes were normal [172].

5.       NERVE GROWTH FACTOR, LEPTIN AND NEUROPEPTIDES

Nerve growth factor (NGF) shares tyrosine kinase receptors with other neurotropic factors that belong to the TNF receptor superfamily and are present in lymphoid cells and cells of the nervous system. NGF stimulates mast cells, haemopoietic colonies and neutrophilic leukocytes and locally exerts a proinflammatory effect. However, systematically applied NGF suppresses inflammation. Lymphocyte-derived NGF protects the nervous system and other tissues from inflammatory damage [173, 174].

          Leptin (LEP) is adipocyte-derived. It belongs to the GLH cytokine family and signals by a class I cytokine receptor. LEP regulates energy metabolism, reproductive function, lymphocyte development and function, and it up-regulates phagocytosis and proinflammatory cytokines. LEP stimulates the production of IL-1ra, which protected mice against LPS toxicity. In murine glial cells LEP stimulated IL-1β, it promoted wound healing and angiogenesis. During the acute phase response (APR), LEP increased rapidly in response to elevated TNF levels. LEP contributes significantly to survival in sepsis by moderating glucocorticoid and IL-6 production, stimulating IL-1ra and potentiating the immune response. [175-184].

          Arginine vasopressin (AVP) V₁ type receptors are present on human PBMC. AVP stimulated the production of prostaglandin E2 (PGE2) by human mononuclear phagocytes, and the production of βEND by human PBMC. AVP is antipyretic and it attenuates fever after central administration [6, 185, 186].

          Substance P (SP) is produced in both the nervous- and in the immune systems. SP is a major mediator of neurogenic inflammation. SP induces mast cell discharge, increases capillary permeability and smooth muscle contraction, stimulates immune phenomena, bone marrow cytokines and the formation of granuloma tissue, increases Fcγ and , ε receptors and decreases C3b receptors on eosinophils, releases TNFα from macrophages and modifies macrophage function during stress. In polymorphonuclear leukocytes SP stimulated the respiratory burst and chemotactic and phagocytic activities [187-194]. SP increased the release of PGE2 and collagenase from rheumatoid synoviocytes [195] and PGA and thromboxane B2 from astrocytes [196]. SP induced IL-3 and GM-CSF secretion by bone marrow cells, which was mediated by the stimulation of IL-1 and IL-6 [197]. SP activated platelet cytotoxicity against Shistosoma mansoni larvae and its receptor was necessary for the normal granulomatous response [198].

          Calcitonin gene related peptide (CGRP) stimulates mast cell discharge and inflammation, inhibits antigen presentation, lymphocyte proliferation, IL-2 production, mRNA synthesis for TNFα, -βb and IFN-γ and suppresses IFN-γ induced H₂O₂ production by human monocytes. T lymphocytes synthesize CGRP. [199-201].

          Somatostatin (SOM) is an antagonist of SP and inhibits inflammatory and immune reactions. SOM is beneficial in models of autoimmune disease and chronic inflammation. Lymphocyte proliferation, endotoxin induced leukocytosis, IgA secretion, and IFNγ production are inhibited by SOM. Its effect on antibody dependent cytotoxicity is variable. SOM exerts a regulatory influence on macrophages [190,191, 202-204].

          Vasoactive intestinal peptide (VIP) receptors are present on monocytes and lymphocytes. VIP stimulated macrophage chemotaxis and inhibited lymphocyte chemotaxis through the activation of adenylate cyclase [205], it enhanced phagocytosis by mouse peritoneal macrophages [206]. Immunoreactive VIP was detected in rat thymus, spleen and lymph nodes in both lymphoid and non-lymphoid cells [207].

          Pituitary adenylate cyclase activating peptide (PACAP) and VIP inhibited the nuclear translocation of NF6B in stimulated macrophages. This antagonised the effect of IFN-( and down-regulated the inflammatory response. The production of TGF-β1, IL-4, -6, -12, TNF-α and NO were inhibited by both peptides [208-213]. IL-6 production was enhanced by VIP/PACAP in unstimulated macrophages [214].

          Various immune cells express β-type adrenergic receptors. Beta-adrenergic agents inhibit allergic and asthmatic reactions and various immune phenomena. Acetylcholine and cholinergic agents, by acting on muscarinic receptors, enhance immune phenomena, including the release of histamine and other mediators from mast cells. Allergic patients show an increased sensitivity to cholinergic stimulation (214).

6.       STEROID HORMONES

6.1.      Glucocorticoids (GC)

Leukocytes express GC receptors (GR) [215,216]. GR are bound to heat shock protein 90 (HSP90) in the cytoplasm and function as nuclear transcription factors. GR modulate the transcription factors AP1, IκBα, and the cAMP-responsive element binding protein (CREB) [217-220]. Membrane GR initiate apoptosis [221]. Macrophage migration inhibitory factor (MIF) counter-acts GC action [222]. Lipocortin 1 is a GC-induced protein, which inhibits neurogenic inflammation. [223,224]. Cytokines that activate AP-1 may induce steroid resistance [225, 226]. The thymic epithelium synthesizes GC, which inhibits T cell antigen receptor (TCR)-mediated apoptosis [227-229]. GC induce thymus involution (36, 230,231]. Cytokines prevent their own toxicity by stimulating GC production, and by modifying target cell sensitivity for GC counter-regulatory action [232].

           In the monocyte-macrophage lineage, metabolism, chemotaxis, phagocytosis, cytotoxic reactions, antigen presentation, IL-1, IL-1ra, IL-6 secretion and the ability to respond to lymphokines are inhibited by GC. In macrophages GC suppressed the production of collagenase, elastase, plasminogen activated TNFα, superoxide and NO [36, 233-235].

           GC increased HLA antigen and IFNg receptor expression [236, 237], and potentiated the induction of Fc-γ receptors in human monocytes by IFNg [238]. Low concentrations of GC induced MIF production by macrophages. MIF could override the GC-mediated inhibition of cytokine secretion by LPS activated monocytes and antagonized the protective effect of GC in lethal endotoxemia [239].

           Dexamethasone (DEX) inhibited the IL-12-induced IFNγ secretion and IFN regulatory factor-1 expression in NK and T cells [240]. GC inhibited NK cell-mediated cytotoxicity and ADCC, and this effect was potentiated by PGE2 and abrogated by IFN-γ or IL-2 [36, 241].

           GC have a powerful anti-inflammatory effect, including action against neurogenic inflammation. This is the result of GC action on cytokine and other mediator secretion, inhibition of leukocyte priming, reduction of vascular permeability, and synergism with other anti-inflammatory mediators, such as catecholamines, βEND and α-MSH [223, 242-244]. The general hypersensitivity to GC is eliminated at the site of inflammation by locally produced cytokines [245].

           GC induced mast cell destruction in rats and depleted cutaneous mast cells in man [246]. GC inhibited mediator release from mast cells and basophilic leukocytes [247]. Neonatal GC treatment reduced the corticosterone response to LPS in adult rats; LPS-stimulated macrophages of such rats produced less TNF-α and IL-1β and splenocytes showed increased mRNA levels for IFNγ and TNF-β [248].

           The GC response peaked 36 hours after murine cytomegalovirus (MCMV) infection, coincident with elevated blood levels of IL-12, IFN-γ, TNF, and IL-6, and was dependent on IL-6 for maximal release. Adrenalectomised (ADX) mice were more susceptible than controls to MCMV-induced lethality, which was mediated by TNF and could be reversed by GC replacement. Lack of endogenous GC resulted in increased IL-12, IFN-γ, TNF, and IL-6 production as well as in mRNA expression for IL-1α; and IL-1γ. [249].

6.2.      Aldosterone

Aldosterone, which is a GR-I agonist, significantly reduced the number of lymphocytes and monocytes and, unlike RU48362, also decreased the number of neutrophils. T helper cells and NK cells were decreased by aldosterone. Corticosterone at physiological doses behaved like a GR-II agonist in these experiments. The GR-II agonist RU48362 decreased T and B and NK cells to a very low absolute level in young adult rats. [250].

6.3.      Sex hormones

6.3.1.   Gonadotropins

Pyrogenic cytokines, especially IL-1β, are significantly influenced by exogenous gonadal steroids and gonadotropins. [251]. IL-1β, generated in the central nervous system (CNS) during inflammation, upregulates opioids and tachykinins in the hypothalamus which cause the suppression of hypothalamic luteinizing hormone releasing hormone (LHRH) and pituitary luteinizing hormone (LH) release [252].

6.3.2.   Sex hormone receptors and signalling

Estrogen (ER) and androgen receptors (AR) are present in lymphoid tissues. The classical estrogen receptor (ERα) is detectable in lymphoid tissue, however, ERβ is more abundant in lymphoid cells. Progesterone (PS) also acts through GC receptors in addition to its own specific receptors (PR). At high concentrations, estrogens and androgens also act on GR [253-256]. Membrane bound steroid receptors, which include the polyglycoprotein (PGP) pump also exist. [257]. ER binds to estrogen response elements (ERE) in target genes, recruits a coactivator complex called CBP-pl60 that mediates the stimulation of transcription, and also activates AP-1 sites that increases the activity of Jun/Fos [258,259]. ER interacts directly with the transcription factors NF-IL6 and NF6B, and inhibits their binding to DNA, which is likely to be the molecular basis for repression of IL-6 gene expression by estrogens. Unlike estrogens, the anti-estrogen, tamoxifen (TX) does not inhibit the induction of the IL-6 promoter [260].

           Three dimeric species of PR, namely A/A, A/B, and B/B may be formed and bind to progesterone response elements (PRE) and subsequently regulate transcription. Receptor dimerization is obligatory for binding to PRE, but it is not sufficient to activate transcription without the hormone. In pure heterodimers, A receptors are dominant negative inhibitors of B receptors [261-263].

6.3.3.   Estrogens

Estradiol (E2) causes thymic involution, suppresses bone marrow function, cell-mediated immune reactions, including the helper, suppressor and effector functions of T lymphocytes. Natural killer cells, neutrophils and mast cell degranulation are also inhibited by E2. Phagocytosis, antibody production and some autoimmune diseases of animals are enhanced by E2 [36, 264, 265].

           In rat alveolar macrophages E2 and PS inhibited NO production [266]. In mice E2 inhibited the homing and activation of inflammatory cells and their production of TNFα and IFNγ [267], and reduced NK cell-mediated cytotoxicity [268]. In ovariectomized rhesus monkeys nine months after E2 replacement killer cell activity was reduced [269].E2 modulated both pro- and anti-inflammatory cytokine activities [270]. In women, ovarian hyperstimulation led to neutrophil activation, which correlated with the degree of luteinization. Neutrophil L-selectin expression negatively correlated with serum progesterone levels [271].

6.3.3.1. Immunomodulation by antiestrogens

The non-steroidal antiestrogen, TX, has an antiproliferative effect on lymphocytes, interferes with the stimulatory effect of E2 on phagocytosis, inhibits giant cell formation by monocytes and blocks H₂O₂ production by human neutrophils. TX enhanced the LPS induced production of TNFα by human monocytes and by rat peritoneal cells [257, 272-274].

           TX and toremifene (TO) decrease serum PRL, GH and IGF-I levels, influence the expression of hormone receptors and their binding proteins, which also affect the immune system. TX-induced immunosuppression in rats could be reversed by treatment with either GH or PRL. TX also antagonized the stimulatory effect of PRL on lymphoid cells [257, 275]. TX antagonized the inhibitory effect of E2 on NK cells. NK, LAK and CTL effector cells maintained their cytolytic activity after treatment with 1 μM TX or 5 μM TO, which are commonly achievable during cancer therapy. In murine tumour systems both TX and TO enhanced the immunotherapy (e.g., by NK, LAK or CTL effectors) of ERα negative tumours, which led to the cure and long-term survival of a significant proportion (50-75%) of animals with lethal cancer [276-282].

           Antiestrogens enhanced the cytotoxic effect of anti-Fas monoclonal antibody in the majority of human ovarian carcinoma cells so examined. In Fas negative K562 target cells the NK mediated perforin/granzyme pathway of immune cytolysis was also enhanced [274, 283, 284].

6.4.      Androgens

Human lymphocytes metabolize sex hormones and are capable of synthesizing androgens [285]. In general, testosterone (TS) suppresses immune reactions. The development of the bursa of Fabricius is prevented by TS in chicken embryos. TS has been proposed to selectively favour the differentiation of suppressor T lymphocytes in the thymus [36, 286]. MHC-linked genes influence the effect of androgens on the immune system [287]. Androgens stimulate haemopoiesis [288]. In the thymus TS is converted to E2 by aromatase and E2 is a powerful inducer of thymic involution [289]. Dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA) restored the capacity of T cells to produce IL-2, IL-4 and IFN-γ in aged mice to the levels of young animals [290].

           TS inhibited NO release and stimulated the release of reactive oxygen intermediates from rat peritoneal macrophages [233], inhibited inducible NO synthesis in the RAW 264.7 murine macrophage cell line [291]. In guinea pigs, androgens (TS, DHT and mesterolone) impaired the clearance of IgG-coated cells by decreasing splenic macrophage FcγR expression. Antiandrogens (flutamide, nilutamide, cyproterone acetate, spironolactone, and finasteride) counteracted the inhibitory effects of androgens [292].

           In women, androgens slightly decreased free urinary cortisol levels and enhanced the mitogen-induced IFNγ/IL-4 ratio and TNFα production. [293]. Bioactive TGF-β₁ fell to approximately 50% after castration of male mice and was normalized 1 week after TS treatment. TS modulated the production of TGF-β by thymocytes [294]. In male mice DHT significantly decreased the releases of IL-1β and IL-6 by splenic and peritoneal macrophages after trauma-haemorrhage. DHT-treated animals exhibited increased IL-10 and Kupffer cell IL-6 release. Estrogen prevented this immunodepression in castrated male mice [295,296].

           Both pregnenolone (PREG) and DHEA are metabolized by the spleen and the derivatives, which include testosterone, DHT, androstenediol (AED) and androstenetriol (AET), are much more potent immunoregulators than DHEA itself. [297]. Human monocytes express receptors for DHEA. In monocytes DHEA enhanced the induction of cytotoxicity, IL-1 secretion, reactive nitrogen intermediate release, and the expression of complement receptor-1 and TNFα protein [298].

           Plasma levels of DHEA-S, AED and TS are suppressed in chronically ill patients and in those treated with DEX. This is corrected by ACTH [299]. DHEA-S is depressed in postmenopausal women with rheumatoid arthritis [300]. In postmenopausal women treated with physiologic doses of DHEA for 3 weeks, CD4⁺ T cells were decreased and CD8⁺/CD56⁺ (natural killer) cells increased. T cell mitogenic and IL-6 responses were inhibited, whereas NK cell cytotoxicity was dramatically increased [301].

6.5.      Progesterone (PS)

PS is immunosuppressive. It suppressed lymphocyte proliferation [302] and the anti-Candida activity of neutrophils from mice [303]. PS significantly inhibited nitrite release and stimulated the release of reactive oxygen intermediates [304] and stimulated TNF release from rat peritoneal macrophages [234]. Human PBMC, stimulated with LPS, produced less IL-1 upon exposure to PS, whereas IL-6 secretion was not altered. However, PS failed to inhibit IL-1 secretion by PBMC from male donors with rheumatoid arthritis [305].

            PS protects the foetus against maternal immune reactions [306, 307] and lymphocyte sensitivity to PS is increased during pregnancy, due to the expression of PR by (* T lymphocytes in response to foetal antigens, leading to the production of a progesterone-induced blocking factor (PIBF). PIBF acts on the phospholipase A2 enzyme, interferes with arachidonic acid metabolism, induces a Th2 biased immune response, and exerts an anti-abortive effect by controlling NK activity [308]. PS treatment of mice suppressed glucocorticoid-induced thymocyte apoptosis [309] and decreased host resistance against viral and fungal infections [310,311].

6.6.      1,25-Dihydroxivitamin-D3 (VD3)

The VD3 precursor, cholecalciferol, is present in the diet and induced in the skin by UV radiation. 25-Hydroxyvitamin D3 is generated in the liver, which is processed further by 1-hydroxylase in the kidney, in monocyte/macrophages, in keratinocytes, bone marrow, placenta, glia cells and pneumocytes [312-314].

            The VD3 receptor (VDR) interacts with VD3 responsive elements (VDRE) on DNA and also with the retinoic X receptor (RXR). Stimulation and inhibition are both possible through VDRE. Protein kinase C is involved in VDR mediated signalling and VD3 regulates the DNA binding subunit of NFkB. Monocyte/macrophages, activated T lymphocytes, and bone marrow cells express VDR [312-315]. The GM-CSF enhancer is transcriptionally repressed [316] and the IFN( promoter is down-regulated by VD3. [317].

            VD3 is a potent anti-proliferative and pro-differentiation mediator for macrophages, lymphocytes and other cells [318]. In monocytes/macrophages, adherence, chemotaxis, phagocytosis, cytotoxicity, H₂O₂, oxygen radicals, and HSP production are stimulated by VD3. Antigen presentation, the production of IL-1, -2, -6, -12, TNFα, IFN-γ and the function of Th-1 cells are inhibited. Suppressor T cell function, B cell proliferation and Ig secretion are inhibited. Natural killer cell cytotoxicity is stimulated [312,313].

            The NK cell number and/or cytolytic activity of healthy subjects greater than 90 years old was positively associated with serum levels of vitamin D, while T3, FT4, i-PTH hormones and lean body mass were associated only with NK cell number [168].

            The regulation by neurohormonal regulatory factors of natural immunity is summarized in Table 1.

Table 1           Major hormonal and neural regulators of natural immunity. ___________________________________________________________________________________________________ Hormone Mediator NK γδT CD5+B /NAb CTK PHAG CTX APP INFL ___________________________________________________________________________________________________ HPT axis TRH IFNγ TSH IL1,2,6,12,IFNβ,TNFα,TGFβ T4/T3     GLH hormones GH IL1,2,6,IFNγ,TNFα;IL5 PRL IFNγ;IL1α IGF-I INS IL1,IFNγ ADCC GLU IFNγ    HPA axis CRF IL18;IL1,2,5 ACTH IL18 GC IL10;IL1,6,TNFα 0 αMSH IL10;IL1,6,TNF.IFNγ βEND CAT Steroid hormones E2 IL6;TNFα,IFNγ TS DHEA IL2,4,IFNγ PS IL1;TNF VD3 I11,2,6,12,TNFα,IFNγ Neurotransmitters /peptides SP IL1,3,6,TNFα,GMCSF CGRP SOM NGF LEP IL1,6,IL1Ra,TNF ___________________________________________________________________________________________________ Legend:=increase;=decrease;=variable effect; 0=no effect Abbreviations not used in the text: CTK = cytokines, PHAG = phagocytosis, CTX = cytotoxicity, INF = inflammation.

7.          IMMUNOCONVERSION IN THE ACUTE PHASE RESPONSE

7.1.        Introduction

The healing power of fever has been recognised in ancient Egypt, Greece the Roman and Persian empires and fever therapy was practised during the first half of the 19^th century [319] using whole Gram-negative bacteria until Boivin et al. [320] purified endotoxin from such bacteria. Numerous beneficial effects can be induced by sublethal doses of endotoxin in animals [321-329].

            Glucosuria, hyperglycaemia and insulin resistance have long been recognised in infectious disease. Subsequently the pyrogenic leukocyte derived endogenous mediator (LEM) was discovered, which induced acute phase reactants in the liver during severe infectious disease. Pyrogens also induced ACTH release. By 1975 infection was known to influence GC, mineralcorticoids, INS, glucagon, GH, and metabolism [330-332]. Subsequently, IL-1 was identified as an endogenous pyrogen. Because endogenous pyrogens released ACTH, the hypothesis was proposed that IL-1 is an immune derived mediator that acts on the pituitary gland [333]. Indeed, IL-1 was shown subsequently to activate the HPA axis [334-338]. Today it is clear that IL-1 and other cytokines induce the neuroendocrine and metabolic responses to infection and to other forms of injury, which is designated as the acute phase response (APR).

            Hans Selye discovered that infection and injury activated the HPA axis. He concluded that various noxious agents elicit stress , which leads to a general adaptation syndrome (GAS). Stressed animals showed an initial alarm reaction, followed by adaptation when the organism was resistant towards various insults including the stressor, and the endocrine and other parameters returned to normal. With lasting stress, exhaustion would occur which could lead to death [339-342]. Selye also discovered the anti-inflammatory effects of GC [343] and the influence of sex hormones on lymphoid tissue [344]. It is now apparent that Selye’s general adaptation syndrome is analogous to the acute phase response [345-348].

7.2.      The response to endotoxin

In a broad sense physical, chemical and biological agents may cause injury. Injured cells release chemokines and cytokines, which in turn attract and activate leukocytes. This leads to immune activation and inflammation and is in most instances followed by regeneration and healing [349].

Figure 1: The molecular structure of baccterial lipolysaccharide  [After Westphal et al.,351].

7.2.1.    Bacterial endotoxin

Lipopolysaccharide, or endotoxin, is present in the outer cell membrane of all Gram-negative bacteria and divided into polysaccharide and core glycolipid, which consists of lipid A (LA) plus the core polysaccharide. The core glycolipid is the “toxic” moiety and it is an obligatory component of the bacterial cell wall (Fig. 1) [350]. The polysaccharide chain exhibits heterologous epitopes that stimulate specific antibodies, which are used for the serological classification of Gram-negative bacteria [246]. LA is highly conserved and shows extensive cross-reactivity amongst all Gram negative bacterial strains. Therefore LA functions as a homologous epitope, or homotope, which identifies all Gram-negative bacteria towards the immune system [351, 352].

            Lipopolysaccharide binding proteins (LBP) bind LA (10^-9 M) in the serum of multiple species [353]. It is a 60 kDa glycoprotein with normal serum level of 0.5-10 μg/ml, but it may surpass 200 μg/ml during APR [354, 355]. LBP mediates the interaction of LPS with CD14, which is present on monocytes, macrophages and neutrophilic granulocytes, and enables them to respond to extremely low levels of LPS. CD 14 lacks a transmembrane sequence [356, 357]. CD14 mediates TNF, IL-6 and IL-8 responses in monocytes and macrophages. VD3 induced CD14 in a premonocytic cell line. IL-4 decreased CD14 expression, and IL-4 or IFN inhibited CD14 release by monocytes. TNF and LPS enhanced CD14 expression by monocytes. In human neutrophils, TNF, GM-CSF, G-CSF and formyl peptides increased CD14 expression [358].

            The CD14 concentration increases in hospitalized patients, especially in those with autoimmune disease. Soluble CD14 inhibits the biological activities of LPS and is assumed to present LPS to endothelial cells [358]. The Toll-like receptor 4 is involved in signal transduction by LPS-CD14 complexes, leading to NF6B activation [359]. An integrin, CD18, also mediates LPS signalling. Three forms exist, CD11a/CD18, CD11b/CD18, and CD11c/CD18. All three bind LPS, participate in phagocytosis but are not essential for cellular responses. CD11b/CD18 expression by human neutrophils was up-regulated by LPS [359].

            Frog and fish show extreme resistance to LPS, whereas mammals are very sensitive [360]. However, in Tilapia oreochnomis mossanbicus (Teleosti) LPS elicits integumental and cortisol responses [361]. In the horseshoe crab (Limulus polyphemus) LPS causes fatal intravascular coagulation by activating clottable proteins in the blood, which is produced by amoebocytes [362]. It seems apparent that lipid A is not inherently toxic to animal cells, but rather, the immune system has evolved in higher animals to recognize LA as a target (homotope) for the purpose of natural immune host defence . Numerous other homotopes are present on microbes, self-components and cancer cells [345, 349].

7.2.2.    The cytokine response to LPS

In normal mice, blood TNF is significantly increased at 1-2 hours after systemic LPS administration, which is followed by a decline and return to normal levels at around 4 hours. In ADX animals the TNF response was 60 times higher and sensitivity to LPS toxicity increased about 500 times compared to controls. Pre-treatment with DEX prevented these excessive responses. The inhibition of cortisone synthesis in the adrenals by metyrapone also increased susceptibility to LPS (~15 times higher than controls) [364-366]. Similar kinetics of TNF release were found in man after LPS infusion [366].

            Blood IL-1 reaches the maximum at 4 hours in mice after LPS administration and remains elevated up to 24 hrs [365]. In man IL-6 peaked at 120 minutes after LPS administration, which was not inhibited by glucocorticoids or by repeated LPS administration [352,367,368]. Leukaemia 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 induced catabolism and had a protective effect if given prior to the administration of bacteria [369]. Additional cytokines/mediators that participate in endotoxin shock include IL-8 [370], IL-10 [371], IFNγ [372], TNF synthesis inhibitor [373], IL-1ra [374], platelet activating factor, colony stimulating factor, prostaglandins and thromboxanes [352,375,376].

7.2.3. Neuroendocrine response to endotoxin

Wexler et al. [378] discovered that in rats that LPS stimulated the release of ACTH. Endotoxin, infectious disease, and various forms of injury elicit a neuroendocrine response by the stimulation of cytokines and chemokines [353,376,377]. Profound changes occur in serum hormone levels (Table 2), which are likely to be much more complicated than is indicated in the table. Dynamic and diurnal changes of hormones also play a role. Although much remains to be elucidated, it is very clear that the HPA axis exerts a powerful suppressive effect on the adaptive immune system and on inflammatory cytokines. Thus adaptive immune reactions are profoundly suppressed, whereas the induction of acute phase proteins in the liver and the production of natural antibodies by CD5+ B cells are stimulated by cytokines glucocorticoids and cathecolamines [349,352,378-380]. PRL and GH are immunostimulatory and usually rise within the first hour after endotoxin injection. This is followed by a decline to low normal to subnormal levels in endotoxin shock. LH, FSH, estrogens, androgens, progesterone, and thyroid hormones also decline, whereas leptin, insulin, glucagon, α-MSH, β-endorphin, and arginine vasopressin are increased during endotoxemia [179, 352,375,376].

Table II   Major neuroendocrine changes induced by endotoxins.
____________________________________________________________________
HPT and GLH hormones	Response	The HPA axis	Response	Gonadotropins and sex hormones	Response
____________________________________________________________________
TRH		CRF		LH
TSH	0	AVP		FSH
T4		ACTH		E2
T3		GC		TS
PRL		αMSH,		DHEA	?
GH		βEND		PS
IGF-I		CAT
INS
GLU
LEP
____________________________________________________________________
Please see legends to Table1. 0=no effect. This table is modified from reference [350].

7.2.4.    The acute phase response induced by endotoxin

Geller et al. [381] discovered that cortisone treatment protected mice against a lethal dose of LPS. Since then, the protective effect of GC against endotoxin shock has been confirmed repeatedly in various species [381-383]. The systemic response to LPS is a typical APR. Moreover, during severe trauma and shock LPS absorbs from the intestines and may aggravate the condition [374]. In mice approximately 7% of genes were activated in the liver by LPS. The pathways for cholesterol, fatty acid, and phospholipid synthesis were suppressed and gene expression for innate defence were enhanced, which resulted in the coordinate induction of the MHC class I antigen presentation machinery, illustrating an interaction between innate and adaptive immunity [384].

               Animals exposed to LPS will produce fewer cytokines in response to a second dose, which is known as endotoxin tolerance. Pathological changes are reduced and resistance to endotoxin, to infectious agents and to toxic and other noxious insults is significantly increased in LPS-tolerant animals [370,385,386]. LPS toxicity and tolerance is mediated by macrophages [387]. The hormones of the HPA axis play an important role in the development of LPS tolerance [358,378,389]. LPS injected into rats ip increased IL-1 levels in the hypothalamus, hippocampus, dorsal vagal complex, cerebellum, posterior cortex, and pituitary 2 hrs after injection [390]. CRP protected mice from a lethal LPS dose by binding to Fcγ-receptors (FcγR)-I and FcγRII, which results in the enhanced secretion of the anti-inflammatory cytokine, IL-10 and in the down-regulation of IL-12 [391].

               In mice transgenic for human CRP and deficient in the C3 or C5 components of complement, there was a diminished induction of CRP and serum amyloid P-component (SAP) by LPS. LPS induced IL-6, but not IL-1 in complement-deficient mice. Human C5a induced IL-1β and caused significant elevation of both serum CRP and SAP in human CRP transgenic mice. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a was ineffective [392]. A transgenic human CRP protected C57BL/6 mice against experimental allergic encephalomyelitis (EAE). Human CRP inhibited the encephalitogenic peptide-induced proliferation of T cells, the production of TNF-α IFN-γ and chemokines (macrophage-inflammatory protein-1α RANTES, monocyte chemoattractant protein-1), and increased IL-10 production [393].

            Newly synthesized acute phase proteins are essential for the development of endotoxin tolerance and these proteins exert anti-microbial and immunoregulatory functions [375]. The importance of the liver in the development of endotoxin tolerance is emphasized by the observation that D-galactosamine (GalN), which profoundly increases endotoxin sensitivity, intoxicates the liver [394]. Moreover, GalN- treated mice cannot develop endotoxin tolerance [389]. Metallothionein (MT), a low-molecular weight, cysteine-rich, metal-binding protein, is also induced in APR. MT-null mice were more sensitive to LPS/GalN-induced lethality than wild-type mice. Messenger RNA levels of APP in response to LPS/GalN were decreased in MT-null mice compared to wild-type mice [167].

            Treatment of rats with the cyclooxygenase inhibitors either attenuated (meloxicam) or abolished (diclofenac) LPS-induced fever, but had no effect on plasma cortisol or IL-6. The TNF response was enhanced by both drugs. Thus the prostaglandin-dependent inflammatory pathway for fever induction is distinct from the pathway of HPA axis activation [396]. The expression of the G protein-coupled prostanoid receptors EP2-R, EP4-R, and DP-R, but not the IP-R, was up-regulated by treatment of rat hepatocytes with IL-6. In such hepatocytes PGE2 attenuated the IL-6-induced alpha-2-macroglobulin formation [397].

            IL-6 induces DNA-binding of STAT transcription factors on regulatory elements in target genes. TNFα is involved in several models of liver failure as a mediator of both cytotoxicity and cell proliferation. It activates NF6B, thereby triggering inflammatory processes [398] IL-1 concomitantly induces NF6B activation and dephosphorylates IL-6-activated STAT1. The latter mechanism could account for the inhibition by IL-1 of the IL-6-dependent induction of type II acute-phase genes [399]. Soluble gp130 is the natural inhibitor of IL-6 responses [400].

            The nuclear hormone receptors, peroxisome proliferator-activated receptor alpha (PPAR) and liver RXR play key roles in regulation of hepatic lipid metabolism. LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPAR target [401]. LPS elicits a dramatic increase in the synthesis and secretion of triglyceride (TG)-rich lipoproteins by the liver and the inhibition of lipoprotein lipase. This cytokine-induced "lipemia of sepsis" was considered to represent the mobilization of lipid stores to fuel the host response to infection. However, since lipoproteins can also bind and neutralize LPS, it is hypothesized that lipoproteins (VLDL and chylomicrons) are also components of an innate immune response to infection [402].

            Elevation of IL-6, soluble TNFα and soluble IL-6 receptors was detected during liver regeneration [403]. In mice with acute liver failure induced by GalN, a single low dose of a hyper-IL-6-encoding adenoviral vector maintained liver function, prevented the progression of liver necrosis, induced liver regeneration, and dramatically enhanced survival [404]. In mice IL-1Ra was up-regulated in the liver after systemic LPS and local turpentine injections. After LPS stimulation, the hepatic production of sIL-1Ra correlated with the increase in plasma IL-1Ra levels. The total amount of LPS-induced soluble IL-1Ra present in the liver was six fold and tenfold higher than in the lung and spleen. In IL-6(-/-) mice exogenous IL-6 mediated the turpentine-induced production of IL-1Ra mRNA by the liver [405].

            In bone marrow donors treated with G-CSF, IL-6 induced bone metabolism and an acute-phase reaction along with mobilization of CD34+ cells in the peripheral blood [406]. IL-6 treatment of rabbits caused an accelerated release of polymorphonuclear cells from the bone marrow [407].

            Serum MIF levels were significantly elevated on day 1 in patients with septic shock, as opposed to trauma patients and controls. MIF paralleled cortisol, but contrasted with ACTH and was significantly higher in non-survivors than in survivors. Patients with septic adult respiratory distress syndrome (ARDS) showed higher MIF levels than those without ARDS. MIF and ARDS were independent predictors of adverse outcome. Significant correlations were established between MIF and cortisol and MIF and IL-6 and disease severity scores. No relation was found between MIF and acute phase proteins (APP, e.g., procalcitonin, CRP, and LBP). In multitrauma patients MIF levels were not elevated. During immune-mediated inflammation (such as septic shock) MIF is a contraregulator of the immunosuppressive effects of glucocorticoids [408].

            Anti-inflammatory cytokines, including the IL-1ra [374], the TNF synthesis inhibitor [375], IL-10 [371] and LIF [369], participate in the down regulation of the noxious effects of endotoxin. Interferon-γ antagonizes the development of endotoxin tolerance [372].

            Previte et al. [409] discovered that ionizing radiation detoxifies endotoxin. Bertok and coworkers [410] demonstrated 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 [411]. Radiodetoxified endotoxin has been tested clinically for the treatment of infectious disease [412]. Monophosphoryl lipid A preparations of low toxicity were also studied in animals and in man for boosting host resistance to infections and traumatic events [413,414].

7.3.      The acute phase response (APR)

The APR is a neuroimmune defence reaction to injury caused by physical, chemical and biological agents. It is characterized by fever, loss of appetite, inactivity and sleepiness. Cytokines, primarily IL-1, -6 and TNFα, which act on the CNS, the endocrine system and virtually on all other tissues and organs initiate the neuroendocrine and metabolic changes characteristic of APR. Later several other cytokines have been found to be involved in APP [311-314]. ACTH and GC, LEP, IN, EP, NEP, GLN, AVP, and ALD are elevated during the APR, whereas GH, estrogens, androgens and thyroid hormones may be either elevated or suppressed, depending on the severity of the condition (Table 2) [345-3548,419,420].

            C reactive protein (CRP) is an important acute phase protein. It binds to C-type pneumococcal cell walls in the presence of Ca²⁺. This protein is present in mammals, birds, fish and crabs. In the serum, 5 identical subunits (23 kDa ) of CRP form a ring-shaped molecule called pentraxin [112]. Pentameric CRP recognizes multiple homotopes, such as phosphocholine, polysaccharides containing galactose. Monomeric CRP binds some biologic polycations, such as protamine, poly-L-lysin and myelin basic protein. These 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 tumouricidal 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 of infectious and inflammatory disease [422-424]. A third conformation is referred to as neoCRP, which functions as galactose-specific receptors on NK cells and macrophages. It also accumulates at sites of injury. Monocyte/macrophages express specific CRP receptors and proteolytic fragments of CRP activate macrophages and neutrophils [421]. Human CRP protected mice from an otherwise lethal S. pneumoniae infection [424]. In patients with APR, CRP concentrations correlated with an increased cortisol/cortisone ratio, which was the result of a shift towards the active cortisol [425].

            LBP opsonizes LPS bearing particles, and thus may be required for the activation of complement by endotoxin through the alternate pathway. LBP-LPS complexes are potent stimulators of cytokines from monocytes and macrophages after combining with CD14 on the surface of these cells [353].

            Other APP are proteinase inhibitors, such as α-macroglobulin, ⓫-acid glycoprotein, antithrombin III, α-1-acute phase globulin, and ⓫-proteinase inhibitor, which are present in the rat. Kupffer cells stimulated ⓬-macroglobulin synthesis by hepatocytes in vitro in the presence of 10^-9 M DEX [426]. Fibrinogen is an important APP. Alpha-macrofetoprotein (αMFP) is a strong inhibitor of inflammatory mediators, such as histamine, bradykinin, serotonin, PGE2 and also polymorphonuclear cell chemotaxis. Catecholamines and GC induce αMFP in normal rats. Some other APP, such as haptoglobin and ⓫-major acute phase protein, were affected differently by these hormones [427]. Haptoglobin is an APP that 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 [428]. ⓫-acid glycoprotein and "1-antitrypsin exert antiapoptotic and anti-inflammatory effects and contribute to the delayed type protection associated with ischemic preconditioning in the kidney and in other insults [429].

In 10 laboratory mouse strains mannan-binding lectins (MBL)-A and MBL-C varied between 4 μg/ml to 12 μg/ml, and 16 μg/ml to 118 μg/ml, respectively. After ip injection of casein or LPS, MBL-A increased approximately 2-fold, with a maximum at 32 h, while MBL-C did not change. Serum amyloid A peaked at 15 h with an approximately 100-fold increase [430]. MBL is characterized by both collagenous and lectin domains. It binds to repeating sugar arrays on microbes. Following binding, MBL activates 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 SLE and rheumatoid arthritis [431].

            IL-6 is a major inducer of APP synthesis. Additional cytokines, namely IFN(, LIF, TGF$ and OSM, were found to be inducers of APP from the liver. IL-6 activates the genes of APP through the DNA binding protein called NF-IL-6, which is a pleiotropic mediator of many inducible genes involved in the acute-phase-, immune- and inflammatory responses, similarly to NFκB. Both NF-IL-6 and NFκB binding sites are present in the inducible genes, such as IL-6, IL-8 and several acute phase genes [418].             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, ⓬-macroglobulin and cysteine protease inhibitor are strongly depressed. Isoprenalin, a ⓶-adrenergic receptor agonist, also causes very high levels of IL-6 [431].

            APR causes a rapid thymus involution. Thymus-derived (T) lymphocytes govern adaptive immune reactions through their regulatory function. The thymus is an endocrine organ and is subject to complex neuroendocrine control mechanisms. During APR, HPA axis activation and the suppression of the GH/PRL-IGF-1 axis result in the suppression of the T-cell-dependent adaptive immune response. Catecholamines and glucocorticoids, which are released in large quantities during APR, induce apoptosis in the thymus with a striking efficiency. The elevated levels of TNF and zinc deficiency, which develop during APR, also contribute to thymic involution and to the suppression of the adaptive immune system [348].

               In patients with APR the GH-IGF-I axis is suppressed and GH action is attenuated. TNF, IL-1 and IL-6 inhibit GH-signalling pathways, which results in the reduced expression of GH-responsive genes [126]. These observations prompted the treatment of acutely ill patients with GH with the aim of preventing the severe catabolic state and improving immunocompetence. However, so far the results are not encouraging. A controlled clinical trial revealed that deaths attributed to “septic shock or uncontrolled infection” occurred nearly four times more commonly in GH treated patients compared to placebo receiving patients [433]. These findings suggest strongly that the suppression of the GH/PRL – IGF-I axis in APR is required for the development of intense catabolism, which must be fundamental to the rapid release of large amounts of nutrients and of energy to support maximally the acute phase immune host defence system. During APR the CNS, the HPA axis, the sympathetic nervous system, the bone marrow, CD5+ B lymphocytes, leukocytes and the liver are metabolically activated and functionally altered [345,347,433]. The adaptive immune system is controlled by T lymphocytes and it needs 7-10 days to develop an effective host defence. During APR no time is available for an adaptive immune reaction, and therefore, this system is shut down, primarily by the cytokine and neuroendocrine alterations that take place. Thymus and T cell function is heavily dependent on the GH/PRL–IGF-I axis and it is suppressed profoundly by the elevated levels of glucocorticoids and catecholamines [345,347,348]. GH inhibited the production of acute phase proteins in rats with burn injury and in human hepatocytes [348,434,435]. On the basis of this information one may argue that the rapid breakdown of bodily tissues is the only way to fuel the acute systemic effort for host survival during APR. Clearly, GH is a powerful anabolic hormone and acts as an antagonist of the HPA axis that promotes APR [332,346,348,433,434,438]. This hypothesis is further supported by the results of GH treatment in burn injury. The catabolic effect and wasting has been reduced [439], serum IGF-I, IGFBP-3 and free fatty acids, constitutive hepatic proteins and P-selectin were increased, whereas TNFα IL-1β CRP and amyloid-A were decreased [438-442].         Chronic APR may lead to cachexia [443] or anorexia of infection [444]. The presence of APP predict future risk for coronary disease in healthy subjects [445]. A minimal APR is present during aging [446, 447], and may underlie the metabolic syndrome that leads to type 2 diabetes [448-453]. Polymorphism of the IL-6 gene influences the relationship among insulin sensitivity, post load glucose levels and peripheral white blood cell count [453]. APR plays a role in the pathogenesis of rheumatoid arthritis [454-458] in IgA nephropathy [457] and in numerous other inflammatory diseases. Various insults, including irritation, trauma or toxic agents, are also capable of immune activation by nonspecific pathways which can lead to APR [448].                Adaptive or natural immune activation will lead to shared host defence reactions that include inflammation, phagocytosis, cytotoxicity and neutralization of viruses, toxins etc. During systemic reactions the immune-effector function is supported by neuroendocrine and metabolic responses. Once the pathogen has been eliminated, the immune system participates in the healing process [9,347-349,448-459].

7.4.      Concluding remarks.<

The current consensus is that endotoxin is harmful. However, LPS itself is devoid of toxicity in some lower animal species. The toxicity of LPS is due to the stimulation of immune-derived cytokines, which kills the host. Many other microbial pathogens induce such "polyclonal lymphocyte activation" [461]. Some are known as "superantigens" [362-364]. Many of these substances are pyrogenic [464] and induce APR.

            In a natural setting immune mechanisms are activated locally at the site of pathogen penetration. This provides instantaneous defence at the site of invasion. The target is promptly identified by one of the innate mechanisms (e.g., natural antibodies, serum proteins, leukocytes) that recognize the homotopes of the pathogen. This is followed by the activation of effector mechanisms that may involve complement, blood clotting and various subsets of the white blood cell series. Blood coagulation at the focus of infection is a defence reaction, whereas disseminated intravascular coagulation may lead to disaster [345,348].

            When the immune system fails to control the infection/insult locally, APR will develop. APR is an emergency reaction, which represents a switch of the immune system from the specific, adaptive mode of reactivity, which is under the control of thymus derived T lymphocytes to a less specific, but very rapid and intense natural immune reaction. Natural antibodies and APP play a major role in the identification of the target and the activation and regulation of immune effector mechanisms during APR. Febrile illness represents the mobilization of all resources of the host in the interest of defeating/eliminating the pathogen and achieving survival/recovery. By and large, APR is very successful, as in the overwhelming majority of cases febrile illness will lead to healing and recovery. On this basis, one may suggest that APR is truly beneficial and only in rare and extreme cases will it result in severe disease, shock and death [345,348]. LPS has affects on the immune system, CNS, endocrine organs and on many other tissues and organs in the body [363-366]. All tissues contain "resident macrophages" or related cells such as the glia cells in the CNS, the Kupffer cells in the liver, Langerhans cells in the skin, etc., which have the capacity to react to LPS with cytokine production [469, 470]. Consequently, systemically applied LPS has the capacity to activate the immune and the neuro-endocrine systems, and also the liver via locally induced cytokines. This is supplemented by the effect of blood borne cytokines and also by neural communication. For instance, the vagus nerve plays a role in the activation of the ACTH-adrenal axis and the initiation of a behavioural response after the intraperitoneal injection of LPS [471,472].

            Endotoxin is always present in the gastrointestinal tract, even in germ free animals [23]. However, bile acids normally destroy LPS and thus prevent its absorption from the gut of rats even when mucosal damage is inflicted [473, 474]. The liver is an important clearance organ of LPS via bile secretion [475]. Therefore, bile provides a physico-chemical defence barrier against LPS toxicity in vertebrate animals [476].             LPS has an enormous potential to boost host resistance by its ability to stimulate immunity and APR. The absorption of LPS from the intestine during acute illness may represent an important pathophysiological mechanism that evolved in higher animals for the rapid conversion of the immune system from the adaptive mode of reactivity to the amplification of natural immune mechanisms. This immunoconversion may be achieved simply by the control of bile secretion. Indeed, there is good evidence to illustrate that intestinal endotoxin is readily absorbed after X-irradiation, trauma or even after stressful situations [348]. Liver regeneration was stimulated by intestinal endotoxin as was granulopoiesis and healing in the central nervous system [26]. However, intestinal endotoxin was blamed for death in trauma patients [345,348].

            The adaptive immune system fails gradually in many people due to aging, during disease or as the result of various insults to the body. Stressful insults initially mobilize the adaptive immune system to enhance immune reactivity in peripheral tissues. If the pathogenic insult continues immunoconversion occurs from adaptive to natural immune host defence [342,345-349,352].

Neuroimmune Biology Volume 5

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