The cytosol also contains an antimicrobial factor called "calprotectin," and just for the record, the nuclear proteins, histones, are microbicidal. The antimicrobial activities of non-granule proteins (histones, calprotectin) have not previously been emphasized due to the intense bias of neutrophil and cell biologists, who believe that only granule proteins are antimicrobial and who forget that many (if not all) proteins are multifunctional. Thus, although histones may play a very important role in the architecture of the nucleic acid-protein complex, they may serve another purpose (killing microbes). When you think about it, why did we start putting proteins around our nucleic acids to begin with? In part, this is a very primitive defensive strategy, protect the genes. Also, why should circulating neutrophils retain their nuclei? Some insight is provided by the recent isolation of three microbicidal proteins from mouse macrophage granules (Heimstra et al.. 1993. Infect. Immun. 61: 3038-3046). These proteins were designated "murine microbicidal proteins 1,2 and 3," or "Mump-1-3." Mump-1 and Mump-2 appear to be members of the highly variable H1 linker histone family. Mump-3 is identical to the mouse H2b core histone. The antimicrobial activity of histones may reflect an ancient biologic role. Perhaps neutrophils retain their nuclei because, unlike macrophages, they are terminally differentiated and do not need to package antimicrobial histones within the granule compartment.
The oxidative antibacterial effects are mediated by two main biochemical entities, the NADPH oxidase system and myeloperoxidase (MPO). MPO localizes into the azurophil granules. The NADPH oxidase system spans the cytoplasm and plasma membrane.
Phagocytosis is the engulfment of a particle which requires active actin "sol-gel transformation" and the formation of a membrane-bound structure called the "phagosome." In contrast, pinocytosis, or receptor-mediated engulfment of solutes, does not require actin polymerization and depolymerization. Phagocytosis requires receptors, and receptor-ligand interactions must occur over the entire surface of the target particle. In contrast to extracellular secretion (above), phagocytosis involves intraphagolysosomal secretion and additionally, isolates the target organism within an extremely stringent environment.
Opsonization. If a particle is in suspension (in blood, for example), the phagocyte will probably not ingest the particle unless it is coated by some substance which lets the phagocyte know that the particle should be ingested. The process of coating the particle such that the phagocyte may ingest it is known as opsonization. There are two main serum-derived pathways for preparing bacterial and fungal particles for phagocytic ingestion (Figure 9). The first involves the nonspecific coating of bacteria by complement proteins as discussed in the preceding chapter. The phagocyte uses CR1 to propel the conversion of C3b to iC3b. Then, iC3b receptors (CR3, CR4) which are stored in gelatinase-containing granules (considered a distinct type of "tertiary" granule) are brought to the surface of the phagocyte if the right stimulatory signals are provided (ie., "chemoattractant receptor" stimulation). CR3 and CR4 binds to iC3b and enables the phagocyte to ingest microbial targets. Blockade of either CR1 or CR3 (using monoclonal antibodies) greatly impairs the ability of neutrophils to engulf encapsulated bacteria; thus proving the importance of the CR1 cofactor to neutrophils (Edwards et al., 1993. Infect. Immun. 61:2866-2871).Occasionally, a microbe has evolved a mechanism of preventing opsonization by the alternative pathway. This can result in a more prolonged infection. The prolonged infection results in the generation of specific antibodies (remember, a prolonged infection is required for the chronic immune system to activate specific immunity). The antibodies do two things. First, by initiating the classical pathway of complement activation, they circumvent mechanisms that the microbe has developed to protect itself against the alternative pathway. If the organism is also resistant to classical pathway opsonization, then the antibodies themselves can serve as opsonins. Fc receptors can also mediate antibody specific endocytosis. Interestingly, there are a variety of Fc receptors possessed by phagocytes. Clearly, the most important opsonic Fc receptors are those which bind IgG (the high affinity FcgRI or CD64 in monocyte/macrophages, and the low affinity FcgRII or CD34 in neutrophils). For dentists, it is also very interesting that neutrophils obtained from the oral cavity possess significant levels of an IgA Fc receptor (FcaR) which are higher than levels found on blood neutrophils (Fanger et al., 1983. Mol. Immunol. 20: 1019-1027). The extent to which these IgA receptors promote phagocytosis, however, is unknown (for example, they may simply promote secretion).
Intraphagolysosomal secretion. All phagocytes are secretory cells. Phagocytic killing and digestion of microbes involves secretion (degranulation) into the phagosome. This is called phagolysosomal fusion. Within 30 seconds after a neutrophil ingests a particle, it begins to secrete specific granule components into the phagosome via phagolysosomal fusion. Within 3 minutes, azurophil granule components are discharged into the phagolysosome. The phagolysosomal fusion process is dependent upon intracellular calcium and requires the activity of a mixture of 3 "synexin-like" cytosolic proteins (MW = 28,000; 47,000; and 67,000) (Meers et al., 1987. J. Biol Chem., 262: 7850-7858), now collectively known as "annexins."
Extracellular Secretion. Extracellular secretion by phagocytes is important in inflammation, receptor regulation, tissue destruction, and antimicrobial effects. Azurophil granules are released when the neutrophils is stimulated and in contact with a surface. Specific granules are released with much less stimulation (soluble molecules can stimulate specific granule release). Extracellular secretion can be stimulated by microbial particles or fluid phase molecules, including most chemotaxins. Usually, a higher concentration is required for secretion than for chemotaxis. For extracellular secretion to occur, about 10 nM of chemotaxin is required. Along with secretion, there are also increases in cellular adherence, chemotactic receptors, and priming for respiratory burst. Secretion by PMN increases as the cell approaches its target (Fig. 10). This is because secretion of various lysosomes is dependent upon the concentration of the secretagogue. As the cell nears its target, the concentration of the secretagogue increases. Specific granule components are released first (light fill pattern). As the stimulus becomes more intense, azurophil granule components are released. Oxygen reduction also occurs at higher secretagogue concentrations. Secretion results in the release (externally or into the phagolysosome) of antimicrobial substances which are discussed below.Cytolysis and apoptosis. Antimicrobial delivery may result from the complete disruption of the cell and the subsequent release of all subcellular components (including those in the nucleus, cytosol, and cytoplasmic granules). Cytolysis (or necrosis) is a disorganized form of death which is not programmed. Cytolysis may result in the release of antimicrobial compounds. Only some of these antimicrobial compounds can function effectively outside of the phagolysosome. Both histones from the nucleus and calprotectin (L1 protein) from the cytosol can exert killing and/or microbiostatic effects. In particular, calprotectin has been shown to function in conditions that completely inhibit the antimicrobial activities of granule-derived antimicrobial substances. No respiratory burst metabolites are released in a purely cytolytic event. Cytolysis may be indicated when dealing with extracellular mucosal pathogens (such as Candida albicans). In such instances, the phagocyte cannot engulf the pathogen (for several anatomic reasons). Indeed, although it is known that phagocyte defects lead to mucocutaneous candidal infection and that phagocytes will ingest the fungi in blood (ie., in diseminated candidiasis), it is also clear from histology that phagocytes never engulf the fungi they are controlling in mucocutaneous candidiasis (McNamaraet al. 1988. Lancet II(8621): 1163-1165). Apoptosis (programmed cell death) may be a mechanism of permitting phagocytes to die in the absence of release of potentially cytotoxic substances. It may be desirable to retard this form of neutrophil death and the host has a number of pathways available to it to retard apoptosis of granulocytes, including the release of cytokines such as IL-1b, TNF, IL-6, IFNg, G-CSF, and GM-CSF (Colotta et al., 1992. Blood. 80: 2012-2020). Also, the bacterial component, lipopolysaccharide, seems to prolong granulocyte survival.
There are many components within the phagocyte granules (lysosomes) which exert antimicrobial effects. Some of these components are herein.
Defensins are major components of the azurophil granules of neutrophils and consist of a group of low molecular weight arginine/cysteine-enriched cationic peptides (3000-4000 daltons) which exhibit three intrachain disulfide linkages (high for a peptide containing 30 or so amino acids). The defensins are encoded on chromosome 8p23 (Lehrer et al., 1990. ASM News. 56: 315-518). Four human human leukocyte defensins are known (HNP-1, HNP-2, HNP-3, and HNP-4). Defensins are found in other cells besides phagocytes, and two defensins have recently been localized to the human gut in specialized secretory cells called "Paneth" cells. Inbred mice lack the leukocyte defensins, but nevertheless maintain Paneth cell defensins, which are referred to as "cryptdins" in mice, are called "HD-5" and "HD-6" in man. HNP-4 has been shown to bind to adrenocorticotrophic hormone receptors and inhibit ACTH-stimulated corticosteroid production (a potentially significant hormonal function with respect to lymphocyte activity). Thus, HNP-4 has also been referred to as a "corticostatin." Evolutionarily, the defensins are quite old and defensin-like molecules have been observed in insects. Defensins assume a homodimeric structure in solution (Figure 11). The antimicrobial activity of the defensins appears to require dimerization. To date, the antimicrobial effects of the gut defensins has not been demonstrated.Bactericidal/permeability increasing protein (BPI) is a 59 kdal cationic protein which is only found in neutrophils. BPI does not require enzymatic activity to function and belongs to a class of proteins which transport or bind cholesterol esters and lipopolysaccharides (Gray et al., 1989. J. Biol. Chem. 264:9505-9509; Schumann et al., 1990. Science 249: 1429-1431). BPI itself can "neutralize" endotoxin (Wright et al., 1990. Science 249:1431-1433). It is exerts microbicidal activity by permeabilizing bacterial membranes (Weiss et al., 1978. J. Biol. Chem. 253: 2664-2672).
Neutral Serine Protease (NSP) Family. The NSP are major components of the azurophil granules. There are at least four members of the NSP family, including elastase, proteinase 3, azurocidin, and cathepsin G (Fig 12). These are 23-36 kdal cationic glycoproteins which share 30-70% primary sequence homology. Elastase, proteinase 3, and azurocidin have been referred to as "serprocidins" and are encoded in a coordinately regulated serprocidin gene complex located on chromosome 19. Cathepsin G is encoded on chromosome 14 (near the TCRa gene), near its close relative, lymphocyte granzyme B. Both elastase and cathepsin G appear to kill bacteria by two distinct mechanisms, one enzyme-dependent and an enzyme-independent manner (Miyasaki and Bodeau, 1991. J. Clin. Invest. 87:1585-1593).Proteinase 3 (also called AGP-7, VII, p29b, Proteinase 4, and the ANCA-C antigen of Wegner's granulomatosus) is relatively poorly characterized elastolytic enzyme. Proteinase 3 is the only other NSP member besides elastase which has been implicated in the immunopathogenesis of emphysema. It is a major component of the azurophil granules along with elastase. It reportedly kills Gram-negative bacteria, Gram-positive bacteria, and fungi. Azurocidin (also called CAP37) has no known enzymatic (proteolytic or esterolytic) activity. The "active site" of azurocidin shares 70% sequence homology with human neutrophil elastase, however the catalytic serine195 is replaced by glycine, and the catalytic histidine has been replaced by a serine. Nevertheless, azurocidin is actively antimicrobial. Azurocidin is synergistic with elastase in the killing of oral bacteria (Miyasaki and Bodeau, 1992. Infect. Immun. 60:4973-4975).
hCAP-18 is a newly described 19 kdal specific granule protein of human neutrophils belonging to the bactenecin family (Crowland et al., 1995. FEBS Lett. 368: 173-178). Bactenecins are antibiotic peptides originally described from bovine leukocytes. Unlike many other antibiotic peptides, bactenecins are stored in an inactive proform, and appear to require the catalytic activities of neutral serine proteases (azurophil granule components) to remove the neutralizing propiece and become active. More intriguingly, the propiece of hCAP-18 is highly homologous to a cysteine protease of the stefin A family called "cathelin." As it turns out, many antibiotic peptides share this characteristic propiece, including defensins, bactenecins, protegrins (not yet found in humans), prophenins (not yet found in humans), and others (Ganz, 1994. CIBA Found. Symposium 186:62-71).
Lactoferrin is an iron-binding glycoprotein protein belonging to the transferrin family. Lactoferrin has a very high affinity for Fe+++, higher than that of transferrin. Because bacteria need iron to grow, one host defense strategy includes iron-deprivation. Iron-binding proteins such as ferritin and transferrin are primarily used for intracellular iron storage and extracellular iron transport, respectively. The unbound iron concentration in the presence of normal levels of transferrin is about 10-18 M, about the minimum level at which certain bacteria can proliferate. Both transferrin and lactoferrin therefore tend to prevent bacterial growth. Additionally, apolactoferrin (iron-less) is bactericidal against certain organisms. Lactoferrin localizes to the specific granules, suggesting that it plays an important role in the antimicrobial effects of extracellular phagocyte secretion. It has also been found that lactoferrin binds to the Lipid A portion of bacterial lipopolysaccharide. It may thereby exert anti-inflammatory effects by opposing the activities of the "LPS-binding protein"- "CD14" interaction (Appelmelk et al., 1994. Infect. Immun. 62:2628-2632).
Lysozyme is a multifunctional protein best known for its ability to (N-acetylmuramide glycanohydrolase) cleave ß-1,4 linkage between N-acetyl-glucosamine and N-acetylmuramic acid in the peptidoglycan layer of the bacterial cell wall. Alone, lysozyme has weak bactericidal activity and is primarily active against Gram-positive bacteria, which have no outer membrane to protect their cell wall. In conjunction with membrane disruptive agents, such as chaotropic ions (ions of low charge density), EDTA, and SDS, the bactericidal activity of lysozyme can be enhanced against Gram-negative bacteria. Lysozyme may also function non-enzymatically to enhance autolysin activity by binding to teichoic acids in the Gram-positive cell wall. Lysozyme also kills fungi (which don't even possess peptidoglycan). In neutrophils, lysozyme is found in both the specific and the azurophil granules.
Prior to adherence to the target microorganism, the phagocyte begins to consume oxygen. This is called the respiratory burst, and in the neutrophil, very little of this consumed oxygen is used for energy needs. Instead, oxygen is consumed to form reduced oxygen metabolites which will be used to kill the target microbe.
Cytochrome b, also called "cytochrome b558" for its absorbance at 558 nm and "cytochrome b-245" for its redox potential of -245 mV) is a heterodimeric structure consisting of a 22 kdal and 91 kdal subunit (gp91[phox]). Cytochrome b is a heme protein found only in neutrophils, monocytes, and eosinophils. Cytochrome b adds electrons to external or intraphagolysosomal dioxygen (with a redox couple of -160 mV). The 91 kdal subunit of cytochrome b is encoded on the X chromosome and the 22 kdal subunit (p22[phox])is probably encoded on chromosome 16. Recombinant 91 kdal subunit possesses primary structure consistent with an FAD-binding protein, and recently gp91[phox] has been shown to bind both NADPH and FAD (Rotrosen et al., 1992. Science 256: 1459-1462). Flavin adenine dinucleotide (FAD)-binding proteins are generally used to couple two electron donors such as NADPH with single electron carriers such as heme iron. The illustration also shows Rap1A. Rap1A is another G protein which is closely associated with cytochrome b, is a substrate for protein kinase A, and may inhibit oxidase activity (Bokoch et al., 1991. Science 254: 1794-1796).
O2- + HO2 + H+ -----> H2O2 + O2 (superoxide dismutase)
Hydrogen peroxide. Most phagocyte H2O2 is thus formed by two sequential univalent reductions, the first is the reduction of dioxygen by cytochrome b to superoxide anion and the second is the dismutation reaction between two superoxide anions, forming H2O2 and O2. However, some H2O2 is formed by the direct, divalent reduction of dioxygen by cytochrome b, using no superoxide intermediate. H2O2 is not very toxic, but it is still the most important reduced oxygen intermediate. There are two reasons for this: (1) it may be further reduced to a very toxic compound, hydroxyl radical (OH€) and (2) H2O2 is a substrate for the peroxidase enzymes (below).
H2O2 is normally stable, but because it can lead to more potent killing compounds, some microorganisms and virtually all host tissues destroy H2O2 using the enzyme, catalase. Catalase catalyzes the disproportionation of H2O2, wherein one molecule of H2O2 is divalently reduced and the other is divalently oxidized.
H2O2 + H2O2 ------> O2 +2 H2O (catalase)
This results in the formation of H2O and O2. Hydroxyl radical. Extracellular H2O2 can oxidize transition metals, resulting in its own univalent electron reduction and the formation of hydroxyl radical (OH€). In such an external milieu, OH€ can cause cell death via lipid peroxidation. But, one of the protective effects of lactoferrin is believed to result from it's ability to block this reaction. More crucial to the bactericidal activity of H2O2 is intracellular. Since H2O2 is stable and uncharged, it can also cross cell membranes freely, and within a metabolically active cell, H2O2 can be reduced by transition metals maintained in a reduced state by the cellular metabolism. Whereas OH€ is quite effective at breaking apart bacterial (and eukaryotic) chromosomal DNA (whereas H2O2 has no in vitro effect). Cells die if the rate of DNA damage exceeds the rate of repair.
Water. Finally, addition of one more electron to the hydroxyl radical (OH.), results in the formation of water (H2O). Water doesn't do too much!
Singlet Oxygen. Another reactive, potentially toxic oxygen species is singlet oxygen (1O2) (Figure 14). 1O2 is a dioxygen molecule which possesses a normal number of electrons but has one electron which has either undergone spin inversion or switched orbitals (two types of singlet oxygen exist) such that the molecule is no longer at its lowest energy state. 1O2 may be generated by a number of reactions, including those catalyzed by superoxide dismutase and catalase (above) and myeloperoxidase (below). At present, there is abundant evidence that phagocytes make singlet oxygen, but no convincing evidence to suggest that this reactive compound has any antimicrobial activity. Interestingly, singlet oxygen gives off photons of light as electrons relax into ground state conditions. Using a photomultiplier tube, it is possible to measure these emissions. Phagocytes truly light-up when they become excited (if O2 is around). This is partially responsible for the chemiluminescence of phagocytes.
Lysis and digestion of bacteria proceeds most rapidly intraphagolysosomally. The neutrophil is not as good at digestion of foreign particles as macrophage, thus, when neutrophil egress from a local site is obstructed, macrophage are always recruited to eliminate neutrophil and bacterial debris. Usually, the neutrophil is eliminated in the form of pus, thereby minimizing the amount of macrophage activity required.
Eicosanoids are oxidatively-derived metabolites of a 20 carbon fatty acid, eicosanoic acid (trivial name: arachidonic acid). Eicosanoids behave as hormones, and enable cells to affect the local tissues surrounding them. Eicosanoids are of some importance in neutrophils, which are relatively limited (both with respect to time and capability) in transcribing proteins which may exert an extracellular effect. Activation of acute inflammatory cells causes a local release of eicosanoic acid from the phospholipids from the plasma membranes of cells. This activity is catalyzed by phospholipase A2 and inhibited by anti-inflammatory glucocorticoids.
The subsequent metabolites that form from arachidonic acid result in modulation of the inflammatory response. The precise metabolites vary according to the cell which has been activated. In general, activation of acute inflammatory cells (ie., neutrophils), results in the production of leukotrienes by the lipoxygenase pathway (initiated by an 80 kdal enzyme, 5-lipoxygenase), and activation of chronic inflammatory cells (ie., monocytes), results in the formation of prostaglandins and thromboxanes via the cyclooxygenase pathway (Figure 17).The 5-lipoxygenase pathway is prominent in granulocytes and mast cells, but there is some evidence that it may also function in certain lymphocytes. It is initated by the translocation of the enzyme, 5-lipoxygenase, from the cytosol to the cell membrane, which requires an 18 kdal membrane-associated protein referred to as "FLAP," an anchronym for 5-lipoxygenase activating protein. Leukotriene B4 (LTB4) is the main product of neutrophil arachidonate metabolism. It may act as an intracellular signal as well as a hormone. Intracellular effects may include promoting the cell for aggregation (adherence), deactivation of chemotaxis, and secretion of granules. The extracellular effects of LTB4 include the stimulation of chemotaxis and chemokinesis activity in neutrophils (Lewis and Austen, 1988. In: Gallin, Goldstein, and Snyderman (eds) Inflammation: Basic Principles and Clinical Correlates, Raven Press, NY. pp 121-128). Certain leukotrienes (especially LTC4) are also known as "slow-reacting substances of anaphylaxis" (SRS-A) and are produced by mucosal mast cells and basophils.
Phagocytes are myeloid-derived leukocytes which are specialized in exiting the blood, migrating through the connective tissues, and finding large, particulate targets (such as bacteria and fungi) to ingest. There are two closely related "professional phagocytes," neutrophils and monocytes. Myeloid cells committed to become neutrophils spend about 14 days in the bone marrow, where they mature completely to a terminally differentiated state; virtually incapable of further differentiation once they arrive at a tissue site.
Neutrophils use selectins and b2-integrins to extravasate from blood. They hunt their targets by chemotaxis, sensing incredibly low and shallow gradients of chemotaxins such as C5a or formyl-methionyl peptides. The receptors for chemotaxis belong to the rhodopsin (G-protein coupled receptor) superfamily. Once the neutrophil contacts its target, it uses the CR1 and CR3 receptors to convert C3b to iC3b and to bind iC3b, respectively, for subsequent phagocytosis.
The function of neutrophils ends in the killing and destruction of the microbial particle. It performs killing by both oxidative and nonoxidative mechanisms. Important nonoxidative mechanisms include defensins, lysozyme, neutral serine proteases, and BPI. Important oxidative mechanisms involve the generation of hydrogen peroxide by the NADPH oxidase system and the generation of hypochlorous acid by myeloperoxidase. Neutrophils have additional microbiostatic molecules, including lactoferrin, hCAP-18, cobalophilin, and calprotectin. Neutrophils also may communicate with the chronic immune cells, but the pathways for such communication are not yet fully elucidated. The eicosanoid, LTB4, enables neutrophils to recruit other neutrophils into an area.
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