In humans, as mentioned earlier, defensins are released by phagocytes and epithelial cells, such as in the skin, respiratory, GI and GU tracts, which provide a first line of defence against invading pathogens (Harder et al., 2007). Human defensin concentrations are usually found to be at 40ng/ml in the blood but in the event of an infection, these concentrations can increase exponentially to 1000ng/ml in localised areas of infection (Yang and Oppenheim, 2003). Defensins perform multiple roles (figure 1.8) with the most important being the potent antimicrobial activity exhibited against various invading microorganisms, such as Gram-negative bacteria (E. coli), Gram-positive bacteria (S. aureus), mycobacteria (M. fortuitum), certain fungi (C. albicans) and enveloped viruses (HSV) (Lehrer et al., 1993) although at high concentrations defensins have been found to be cytotoxic to mammalian cells such as in lung epithelia and tumour cell lines (Ganz, 2003). The antimicrobial activity displayed by Î±- and Î²-defensins is markedly reduced and potentially inhibited in the presence of serum, plasma proteins, excess divalent cations such as Mg2+ and Ca2+ and ionic salts at their physiological concentrations (i.e. 150mM of NaCl) thus consequently optimal antimicrobial effects occur in phagosomes (phagocytic vacuoles) in phagocytes and mucosal and epithelial surfaces such as the skin, where ionic salt concentrations are low, as opposed to blood and extracellular fluid (Yang and Oppenheim, 2003).
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The exact method by which defensins apply their microbicidal properties is not completely understood, but it is widely accepted that defensin-induced microbial eradication is due to the disruption of microbial cytoplasmic membrane integrity (De Smet and Conreras, 2005). To perform antimicrobial actions, the exposed cationic regions of defensins initially attract and subsequently bind electrostatically to the electronegative microbial surfaces, which constitute of anionic molecules (PAMPs) such as the anionic moieties in LPS, negative phosphate groups and anionic lipids on the outer membranes of Gram-negative bacteria, and teichoic acids on the surface of Gram-positive membranes (Brogden, 2005). In the case of Gram-negative bacteria, to access the inner cytoplasmic membrane, defensins must first overcome the outer membrane which is covered by divalent Mg2+ and Ca2+ ions (figure 1.3); as defensins have a higher affinity for LPS they are able to displace these cations with ease, permeate the outer membrane and consequently interact with the inner membrane (Zasloff, 2002). Defensins do not display cytotoxic microbicidal activity to host and other mammalian cells (unless at high concentrations) as, in contrast to prokaryotic membranes, the host (eukaryotic) plasma membranes are composed predominantly of zwitterionic (neutral) phospholipids such as sphingomyelin, and neutral membrane-stabilising cholesterol molecules thereby reducing defensin interactions (Zasloff, 2002; Shai, 2002).
The amphipathic nature of defensins causes the hydrophobic and (charged) hydrophilic regions on the peptide to be spatially separated, therefore allowing the polar cationic hydrophilic regions of defensins to interact with anionic phospholipid heads and water, and the hydrophobic regions to interact and bind accordingly to the phospholipid bilayer, with ease (Yang et al., 2004; Shai, 2002). Once bound to microbial plasma membranes, defensins usually undergo conformational changes which are energetically favourable, where defensin peptide monomers combine to form oligomers and consequently orientate, either parallel or perpendicular to the membrane, to insert into the lipid bilayer thereby leading to membrane permeablisation (Diamond et al., 2009). It is generally thought that defensin-induced membrane permeation occurs due to formation of multimeric voltage-dependent ion-permeable pores. Though the exact mechanism has not yet been established, three theoretical models exist to explain the permeation of microbial cytoplasmic membranes: barrel-stave (figure 1.9-A), toroidal-pore (figure 1.9-B) and carpet model (figure 1.9-C) (Brogden, 2005).
The barrel-stave model (figure 1.9-A) supports the view that the bound defensin peptides first assemble in a helical structure on the pathogen outer membrane, orient perpendicularly and consequently insert in the lipid bilayer where the hydrophobic regions of defensins align with the lipid core and hydrophilic regions face outwardly, interacting with water, thus forming a pore. Initially, a minimum of two defensin monomers traverse the membrane, which initiate further recruitment of monomers, leading to an increase in pore size. The organisation forms a permeation pathway, a stable transmembrane channel-like pore, which allows leakage of intracellular water and electrolytes (Oren and Shai, 1998; Shai, 2002).
According to the toroidal-pore model (figure 1.9-B) defensins initially insert similar to the barrel-stave model, in that the peptides accumulate and adsorb on the membrane surface and insert perpendicularly into the membrane surface. Unlike the barrel-stave model, after the polar regions of the defensins align with the polar lipid head groups, the defensin peptides induce a positive curvature in the membrane monolayers so that eventually both phospholipid monolayers connect with each other in a continuous bend, resulting in an opening, named a toroidal-pore. As a result, the lumen of the resultant pore is not only lined with defensin peptides but by polar phospholipid head groups as well (Brogden, 2005; Diamond et al., 2009).
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The carpet model (figure 1.9-C) proposes that defensins aggregate either as monomers or oligomers on the microbial membrane surface, where they align parallel to the membrane positioning themselves so that the hydrophobic regions interact with the lipid core and hydrophilic regions with the polar phospholipid heads, consequently forming an extensive carpet-like layer (Shai, 2002). Once a threshold concentration is reached (i.e. a particular concentration of defensin peptides accumulate upon the bilayer surface), the target membrane destabilises, forming large transient holes similar to toroidal-pores which cause the loss of cytoplasmic contents. Ultimately defensins disintegrate the membrane curvature, similar to a detergent, eventually leading to micelle formation (Oren and Shai, 1998; Brogden, 2005). Disruption of microbial membrane integrity by defensins in either of the models eventually leads to loss of the electrochemical gradient and membrane potential, interruption of cellular respiration and consequential leakage of intracellular contents such as electrolytes, water and ATP, ultimately resulting in osmolysis and cell death (Shai, 2002).
Studies on E.coli have shown that once internalised Î±-defensins such human neutrophil peptide (HNP) 1 and 2 interfere with and inhibit several intracellular processes such as DNA, RNA and protein synthesis, thus suggesting the possibility that defensin-induced membrane permeation is not the only mechanism by which defensins are able to kill microbes, though further studies are necessary (Brogden, 2005).
Figure 1.8 - General properties of mammalian defensins. Human Î±- and Î²-defensins, apart from exhibiting potent antimicrobial activity against many organisms, are chemotactic to many cells of the innate (monocytes, neutrophils) and adaptive (T cells, DCs) immune system, cause mast cell degranulation and subsequent histamine release, repair wounds, promote cellular growth, interact with complement cascades and inhibit ACTH and associated glucocorticoid synthesis. Rabbit defensins alone display opsonisation properties (Lehrer, 2004).
Figure 1.9 - The plausible mechanisms of defensin-induced microbial membrane disruption and consequential killing. The models that exist are the barrel-stave (a) and toroidal-pore (b) where pores are formed, and the carpet (c) model where membrane is disintegrated in a detergent-like manner, eventually leading to micelle formation. Hydrophilic regions of defensin peptides are shown in red; hydrophobic regions are shown in blue (Brogden, 2005).
Defensins are also responsible in the upregulation of the innate immune response; this upregulation is carried out by a variety of mechanisms (figure 1.10). Î±-defensins such as human neutrophil-derived peptides (HNP) 1 and 2 when released by azurophilic neutrophil granules are chemotactic to monocytes and macrophages and thus recruit them to sites of infection and inflammation (Lehrer et al., 1993). Î±-defensins also enhance phagocytosis thus facilitating the innate immune system in the swift removal of invading pathogens (Yang et al., 2004) whilst Î²-defensins, in particular human beta defensin (HBD) 2, promote migration of mast cells and induce mast cell activation and consequential degranulation, causing the release of histamine and prostaglandin-D2 which increase vascular permeability, thereby facilitating the influx of neutrophils and other phagocytes to inflammatory sites (Yang et al., 2001). Defensins (such as HNPs) have also been shown to stimulate epithelial cell production of IL-8, especially in the lungs, which alongside mast cell products such as histamine, are potent neutrophil chemoattractants; therefore indirectly defensins promote further accumulation of neutrophils to sites of infection, where they in turn release additional defensins, which consequently induce the generation of more IL-8 resulting in a 'positive-feedback loop', amplifying the inflammatory innate immune response (Yang et al., 2001; Yang et al., 2002). Furthermore it has been observed that human defensins such as HNP-1 have the ability to regulate the classical pathway of the complement system by binding to complement molecule C1q, thereby decreasing or increasing complement activation, an essential component of innate immunity (Yang et al., 2004).
In addition to upregulating innate immunity, Î±- and Î²-defensins are also chemotactic to various cells of the adaptive immune system such as immature DCs, CD4+ T cells and resting memory T cells and therefore alert and link the adaptive immune response (the second line of defence) to the innate immune response (figure 1.10). Certain defensins such as HBD-1 to 3 bind directly to chemokine CCR6 receptor consequently enhancing recruitment of the aforementioned cells at sites of infection and inflammation, leading to the maturation of iDCs into antigen presenting mDCs, which display MHC II complexes on cell surfaces ready for antigen uptake, and initiation of the effector phase of the adaptive immune response. Defensins have also been shown to stimulate DC maturation by enhancing synthesis of potent DC maturation inducers such as IL-8 and TNF-Î± and indirectly promote migration of mDCs to spleen and lymph nodes where they present antigens to naÃ¯ve T cells, thereby facilitating and speeding the activation of the adaptive immune response (figure 1.11) (Yang et al., 2002). Defensins are able carry out their chemotactic properties at extremely low nanomolar concentrations in the body, and unlike antimicrobial activity, defensin-induced chemotaxis is not inhibited by ionic salts (NaCl), divalent cations (Mg2+ and Ca2+) and serum (Yang and Oppenheim, 2003).
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At low concentrations defensins have also been shown to be mitogenic to fibroblasts and stimulate DNA synthesis and subsequent proliferation in epithelial cells (Murphy et al., 1993), especially seen in the lungs, suggesting that defensins participate strongly in airway tissue remodelling (in certain inflammatory conditions) (Guani-Guerra et al., 2010). Defensins have also been shown to stimulate mucin gene expression in lung epithelia, thereby promoting the synthesis of mucins which contribute to epithelial regeneration and assist in the clearance of invading organisms (Diamond et al., 2009). Moreover defensins also play an important role in wound repair as studies have determined that Î±-defensins such as HNP-1 to 3 stimulate epithelial cell migration (in a time- and dose-dependent manner) thereby increasing the rate of wound closure (Lehrer, 2004). Increased cellular migration and proliferation is also induced by Î²-defensins (HBD-2, 3 and 4) in skin epithelial cells (epidermal keratinocytes) (Harder et al., 2007). In vitro models have also revealed that HNP-1 "increases the expression of pro-collagen mRNA and protein in dermal fibroblast cultures" therefore enhancing cutaneous wound healing by increasing the deposition of extracellular matrix in injured tissues (Steinstraesser et al., 2008). In addition, increased expression of Î²-defensins has been observed in injured tissues after wounding, yet in the instances of chronic wounds and burns, decreased levels of defensins and other AMPs, and excessive growth of bacteria such as S. aureus has been seen; thus it has been suggested that Î²-defensins such as HBD-2 and 3 are vital in wound repair as they are required to facilitate and accelerate wound closure, which is achieved by maintaining a barrier against infection by reducing growth and subsequent colonisation of bacteria and other disease-causing microbes (Harder et al., 2007; Radek and Gallo, 2007).
Additionally, human defensins such as HNP-2 are capable of inhibiting fibrinolysis, by modifying tissue-type plasminogen activator (TPA) and plasminogen from adhering to fibrin and blood vessel endothelial cells. Defensins achieve this as a result of competing for fibrin binding sites and thus inhibiting TPA (Risso, 2000). TPA converts plasminogen to plasmin and consequently causes the degradation of fibrin; therefore inhibition of fibrinolysis is thought to allow the clotting cascade to successfully continue in infectious and inflammatory situations, thereby helping to control microbial infections (Eales, 2003, Risso, 2000).
Finally it has also been shown in humans, that certain defensins (such as HNP-4) are antagonists to ACTH i.e. inhibit adrenal steroid hormone ACTH synthesis at nanomolar concentrations, by reversibly binding to ACTH receptors and subsequently blocking ACTH and associated steroid hormones from binding to the complementary receptors, thereby demonstrating corticostatic activity. ACTH and associated steroid hormones are exceptionally immunosuppressive thus this inhibitory defensin activity is beneficial as it enhances the initial innate immune response. Effective inhibition of adrenal glucocorticoid synthesis has been observed when defensin levels increase significantly (to 100Î¼g/ml) in infections (Yang and Oppenheim, 2003).
Figure 1.10 - Representation of the potential methods by which human defensins activate and link innate and adaptive immunity.
When host epithelial barrier is breached, invading microorganisms cause the production of defensins which affect the innate immune response in five possible ways (as mentioned in diagram). This can result in the recruitment of T cells and activation of DCs in the adaptive immune response, directly by defensins themselves, or indirectly, by cytokines released in the innate response (Yang et al., 2004).
Figure 1.11 - The possible mechanisms by which human defensins can influence host adaptive immune response.
Defensins are produced by epithelial cells (Ep) and neutrophils (N) at sites of infection. Defensins promote recruitment of immature DCs (iDC) to infected tissues (a) and enhance antigen (Ag) uptake by iDCs (b). They are also able to cause iDCs to mature into mature DCs (mDC) by promoting synthesis of potent DC maturation inducers such as IL-8 and TNF-Î± (c) and facilitate recruitment of T cells (d) thereby speeding the activation of the adaptive immune response (Adapted from Yang et al., 2002).
1.5 Human Monocytic Mono Mac 6 (MM6) cell line
To isolate monocytes from blood can be quite difficult and can produce cells of varying conditions, hence using cell lines in vitro which can mimic properties of monocytes in vivo is useful to use as an experimental model for investigating pathological conditions and observing effects of many stimuli such as drugs, or in this project, bacterial LPS (Wright et al., 1996).
There are several monocyte-like cell lines available; the selected cell line to be used in this study is the human monocytic Mono Mac 6 (MM6) cell line. This particular cell line was acquired from a German cell culturing company DSMZ-German Collection of Microorganisms and Cell Cultures and derived in 1985, from the peripheral blood of a 64-year old male patient suffering from acute monoblastic leukaemia (AML M5) (Zeigler-Heitbrock et al., 1988). Alongside MM6, another clone, Mono Mac 1 (MM1) was isolated from the patients' blood (a month before the patient's death); both were assigned, morphologically, cytochemically and immuno-biologically, to the monocyte lineage.
MM6 cell line, in comparison to the other human monocytic cell lines such as MM1, U937 and THP-1, is the only cell line at present which exhibits phenotypic and functional properties of mature monocytes and hence provides a very suitable in vitro model for mature monocytes (Zeigler-Heitbrock et al., 1988; Wright et al., 1996).
These properties include the expression of LPS receptor CD14 on MM6 cells' extracellular surface, ability to phagocytose antibody-coated erythrocytes and mycobacteria, reacting with a panel of monoclonal antibodies specific for macrophages, antigen expression in response to IFN-Î³, releasing reactive oxygen species (Zeigler-Heitbrock et al., 1988) and the production of an inflammatory response by secreting prostaglandins and cytokines such as IL-1Î², IL-6 and TNF-Î± (Zeigler-Heitbrock et al., 1994).
Other advantageous properties of human MM6 cell lines are that they are grown in suspension thus do not require a surface to grow upon and are subsequently easily transferred and manipulated and that the MM6 cell line is a continuous cell line therefore providing an unlimited supply of homogenous cells quickly which would be useful in generating reliable results (Drexler et al., 2000).
MM6 cells grow as single or round cells or as a small cluster of cells in suspension, though occasionally can adhere loosely to vessel walls (MacLoed et al., 1993). Morphologically the cells have an approximate diameter of 16Î¼m, villous cellular surface, a large round or kidney shaped nucleus with a few nucleoli, many organelles such as mitochondria, Golgi apparatus, rough endoplasmic reticulum as well as numerous lysosomes, granules and vesicles present in the cytoplasm (figure 1.12) (Zeigler-Heitbrock et al., 1988).
MM6 cells, though widely used as in vitro cell models, have not been used frequently as models in studies investigating the expression of defensin genes.