Doctoral Thesis / Dissertation, 2014
Introduction and Aim of Work
Review of Literature
Materials and Methods
P. aeruginosa is a gram-negative bacterium that causes a variety of diseases in compromised hosts.Bacterial endotoxins such as LPS are the major outer surface membrane components present in almost all gram-negative bacteria and act as extremely strong stimulators of innate immunity and inflammation of the airway. The present study was undertaken to determine the effect of combined administration of Gentamicin (GENT) as antibiotic and Dexamethasone (DEXA) as ant-inflammatory drug on some physiological, immunological and histological parameters. After determination of LD50 of P. aeruginosa, mice groups were injected with DEXA, GENT and LPS alone or in combination. LPS single injection caused a significant increase of total protein, globulin, total leukocyte count, lymphocytes, neutrophils and level of IgM and IgG. DEXA induced an increase of serum total lipid, ALT and AST levels, neutrophilia and lymphopenia. GENT administration increased serum total protein, globulin, total lipid, ALT and AST levels. Physiological and immunological examination demonstrated that combined treatment has a significant effect as decreaseing serum total protein, globulin, lymphocytes and IgG level than single treatment. Histological examination demonstrated that the inflammation of thymus, spleen, lymph node and liver decreases in mice received combined treatment than those received individual treatment. Concurrent administration of DEXA and GENT has greatest effect in protecting organs against damage in case of endotoximia.
Keywords: P. aeruginosa, LPS, Dexamethasone, Gentamicin, immunoglobulin, total protein, albumin, globulin, total lipid, AST, ALT , total leukocyte count.
Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen that causes a wide range of acute and chronic infections (Upritchard et al., 2008). Endotoxin or lipopolysaccharide (LPS) is a component of the outer membrane of gram-negative bacteria and it has been implicated as an important inducer of the local and systemic responses to such a bacterial infection (Kohn and Kung, 1995). It releases in excess during antibiotic therapy, activates the immunological and inflammatory reaction (Ngeleka et al., 1990). However, in conditions where the body is exposed to bacterial endotoxin excessively (during severe infection and sepsis with gram-negative bacteria) or systemically (when endotoxin enters the blood stream "endotoxemia"), a systemic inflammatory reaction can occur, leading to tissue injury, metabolic and neuroendocrine changes, multiple organ damage and/or dysfunction, circulatory shock, and potentially death (Amersfoort et al., 2003). The host response to LPS is crucial in the defence against gram-negative bacterial infection.Cells of the myeloid lineage are capable of recognizing picomolar (equal 10−9 mol/m3) quantities of LPS and respond, via several signal transduction cascades, with the release of a wide range of pro-inflammatory cytokines (Rietschel et al., 1994).
Glucocorticoids (DEXA) are important modulators of immune reactions. They are capable of antagonizing several effects of the bacterial endotoxin by inhibiting endotoxin-induced leukocyte activation, and the production of cytokines as inflammatory mediators (Szakács et al., 2000). Dexamethasone (DEXA) is a synthetic glucocorticoid used in both humans and animals (Aengwanich, 2007).
Gentamicin (GENT) is an aminoglycoside antibiotic, which has wide utility in many bacterial infections. It has a broad spectrum of activity against some common pathogens, both gram-positive and gram-negative. It has a strong activity against P. aeruginosa (Beale, 2003).
This study aims at evaluating the effect of administration of an antibiotic (GENT) and an anti-inflammatory drug (DEXA), as singly or concurrently on mice affected by bacterial infection (LPS) through assessment of various physiological, immunological and histological parameters.
The bacteria are a large group of unicellular microorganisms. Typically a few micrometers in length, bacteria have a wide range of shapes, spheres, cocci, rods, filament and spirals. Bacteria are ubiquitous in every habitat on Earth, growing in soil, acidic hot springs, radioactive wastes (Fredrickson et al., 2004; Young, 2006) water, and deep in the Earth's crust, as well as in organic matter and the live bodies of plants and animals. There are typically 40 million bacterial cells in a gram of soil and a million bacterial cells in a milliliter of fresh water; in all, there are approximately five nonillion (5×1030) bacteria on Earth (Whitman et al., 1998). There are approximately ten times as many bacterial cells in the human flora of bacteria as there are human cells in the body, with large numbers of bacteria on the skin and as gut flora (Sears, 2005). The intestinal flora of an adult alters with lifestyle, diet and age (Hopkins et al., 2001). Bacterial density increases in the distal small intestine, and in the large intestine rises to an estimated 1011–1012 bacteria per gram of colonic content, which contributes to 60% of faecal mass (Eckburg et al., 2005).
Bacterial cells are about one tenth the size of eukaryotic cells and are typically 0.5–5.0 µm in length. However, a few species–for example Thiomargarita namibiensis are up to half a millimetre long and are visible to the unaided eye (Schulz and Jorgensen, 2001). Very small forms of bacteria have been reported from marine and freshwater systems, soils and kidney stones. Ultramicrobacteria are very small coccoid forms less than 0.3 µm in diameter. Nanobacteria are extremely small cellular forms with diameters range 0.02 µm – 0.128 µm. They are closely associated with the formation of inorganic precipitates and geological strata (Velimirov, 2001).
More recently, bacteria were discovered deep under the Earth's crust that grows as long rods with a star-shaped cross-section. The large surface area to volume ratio of this morphology may give these bacteria an advantage in nutrient-poor environments (Wanger et al., 2008). This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton, and is important because it can influence the ability of bacteria to acquire nutrients, attach to surfaces, swim through liquids and escape predators (Young, 2006).
The basic function of the bacterial cell wall maintaining cell shape and rigidity (Weidel and Pelzer, 1964). It also protects against osmotic lysis. This strength and rigidity conferred by the cell wall results from a layer of peptidoglycan, which is a covalent macro molecular structure of stiff glycan chains that are crosslinked by flexible peptide bridges (Labischinski et al., 1979). Gram-negative species can form peptide interbridges between peptides that are attached to adjacent glycan strands so strong mesh is created that protects the cell from osmotic lysis (Cabeen and Jacobs, 2005). Gram-negative cell walls include an outer membrane that surrounds a thin 1–7nm, peptidoglycan layer, with a periplasmic space between the inner and outer membranes. The outer membrane and peptidoglycan are linked to each other with lipoproteins ( Braun and Rehn, 1969; Braun, 1975). Gram-positive cell walls are composed of a thick (20–80 nm), multilayered peptidoglycan sheath that includes embedded teichoic and lipoteichoic acids (Bhavsar et al., 2004).
Eukaryotic cells contain three major cytoskeletal systems microfilaments, microtubules and intermediate filaments, which are assembled from actin, tubulin and intermediate filament proteins, respectively, which help maintain cell shape, integrity, motility, chromosome segregation, signal transduction and cytokinesis (Cabeen and Jacobs, 2005).
These differences in structure can produce differences in antibiotic susceptibility; for instance, vancomycin can kill only gram-positive bacteria and is ineffective against gram-negative pathogens (Walsh and Amyes, 2004).
Many bacterial species exist simply as single cells, others associate in characteristic patterns: Neisseria form diploids (pairs), Streptococcus form chains, and Staphylococcus group together in "bunch of grapes" clusters. Bacteria can also be elongated to form filaments, for example the Actinobacteria (Douwes et al., 2003). Bacteria are now classified as prokaryotes. Unlike cells of animals and other eukaryotes, bacterial cells do not contain a nucleus and rarely harbour membrane-bound organelles (Woese et al., 1990).
The bacterial cell is surrounded by a lipid membrane, or cell membrane, which encloses the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential components of the cytoplasm within the cell. As they are prokaryotes, they do not tend to have membrane-bound organelles in their cytoplasm (Berg et al., 2002). There are compartments within bacteria that are surrounded by polyhedral protein shells rather than lipid membranes which provide a further level of organization. The building blocks of bacterial microcompartments (carboxysome) are exclusively proteins and glycoproteins. It contains 5–10 different polypeptides that form a polyhedral shell 100–200 nm across (Bobik, 2007).
Bacteria often attach to surfaces and form dense aggregations called biofilms or bacterial mats. Biofilm is an assemblage of microbial cells that is irreversibly associated with a surface and enclosed in a matrix of primarily polysaccharide material (Donlan, 2002). These films can range from a few micrometers in thickness to up to half a meter in depth, and may contain multiple species of bacteria, protists and archaea. Bacteria living in biofilms display a complex arrangement of cells and extracellular components, forming secondary structures such as microcolonies, through which there are networks of channels to enable better diffusion of nutrients (Branda et al., 2005). Biofilms are also important in medicine, as these structures are often present during chronic bacterial infections or in infections of implanted medical devices, and bacteria protected within biofilms are much harder to kill than individual isolated bacteria (Donlan, 2002). Biofilms also provide an ideal niche for the exchange of extrachromosomal DNA (plasmids) (Ehlers and Bouwer, 1999).
Certain genera of gram-positive bacteria, such as Bacillus can form highly resistant, dormant structures called endospores. Spores have since been recognized as the hardiest known form of life on Earth. Examples of sporeforming bacteria such as aerobic heterotrophs Bacillus in gram-positive bacteria and Sporohalobacter spp. in gram-negative bacteria (Nicholson et al., 2000). Endospores show no detectable metabolism and can survive extreme physical and chemical stresses, such as high levels of wet, dry, UV light, gamma radiation, oxidizing agents, detergents, disinfectants, heat, pressure and desiccation. Spore resistance properties important protection of vital spore macromolecules during dormancy and repair of damaged macromolecules during germination (Nicholson et al., 2002).
Immune response against bacteria
Invasion of the bloodstream by both gram-positive and gram-negative bacteria cause the sepsis syndrome in humans.This syndrome results from the induction of cytokines and other inflammatory mediators and is characterized by alterations in temperature, pulse, hemodynamic instability, and end organ damage (Yoshimura et al., 1999).
Numerous bacteria have made the transition from being intracellular parasites of freshwater amoeba or other protozoa and have become intracellular parasites of phagocytic cells of the immune system (Greub and Raoult, 2004). The intestinal microflora is a positive health asset that crucially influences the normal structural and functional development of the mucosal immune system. Mucosal immune responses to resident intestinal microflora require precise control and an immunosensory capacity for distinguishing commensal from pathogenic bacteria (O’Hara and Shanahan, 2006). Innate immune responses to the commensal flora educate the immune system and influence adaptive responses to exogenous antigens (Rook and Brunet, 2005).
The epithelium provides the first sensory line of defence and active sampling of resident bacteria, pathogens and other antigens is mediated by three main types of immunosensory cell. First, surface enterocytes serve as afferent sensors of danger within the luminal microenvironment by secreting chemokines and cytokines those alert and direct innate and adaptive immune responses to the infected site (Shanahan, 2005). Second, M (microfold) cells (specialized intestinal epithelial cells) that overlie lymphoid follicles sample the environment and transport luminal antigens to subadjacent dendritic cells and other antigen-presenting cells. Third, intestinal dendritic cells themselves have a pivotal immunosensory role and can directly sample gut contents by either entering or extending dendrites between surface enterocytes without disrupting tight junctions (Rescigno et al., 2001).
Bacterial infections with gram-positive and gram-negative bacteria typically results in activation of the innate immune system. Although bacteria differ in the composition of their cell wall the host reaction to invasion is remarkably similar regardless of the species or type of bacterium (Yoshimura et al., 1999). Several bacterial components (endotoxin, teichoic and lipoteichoic acids, peptidoglycan, DNA, and others) can induce or enhance inflammation and may be directly toxic for host cells (Nau and Eiffert, 2002). A gram-negative bacterial infection localized at a particular infection localized at a particular tissue site typically elicits an orchestrated series of host defense responses manifested as an acute inflammatory reaction (Kohn and Kung, 1995).
Infections with encapsulated pyogenic bacteria could indicate a defect in the B-cell system (immunoglobulin deficiency), the phagocyte system or the complement system. The major types of infections are pneumonia caused by pneumococci or staphylococci and urinary tract infections caused by gram-negative organisms (E. coli, Pseudomonas and Proteusand Kelbsiella) (Heise, 1982). The major immunologic defect observed in myeloma patients is in the humoral system (Broder et al., 1975). Pyogenic bacteria, especially Staphylococci cause infections with yelonephritis, pneumonia and septicemia (Heise, 1982).
Diagnosis of bacteria
The thick layers of peptidoglycan in the "gram-positive" cell wall stain purple, while the thin "gram-negative" cell wall appears pink. By combining morphology and gram-staining, most bacteria can be classified as belonging to one of four groups (gram-positive cocci, gram-positive bacilli, gram-negative cocci and gram-negative bacilli) (Woods and Walker, 1996).
Culture techniques are designed to promote the growth and identify particular bacteria for example; a sputum sample will be treated to identify organisms that cause pneumonia, while stool specimens are cultured on selective media to identify organisms that cause diarrhea. Also, there are antigen detection and polymerase chain reaction (PCR) tests for bacterial identification (Weinstein, 1994; Thomson and Bertram, 2001).
Bacteria, often lactic acid bacteria such as Lactobacillus and Lactococcus, in combination with yeasts and molds, have been used for thousands of years in the preparation of fermented foods such as cheese, pickles, soy sauce, sauerkraut, vinegar, wine and yoghurt. The use of adjunct microorganisms, specifically yeasts and lactobacilli strains, has already gained acceptance as the preferred means to introduce specific flavor attributes in a variety of cheeses (Cohen, 2002; Johnson and Lucey, 2006). In the chemical industry, bacteria are most important in the production of enantiomerically pure chemicals for use as pharmaceuticals or agrichemicals (Liese and Filho, 1999). Bacteria can also be used in the place of pesticides in the biological pest control. This commonly involves Bacillus thuringiensis (also called BT), a gram-positive, soil dwelling bacterium. Subspecies of these bacteria are used as a Lepidopteran-specific insecticides under trade names such as Dipel and Thuricide (Aronson and Shai, 2001). Because of their specificity, these pesticides are regarded as environmentally friendly, with little or no effect on humans, wildlife, pollinators and most other beneficial insects (Chattopadhyay et al., 2004; Bozsik, 2006).
Class: Gamma Proteobacteria
Species: Pseudomonas aeruginosa
P. aeruginosa is a gram-negative bacterium with a ubiquitous within the biosphere; it is capable of establishing opportumistic injections (Cripps et al., 2006). It is obligating aerobic rod-shaped bacterium with minimal nutritional requirements (Iversen et al., 2007). It has one or more polar flagella to provide motility and it is non-spore forming (Warghane et al., 2011).
All species and strains of Pseudomonas are gram-negative rods, and have historically been classified as strict aerobes. Exceptions to this classification have recently been discovered in Pseudomonas biofilms ( Hassett et al., 2002). A significant number can produce exopoly-saccharides that are known as slime layers. Secretion of exopoly-saccharide makes it difficult for Pseudomona to be phagocytosed by mammalian white blood cells (Ryan and Ray, 2004).
Since the mid 1980s, certain members of the Pseudomonas genus have been applied to cereal seeds or applied directly to soils as a way of preventing the growth or establishment of crop pathogens. This practice is generically referred to as biocontrol. Theories include: that the bacteria might induce systemic resistance in the host plant, so it can better resist attack by a true pathogen ( Haas and Defago , 2005).
Some members of the genus Pseudomonas are able to metabolise chemical pollutants in the environment, and as a result can be used for bioremediation such as P. alcaligenes, which can degrade polycyclic aromatic hydrocarbons ( O'Mahony et al., 2006), P. mendocina, which is able to degrade toluene ( Yen et al., 1991) and P. pseudoalcaligenes is able to use cyanide as a nitrogen source (Huertas et al., 2006).
As a result of their metabolic diversity, ability to grow at low temperatures and ubiquitous nature, many Pseudomonas can cause food spoilage as Pseudoalcaligene lundensis, which causes spoilage of milk, cheese, meat and fish (Gennari and Dragotto , 1992).
P. aeruginosa flourishes in hospital environments, and is a particular problem in this environment since it is the second most common infection in hospitalized patients. This pathogenesis may in part be due to the proteins secreted by P. aeruginosa (Hardie et al., 2009).
Endotoxin or LPS is a component of the outer membrane of gram-negative bacteria, has been implicated as an important inducer of the local and systemic responses to such a bacterial infection (Kohn and Kung, 1995). It released in excess during antibiotic therapy, activates the immunological and inflammatory process (Ngeleka et al., 1990). The toxicity is now known to be a consequence of the host inflammatory response, which appears to be optimally adapted for the clearance of most local infections. The inflammatory response can lead to septic shock and death that occur between the presentation of endotoxin to the myeloid cells of the immune system and the production of inflammatory cytokines have utilized LPS from gram-negative bacteria (Bishop, 2005). The intense activation of the immune system that companies LPS administration affects a variety of cells, such as neutrophils and lymphocytes (Bauer et al., 2000).
Chemically endotoxins are LPS consisting of an O-specific chain, a core oligosaccharide and a lipid component termed Lipid A which determines the endotoxic activities and together with the core constituent have essential function for bacteria. Lipid A has been proved to represent the toxic and immunomodulatory principle of endotoxins (Rietschel et al., 1994). The O-specific chain constitutes a polymer of oligosaccharides that so-called repeating units consisting of one to eight glycosyl residues. Its structure differs from strain to strain within a serotype, and thus exhibits an enormous structural variability, determines the serological specificity of the LPS and of bacteria containing it, and therefore functions as an important surface antigen (Lüdertiz et al., 1982).
illustration not visible in this excerpt
Figure 1: The structure of lipopolysaccharide (Rietschel et al., 1992)
Today, LPS is known as a major factor responsible for toxic manifestations of severe gram-negative infections and generalized inflammation (Rietschel et al., 1992). Therefore, several current programs of research in the field of infectious diseases aim at the neutralization of endotoxin or its elimination from the circulation. On the other hand, LPS represents a highly active immunomodulator that is capable of inducing nonspecific resistance to viral and bacterial infections (Alving, 1993).
Physiological effect of endotoxin
Endotoxin LPS is a component of the bacterial cell wall, is known to have various biological and immunological activities and induces acute kidney and liver dysfunctions (Hewett and Roth, 1993). It has also been reported that LPS causes cholestatic jaundice in patients with gram-negative bacterial infections concomitant with elevations in plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels between 3 and 6 hour (hr) after LPS administration (Nadai et al., 1998). The synthesis rate of proteins increased by endotoxin treatment including acute increase in fibrinogen concentrations and albumin synthesis rates increased with sublethal doses (Koj and Mcfarlane, 1968).
The great majority of investigators found that normally high density lipoproteins (HDL) are the main LPS-binding lipoproteins in blood sera of experimental animals (Freudenberg and Galanos, 1992). LPS–HDL complexes were found in blood sera of various animals including humans (Freudenberg et al., 1980; Freudenberg and Galanos, 1988). The binding of LPS to HDL was shown to retard LPS clearance, inhibit LPS binding to cells and prevent development of LPS-induced lethal effects (Freudenberg and Galanos, 1992). Kinetic studies have shown that LPS/LBP (LPS binding protein) complexes bind to Cluster of differentiation 14 (CD14) before LPS is transferred to HDL (Yu and Wright, 1996). LPS binding domain of LBP is located in the N-terminal part of the molecule including basic lysine and arginine residues (Inagawa et al., 2002). Under hypercholesterolemia conditions, the atherogenic LPs, such as low density lipoproteins (LDL) can bind excessive amounts of endotoxins entering the blood (Schwartz and Dushkin, 2002). Hypercholesterolemia is known to be the main risk factor of atherosclerosis, and the increase in LDLs in blood leads to their elevated uptake and accumulation in arterial wall, to the stimulation of pro-inflammatory cytokine production in vessels, and a formation of atherosclerotic plaques and atheromas (Libby et al., 2002).
It was found that dexamethasone (DEXA) increased the concentration of plasma free fatty acid, total triglyceride, and very low density lipoprotein (VLDL) protein, triglyceride, phospholipid, and free cholesterol. No changes were observed in the concentration or composition of plasma LDL. The concentration of plasma HDL, lipid, and plasma apolipoprotein (apoA-1) tended to increase the ratio of total HDL cholesterol to LDL cholesterol was elevated with DEXA treatment (Cole et al., 1982).
In vitro and partially in vivo studies have demonstrated that protein synthesis is inhibited with gentamicin (GENT). Phospholipid degradation was inhibited at early time points after GENT treatment. Increase in phospholipids content observed as a result of GENT treatment was predominantly the result of the inhibition of degradation (Sundin et al., 2001).
Immunological response against P. aeruginosa
It is important to recognize the immunological responses to P. aeruginosa during the progress of disease to identify the early stages of chronic infection and determine the appropriate time to begin antibiotic therapy (Shand et al., 1988). Immunoglobulin M and G (IgM and IgG) isotypes presumably sharing a single idiotype corresponding to a common epitope on the O side chain of LPS from P. aeruginosa serogroup O6, the Pseudomonas serogroup most commonly implicated in human infections (Pollack et al., 1995). IgG and its subclasses, which comprise 80% of the antibodies in serum and are important in the secondary response in cystic fibrosis patients, such as opsonization of bacterial antigens, are of particular significance. IgGs are produced in very high titer against specific P. aeruginosa antigens during a chronic infection (Upritchard et al., 2008). Innate immune responses of human tracheal epithelium to P. aeruginosa flagellin, tumor necrosis factor alpha (TNF-α), and interlukin 1-B (IL-1β). Flagellin, TNF-α, and IL-1β activated NF-κB in columnar cells. Flagellin is necessary and sufficient to trigger inflammatory responses in columnar cells during accumulation of P. aeruginosa in the airway surface liquid (ASL); columnar cells express toll-like receptor 5 and Myeloid differentiation primary response gene 88 (MyD88), often associated with flagellin activated cell signaling. IL-1β and TNF-α in the ASL also activate columnar cells (Tseng et al., 2006). Quorum sensing (QS), cell-to-cell signaling, may influence the success of an initial P. aeruginosa infection. QS involves the production of compounds that allow a bacterial population to “sense” its own status, as well as that of other bacteria in the environment (Klemm and Schembri, 2000). QS genes in P. aeruginosa could be divided into genes that are expressed early or late during chronic P. aeruginosa infection (Whiteley et al., 1999). P. aeruginosa outer membrane proteins enhance QS virulence expression by binding to gamma interferon (IFN-γ) (Wu et al., 2005). P. aeruginosa produces a number of virulence factors, which are cell surface components or secreted toxins. An adenosine diphosphate (ADP)-ribosylation exotoxin (exotoxin A-adenosine diphosphate-ribosylation), exotoxin A (ETA), is the most toxic protein secreted by P. aeruginosa (Yates et al., 2005). The type III system of P. aeruginosa consists of three coordinately functional protein complexes: the secretion apparatus; the translocation or targeting apparatus; the secreted toxins and cognate chaperones (Hueck, 1998). P. aeruginosa secretes four cytotoxins via the type III secretion system exoenzyme S, T, U and Y: (ExoS, ExoT, ExoU, and ExoY) (Yahr et al., 1997). These cytotoxins have been implicated in increased cellular and animal toxic effects in experimental models of P. aeruginosa infection as children with cystic fibrosis (CF) have serum antibodies to the type III apparatus at an early stage of P. aeruginosa infection. This early antibody response implies expression of type III cytotoxins during the initial and acute phases of P. aeruginosa infection in the lungs of children with CF (Corech et al., 2005).
Immunological response against bacterial endotoxin
While P. aeruginosa has been appreciated as a significant bacterial pathogen since the 19th century (Doggett, 1979), it initiates an overwhelming host inflammatory response that also contributes significantly to morbidity and mortality of infected patients. The P. aeruginosa LPS is a typical gram-negative bacterial LPS, with a basic lipid A structure containing an N - and O -acylated diglucosamine bisphosphate backbone (Pier, 2007). When gram-negative bacteria multiply or die, LPS is released either as a free form, or as a complex of LPS with bacterial surface proteins (Hellman et al., 2000). If LPS is one of the most powerful microbial products in terms of the pro-inflammatory property, most, if not all, components of the gram-negative bacteria can activate host cells (Heumann and Roger, 2002). The contribution of LPS to pathogenesis and immunity varies depending on the underlying patient basis for increased susceptibility to infection. The isoform of the LPS, particularly the lipid A component, and structural variation in the O-antigen side chain that impacts host immunity (Pier, 2007). Inflammation plays a profound role in health and disease as it is an essential response of the host upon infection or tissue invasion. This response is rapidly initiated by innate immune cells and usually does not require participation of adaptive immune system (Heumann and Roger, 2002). The innate immune molecules can be generally classified into inflammatory mediators, acute phase proteins, complement system components, natural antibodies, Toll-mediated signaling molecules, and receptors of natural killer cells (Ng et al., 2004).
Once bacterial components have been shed into the host, they activate host cells, such as monocytes, macrophages, neutrophils and endothelial cells, to generate an inflammatory response (Heumann and Roger, 2002). One of the initial endotoxin-induced signals is the activation of the transcription factor nuclear factor-κB (NF-κB), which in turn activates several other genes that are major players in the inflammatory process, such as nerve growth factor (NGF), cytokines and extracellular matrix (ECM)-degrading proteases (Safieh-Garabedian et al., 2004). LPS exerts many of its biologic effects by stimulating host cells to produce bioactive inflammatory mediators. Monocytes and macrophages respond to LPS with the synthesis and release of proinflammatory cytokines, such as TNF-α, interleukins 1, 6 and 8 (IL-1, IL-6, and IL-8) (Nathan, 1987; Tannenbaum et al., 1988). At high concentrations, LPS may induce significant morbidity due to gram-negative infections by its capacity to induce shock, fever, and severe inflammatory reactions during gram-negative sepsis, through the release of endogenous mediators from host cells (Mayeux, 1997). The presence of LPS stimulates the synthesis of prostaglandins by macrophages, monocytes and endothelial cells (Andreasen et al., 2008).
PAF (platelet-activating factor) is produced by human neutrophils in response to endotoxic stimulation. It was initially described as a platelet agonist but is now recognized as an agonist for all elements of the inflammatory or innate immune system. PAF receptor ligands have a critical role in the response to endotoxin, because increasing sensitivity to PAF by overexpressing its receptor worsens the effects of endotoxin (Watanabe et al., 2003).
Mechanism of recognition of LPS by macrophages
LPS aggregates are dissociated by the LPS-binding protein (LBP) to form LPS/LBP complexes which are transferred to cell membrane CD14 marker (mCD14) present on monocytic cells, leading to cell activation, or transferred to soluble CD14 (sCD14) present in blood and fluids (Heumann and Roger, 2002). CD14 is a 55-kDa glycosyl phosphatidylinositol (GPI)–anchored glycoprotein identified on the surface of monocytes, macrophages, and polymorphonuclear leukocytes (PMNs) (Echchannaoui et al., 2005). During gram-negative bacterial infections, LPS is released and complexes with the serum-derived LBP, which acts as a lipid transfer protein that facilitates the binding of LPS monomers to the CD14 LPS receptor (Schumann et al., 1990; Wright et al., 1990). LPS released from gram-negative bacteria is present as aggregates due to the amphiphilic structure of the molecule. Spontaneous diffusion of LPS monomers from these aggregates to CD14 occurs at a very low rate (Hailman et al., 1994). LPS transformed into monomers through the action of plasmatic LBP which to both mCD14, sCD14 and phospholipids especially HDL (Gallay et al., 1994). mCD14 is a 53-kDa glycoprotein present on the surface of the myelomonocytic cells embedded in the plasma membrane via glycerophosphate inositol anchor (Heumann and Roger, 2002). LPS signal transduction and LPS clearance utilize both LBP and mCD14. LPS internalized within minutes into monocytic cells (Gallay et al., 1993). LPS-induced cell activation involves the formation of a ternary complex with LBP and mCD14 at the surface of monocytic cells (Gegner et al., 1995). sCD14 resulting either from the shedding of membrane-bound CD14 or form the production of GPI-free CD14 molecules and it is able to interact directly with LPS, a process that is catalyzed by LBP (Hailman et al., 1994). LPS-induced activation of CD14-negative cells, sCD14 can also antagonize LPS/LBP-induced activation of CD14-positive cells (Haziot et al., 1994). LPS, sCD14 also facilitates the transfere of LPS to HDL resulting in the neutralization of LPS (Heumann and Roger, 2002). Toll-like receptor 4 (TLR-4) is essential for the recognition of the lipid A portion of bacterial LPS and mediates both effective host resistance to infection as well as some of the pathology associated with LPS-induced shock (Fitzgerald and Chen, 2006; Schnare et al., 2006). TLRs, and in particular TLR-4, are likely candidates to transmit the LPS signal from the membrane-bound CD14 to the cytoplasm (Anderson, 2000). LPS bound to CD14 can interact with the extracellular or intraluminal domains of TLR4 in the presence of a co-factor, Myeloid differentiation protein-2 (MD2). Whether this occurs on the cell surface as thought by many, or within the lumen of endosomes following phagocytosis or endocytosis of whole bacteria or free LPS (Blander and Medzhitov, 2006). Binding of LPS to TLR4 brings adaptor molecules containing Toll/IL-1 receptor (TIR) domains to interact with the cytoplasmic portion of TLR4. These adaptors include Toll-Interleukin 1 receptor domain-containing adapter protein/ Myelin and lymphocyte protein (TIRAP/Mal) and TRIF-related adaptor molecule (TRAM), which function to sort out the signals to be transduced by TLR4-MD2-bound LPS (Fitzgerald and Chen, 2006). The next set of molecules essential for activating cellular responses to LPS are MyD88 and (Toll/interleukin-1 receptor) domain-containing adaptor protein inducing interferon beta (TRIF), which themselves interact with additional adaptors to lead to activation of transcription factors (Pier, 2007). Signaling by TLR4 occurs through sequential recruitment of the adapter molecules MyD88 and IL-1 receptor-associated kinase (IRAK) (Muzio et al., 1998). IRAK dissociates from the receptor and interact with TNF and receptor-associated factor 6 (TRAF6), which resulted in cell activation through nuclear NF-κB or c-jun N-terminal kinase/stress-activated protein kinase (JNK/SAP) (Heumann and Roger, 2002). TLR2 was in the apical membrane of the cells whereas TLR4 had a more basolateral distribution, and there was no cytokine response to LPS by these cells (Muir et al., 2004).
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