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Published online before print August 3, 2004
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Department of Medicine, Division of Infectious Diseases, Children’s Hospital & Harvard Medical School, Boston, Massachusetts
1 Correspondence: Division of Infectious Diseases, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115. E-mail: ofer.levy{at}tch.harvard.edu
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ABSTRACT |
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Key Words: neutrophil • polymorphonuclear leukocytes • defensins • cathelicidins • bactericidal/permeability-increasing protein (BPI) • lactoferrin • lysozyme • saponins • calprotectin • serprocidins • phospholipase A2 • Toll-like receptors
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INTRODUCTION |
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However, a number of "oxygen-independent" agents with direct microbicidal activity were discovered before and after the identification of the phagocyte oxidase enzyme. Alexander Flemming described lysozyme (Lz), which is found in phagocytes and secreted from mucosal epithelia, in the 1930s [4 ]. Hirsch [5 ] and Zeya and Spitznagel [6 ] examined the microbicidal activity of crude neutrophil extracts and began characterizing some antimicrobial cationic (poly)peptides. Groves et al. [7 ] isolated lactoferrin (Lf) from milk in the 1960s. With the advent of improved tools for molecular research, including novel chromatographic techniques, several groups in the late 1970s and 1980s began reporting the purification of leukocyte-derived antimicrobial proteins and peptides (APP; Table 1 ), including the bactericidal/permeability-increasing protein (BPI) [8 ], Lf [9 ], defensin peptides [10 ], and serine protease homologues with microbicidal activity (serprocidins) [11 ].
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With few exceptions [29 , 30 ], the APP are cationic granule-associated (poly)peptides whose relatively selective cytotoxic action toward microorganisms has been ascribed to their relatively high affinity for the anionic phospholipids in microbial membranes [31 , 32 ]. Although some APP, such as Lz and phospholipase, function enzymatically, most others are solely "membrane active." Initial binding of cationic APP to microbes is driven by electrostatic interactions with the negatively charged outer layer of the bacterial membrane, followed in many instances, by insertion into the membrane bilayer based on hydrophobic interactions [33 ]. Consistent with this scheme, isogenic bacterial mutants with increased negative surface charge demonstrate enhanced susceptibility to cationic APP and decreased virulence, whereas modifications that increase cationicity of bacterial surface components reduce susceptibility to APP and increase bacterial virulence [34 ]. Further penetration of some APP into the cytosol (i.e., via the inner membrane in the case of Gram-negative bacteria) may occur, thereby allowing APP to reach intracellular targets (e.g., nucleic acids) [33 ]. Consistent with the importance of penetration of some APP to the bacterial cytosol, mutants of Neisseria gonorhoeae with defects in the mtr efflux system, which remove microbicidal compounds from bacteria, are substantially more sensitive to the antimicrobial peptide protegrin [35 ]. Some have suggested that APP may generally function by activating bacterial autolysis [36 ], and evidence in favor of this hypothesis has been published recently with respect to the action of PLA2 against target bacteria [37 ]. However, the "autolysis" hypothesis remains to be tested systematically with a range of purified APP in isolation. Of note, some APP demonstrate more than one mechanism of action. For example, the muramidase Lz and the protease CatG possess enzymatic and nonenzymatic antimicrobial activity.
Recent advances in the study of APP include: identification of two novel families of neutrophil-derived APP, the PGRP and NGAL; further expansion of the range of tissues, including epithelial cells and reproductive glands, which express APP; appreciation of additional functional activities, including chemotactic and immune-modulating activities; identification of novel inducers of APP expression, including TLR ligands and endogenous anti-inflammatory lipids (lipoxins); demonstration that activation of the respiratory burst oxidase serves to enhance the activity of APP; evidence in support of a host defense function of APP in vivo, including studies of mice with targeted deletions in genes encoding APP; and advances in the efforts to apply APP as novel anti-infectives in the clinical arena.
Substantial conceptual and applied progress has thus been made over the past 4 years since I last reviewed the topic of mammalian APP [38 ]. Herein, I update the progress about the study of APP derived from mammalian leukocytes with emphasis on their anti-infective properties.
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PMN |
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By inhibiting viral infection of host cells, Lf possesses antiviral activity toward hepatitis C virus (HCV), poliovirus, rotavirus, herpes simplex virus, and human immunodeficiency virus (HIV) [46 ]. Lf binds the E1 and E2 envelope proteins of HCV via a 33 amino acid C-terminal region of Lf and thereby prevents HCV infection of human cultured hepatocytes in vitro [47 ]. Another antiviral activity of Lf relates to its ability to bind heparin sulfate proteoglycans [48 ], onto which viruses often dock prior to interaction with specific receptors.
Although most studies of the interaction of Lf with microorganisms focus on host defense roles of Lf, others demonstrate that Lf actually enhances the growth of some organisms. Neisseria spp. and Helicobacter pylori can bind Lf and use it as an iron source [49 , 50 ]. Lf attaches to the surface of Streptococcus pneumoniae by binding pneumococcal surface protein A, an interaction that may be of benefit to the bacterium [51 ]. Lf also enhances growth of the parasite Tritrichomonas fetus [52 ].
In addition to its direct antimicrobial activities, Lf binds to and modulates the activity of host cells. Lf, via its N-terminal lactoferricin domain, enhances the phagocytic activity of neutrophils, apparently via opsonic and neutrophil stimulatory mechanisms [53
]. Specific receptors on host cells have been detected for Lf [54
], and several studies have documented immunomodulatory activity of Lf. For example, Lf modulates cytokine release from host cells, enhancing interleukin (IL)-6 and tumor necrosis factor 
(TNF-) release from peripheral blood mononuclear cells (PBMC) [55
] but inhibiting release of IL-1 and IL-2 [12
]. Oral administration of Lf to mice enhances secretion of immunoglobulin (Ig)A and IgG from Peyer’s patches and spleen [56
]. Lf modulates NK cell cytotoxicity [57
] and reduces dermal cytokine production [58
].
BPI
Prompted by the observation that Gram-negative bacteria ingested by PMN are rapidly growth-inhibited but remain metabolically and structurally intact for some time [59
], Elsbach and Weiss pursued purification of a putative factor with discreet cytotoxic activity toward these microorganisms. In 1978, they reported the purification of the BPI [8
].
BPI is a 55-kDa constituent of the primary (azurophilic) granules of PMN with a bipartite "boomerang" structure that comprises a cationic (lysine-rich) N-terminal half-linked by a proline-rich hinge to a hydrophobic C-terminal half [60 ]. The high affinity for lipopolysaccharides (LPS) of BPI targets its anti-infective properties: bacterial membrane permeabilization [61 ], LPS-neutralizing activity [62 ], and opsonic activity toward Gram-negative bacteria [63 ]. Binding of BPI to target bacteria is followed by time-dependent progression of damage from the outer to the inner bacterial membrane, coinciding with bacterial killing [61 ].
Structure-function analysis of BPI has been greatly facilitated by cloning and sequencing of the BPI cDNA, isolation of a bioactive proteolytic fragment of BPI [64 ], and analysis of the BPI crystal structure [65 ]. The antibacterial and LPS-neutralizing activity of BPI is localized to the N-terminal half of the protein [64 ], whereas the C-terminal half enhances the opsonic activity of the molecule [66 ]. BPI bears structural homology to the LPS-binding protein (LBP), a liver-derived plasma component that serves to deliver LPS to its receptor on host cells [67 ].
Consistent with the high affinity of BPI for the lipid A region of LPS, the antimicrobial action of BPI is most potently expressed against Gram-negative bacteria including E. coli, Salmonella typhimurium, Shigella, and Enterobacter spp. [68 ]. Of note, the bactericidal activity of BPI against these bacteria is manifest at nM concentrations in biologic fluids such as serum, plasma, and whole blood [69 ] as well as inflammatory peritoneal exudates generated in vivo [70 ]. Some Gram-negative bacteria, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Serratia spp., are relatively resistant to the bactericidal activity of BPI (but fully susceptible to its endotoxin-neutralizing activity) [68 , 69 ]. At relatively high concentrations, BPI or BPI-derived peptides also have in vitro microbicidal activity against L forms of Gram-positive bacteria [71 ], fungi such as Histoplasma capsulatum [72 ], and T. gondii [73 ].
Histones
Histones are highly conserved, 11–21 kDa cationic proteins that bind DNA to form chromatin, the nucleoprotein chromosomal material. The microbicidal activity of histones, when tested in vitro against a broad range of microorganisms, has been appreciated since the work of Hirsch in the late 1950s [74
]. Park and co-workers [75
] have studied the mechanism of action of the histone-derived antimicrobial peptide bufforin II. A proline-rich hinge region of bufforin II is responsible for the ability of this peptide to penetrate and thereby kill target cells. Despite such evidence for in vitro microbicidal activity of histones, their subcellular localization to the nucleus, where they are closely bound to DNA, made it difficult to conceptualize how they might contribute to antimicrobial defense. However, recent work by Zychlinsky’s group [19
] has demonstrated that activated neutrophils extend DNA-based NETs, which contain histones and several other APP, including elastase, CatG, and Lf . NETs entrap bacteria and deliver high local concentrations of several APP. Neutralizing antihistone monoclonal antibodies were found to block the bactericidal activity of NETs.
PGRP
PGRP are components of innate-immune defense in Drosophila, wherein they serve to recognize bacterial invasion and trigger the protective prophenoxidase cascade of insects [76
]. The cloning of homologues of PGRP in mice and humans revealed that this innate defense molecule has been conserved through evolution. Murine PGRP is localized to tertiary granules of PMN and expresses direct antibacterial activity against Gram-positive bacteria of low pathogenicity [15
]. Murine PGRP rapidly bind peptidoglycan (PGN) with nM affinity and are able to inhibit the growth of Staphylococcus hemolyticus and Bacillus megaterium [77
]. PGRP inhibits soluble PGN-induced cytokine production by macrophages and Micrococcus- and Bacillus (but not E. coli-)-induced phagocytosis and oxidative burst responses by murine macrophages. A bovine PGRP ortholog expresses antimicrobial activity at the 0.5–20 µM range against Salmonella and Candida albicans [78
]. As the PGN of Salmonella is shielded by outer membrane, and Candida do not express PGN, it was suggested that the PGRP may be able to bind microbial oligosaccharides, prompting these authors to name the protein bovine oligosaccharide-binding protein (bOBP). Some mammalian PGRP apparently express N-acetylmuramoyl-L-alanine amidase activity, cleaving the lactylamide bond between muramic acid and the peptide chain in PGN [79
]. Much remains to be learned about the function and specificity of mammalian PGRPs.
Serprocidins
A cluster of genes on human chromosome 19 encodes a family of serine protease homologues with microbicidal activity (serprocidins), localized to the primary granules of neutrophils. Among the serprocidins are the proteases NE, CatG, and PR3, each containing a His-Asp-Ser catalytic triad common to serine endopeptidases. Although NE has been shown to participate in the digestion of phagocytosed bacteria, evidence of direct microbicidal activity of NE in vitro has been limited. However, Belaaouaj and co-workers [16
, 80
] have recently demonstrated that NE degrades outer membrane protein A of E. coli, thereby contributing to PMN-mediated microbicidal activity against this pathogen in vitro and in vivo. CatG, which possesses enzymatic and nonenzymatic antimicrobial activity in vitro [81
], contributes to the killing of Staphyloccus aureusin vivo [82
].
Azu is a 29-kDa serine protease homologue possessing alterations in amino acids that comprise the proteolytic active site such that it lacks protease activity [17 ]. Azu is stored in azurophilic granules and secretory vesicles and demonstrates direct microbicidal activity against E. coli, Streptococcus faecalis, and C. albicans [83 ]. A double-loop mutant of Azu in which all eight basic residues were replaced with glutamines demonstrates decreased ability to bind heparin and to kill E. coli and C. albicans [84 ]. Azu acts in synergy with NE and CatG to kill Capnocytophaga sputigena [85 ] and has opsonic activity toward S. aureus [86 ].
In addition to direct microbicidal effects, serprocidins play additional roles in the inflammatory response. In a porcine wound chamber model, NE was found to play important adjunctive roles in generating antimicrobial activity by cleavage of cathelicidin proforms to release active antimicrobial peptides [87 ] (Fig. 1 ). In contrast, the human cationic antimicrobial peptide 18 (hCAP18) is proteolytically activated by PR3 [88 ]. NE also degrades granulocyte-colony stimulating factor (G-CSF), thereby regulating granulopoiesis [90 ] and binds CR3, thereby reversing leukocyte adhesion [91 ]. Upon adhering to endothelial cells, neutrophils release Azu, which triggers calcium-dependent, endothelial cytoskeletal rearrangement; intercellular gap formation; and increased macromolecular efflux in microvessels [92 ], suggesting that Azu is a key agent of neutrophil-enhanced vascular permeability.
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Lz
Lz is a 14.5-kDa enzyme found in the primary and secondary granules of PMN, which degrades bacterial PGN by cleaving the glycosidic bond that connects N-acetylmuramic acid and N-acetyl glucosamine. As the first of the APP to be discovered, Lz has been under study for over 80 years [96
]. More recent studies have focused on the genetic regulation of Lz expression [97
]. Lz is broadly expressed and is found in the primary and secondary granules of PMN as well as in most mucosal secretions. However, PGN that is heavily cross-linked via O-acetylation, such as that of S. aureus, is resistant to the action of Lz. When Lz is tested in isolation, the hydrophobic outer membrane of Gram-negative bacteria excludes Lz and thereby renders these organisms resistant to its direct microbicidal action. Thus, tested in isolation, the microbicidal activity of Lz is generally limited to bacteria of low pathogenicity such as Bacillus subtilis. Lz also possesses nonenzymatic antibacterial activity, as recombinant Lz congeners devoid of enzymatic activity retain some bactericidal activity in vitro [98
]. Lz, at µg/mL concentrations, also possesses in vitro activity against C. albicans [21
].
Calprotectin
A member of the S100 protein family of calcium-binding proteins, the calprotectin protein complex (Clp) is an abundant constituent of PMN cytosol (
30% of PMN cytosolic protein), comprised of 8 and 14 kDa components. Clp is released at inflammatory sites after death and dissolution of PMN in a process referred to as "holocrine secretion" [99
]. Clp expresses antimicrobial activity when tested at µM concentrations in vitro, and Clp expresses antimicrobial activity against bacteria, including the spirochete Borrelia burgorferi, which causes Lyme disease [23
], and fungi. Clp binds zinc, thereby depriving microorganisms of this nutrient [24
]. Zinc inhibits the activity of Clp and accordingly, zinc-reversible, antifungal activity has been detected in abscess fluids [100
] and in plasma during bacterial infection and other inflammatory states [101
]. Transfection of Clp into a Clp-negative, oral epithelial cell line confers protection against binding and invasion by Porphyromonas gingivalis [102
].
Membership of Clp in the S-100 protein family containing members with roles in regulation of cell-cycle progression, cell differentiation, and cytoskeletal-membrane interactions raises the possibility that Clp may have functions in addition to antimicrobial activity. Consistent with such a view, Clp inhibits fibroblast cell growth and can induce apoptosis in a variety of host cells [103 ].
Cathelicidins
Cloning and structural characterization of antimicrobial peptide precursors of bovine, rabbit, and human PMN revealed a novel family of proteins with N-terminal homology to the protease inhibitor cathelin and highly divergent antimicrobial C-terminal peptides and have been therefore named cathelicidins [20
, 104
]. Cathelicidins are stored as proforms in the secondary granules of PMN (Fig. 1)
. Bactenecins are arginine-rich cathelicidin peptides, which are synthesized and stored as 10–20 kDa precursors in the large, tertiary granules of ruminant PMN [105
]. Bovine PMN also express indolicidin, a tryptophan-rich, linear, non--helical, antimicrobial peptide [106
]. Protein isoforms (15 kDa, p15s), derived from rabbit PMN, bear homology to cathelin but apparently do not require cleavage for their antimcirobial activity [107
]. Secretion of high concentrations of p15s into a rabbit peritoneal exudate [70
] is consistent with their localization in the secondary granules of PMN [108
] and enhances synergy with primary granule components, including BPI [89
]. Rabbits also express CAP18, an amphipathic, -helical cathelicidin peptide [109
]. Porcine PMN contain cathelicidin proforms of protegrins, broadly antimicrobial 2 kDa peptides composed of 18 amino acids possessing two internal disulfide bonds important for maintaining their "hairpin" structure and bioactivity [110
]. Humans express hCAP18/LL-37, a 19-kDa protein that is processed to the linear cationic peptide LL-37 upon cleavage by proteinase 3 in the extracellular space [88
] (Fig. 1)
. hCAP18 is bound to lipoproteins in plasma, accounting for the fact that relative to levels in circulating PMN, the plasma level of hCAP18 is >20-fold higher than that of other specific granule proteins [111
].
Cathelicidin-derived peptides express microbicidal activity against bacteria, fungi, viruses, and parasites. Tested in vitro, several cathelicidin-derived peptides are bactericidal toward P. aeruginosa in the presence of physiologic saline concentrations [112 ]. Protegrin-1 has broad-spectrum activity versus P. aeruginosa, S. aureus (including methicillin-resistant strains), and Enterococcus faecium [113 ]. Protegrin-1 and the ovine cathelicidin SMAP-29, a cathelicidin-derived antimicrobial peptide deduced from the N-terminal sequence of sheep myeloid mRNA, as well as cathelicidin-derived congeners novispirin and ovispirin are active in vitro and in vivo against the vector-borne African trypanosome parasite Trypanosoma brucei in a murine model [114 ]. The human cathelicidin LL-37 expresses antiviral activity in vitro and in vivo against the orthopox virus (vaccinia), which is used for immunization against smallpox [115 ]. The rationale for this study is based on the clinical observation that patients with atopic dermatitis, who are deficient in the expression of LL-37 [116 ], suffer a relatively high frequency of eczema vaccinatum, a complication of vaccinia immunization.
Additional roles have been identified for LL-37. Yang and co-workers [117
] discovered that LL-37 is a chemoattractant for human peripheral blood neutrophils, monocytes, and T cells via interactions with the formyl peptide receptor-like 1. Davidson and colleagues [118
] report that that LL-37, added to cell cultures at 50 µg/mL, modulates dendritic cell (DC) differentiation and DC-induced T cell polarization. LL-37 derived DC were found to have increased endocytic activity, modulation of phagocytic receptor expression, and enhanced release of T helper cell type 1 (Th1)-inducing cytokines (including TNF-) and were capable of generating an enhanced Th1 response. LL-37 can also transactivate the epidermal growth factor receptor to enhance epithelial IL-8 release [119
] and up-regulated expression of chemokines and CC chemokine receptors (CCRs) [120
]. Human LL-37/hCAP18 has been implicated as an agent important to the re-epithelialization of wounds [121
] and also demonstrates angiogenic activity in vitro and in vivo [122
, 123
].
Phospholipase
The 14-kDa group II PLA2 is a disulfide-rich enzyme that hydrolyzes phospholipids at the 2-acyl position. PLA2 was found to be an important cofactor in the digestion of Gram-negative bacteria by whole PMN [124
]. Studies of the PLA2 derived from intestinal Paneth cells identified direct calcium-dependent microbicidal activity against Listeria monocytogenes [125
]. Analysis of the microbicidal activity of an evolving inflammatory fluid derived from a rabbit peritoneal exudate model identified PLA2 as a remarkably potent agent with direct bactericidal activity at nM concentration against a range of Gram-positive bacteria, including methicillin-resistant S. aureus [126
]. The PLA2 is an acute-phase reactant, and studies of baboons injected with LPS demonstrate a brisk elevation of potent plasma PLA2 activity [127
].
Progress has been made in characterizing the mechanism by which PLA2 acts on target bacteria. The activity of PLA2 against S. aureus is promoted by cationic residues in the enzyme, which enhance binding to the bacterial cell wall [128 ]. Binding is followed by penetration of the enzyme to the cytoplasmic membrane, where the preference of PLA2 for anionic phospholipid interfaces, typical of bacteria as opposed to zwitterionic eukaryotic cell membranes, which may serve to target the enzyme’s specificity toward anionic bacterial membranes [129 ]. Rapid degradation of bacterial phospholipids and bacterial autolysin activation are believed to be important to the bactericidal effect of the enzyme. In accordance with this model, the action of PLA2 on Gram-positive bacteria such as S. aureus is modulated by the extent of bacterial cell-wall cross-linking and autolytic activity [22 ].
Defensins
Defensins are broadly microbicidal, cationic, 3–4 kDa peptides characterized by three conserved disulfide bonds, which express cytotoxic activity by the formation of multimeric pores in microbial membranes [26
]. Defensins have been identified as a broadly expressed family of innate-immune defense molecules expressed by leukocytes and epithelial cells. Humans express four -defensins, HNPs 1–4, in the primary (azurophilic) granules of PMN. -Defensins express broad-spectrum microbicidal activity at µM concentrations against bacteria, fungi, enveloped viruses (including HIV; ref. [130
]), and parasites. The microbicidal activity of -defensins is limited by physiological concentrations of mono- and divalent cations (i.e., is "salt-sensitive") as well as by some components of plasma/serum (e.g., albumin, 2-macroglobulin). Nevertheless, the relatively cationic, rabbit -defensins retain some microbicidal activity against encapsulated, serum-resistant E. coli when tested in citrated whole blood in vitro [89
]. Of note, although mice express defensins in mucosal epithelia, murine PMN lack defensins [131
].
The ß-defensins are characterized by a distinct pairing of cysteines and have a higher lysine-to-arginine ratio than the -defensins [26
]. Whereas bovine PMN express ß-defensins, expression of human ß-defensins is apparently restricted to epithelial cells, including those of the respiratory tract [132
]. Of note, some ß-defensins, such as human ß-defensin-3, retain antimicrobial activity in the presence of isotonic saline.
A third defensin subfamily, 
-defensins, was discovered upon characterization of the antimicrobial peptides of the rhesus macaque (Macaca mulatta) [133
]. -Defensins are synthesized by an unusual mechanism by which nonapeptide demi-defensin precursors are joined by head-to-tail splicing and are characterized by an internal trisulfide ladder. Two peptide precursors are combined to form three different microbicidal peptides [134
]. The cyclic conformation of -defensin confers relative salt insensitivity with respect to its microbicidal action [135
]. Of note, a mutation has silenced Homo sapiens gene expression such that in humans, the -defensin sequence is a pseudogene. Retrocyclin-1 is a hominid -defensin whose lectin-like properties allow it to bind O- and N-linked sugars on gp120 and CD4 with a dissociation constant (Kd) of 30–35 nM, thereby blocking infection of human PBMC by HIV-1 [136
, 137
].
It is increasingly appreciated that in addition to their direct microbicidal activity, defensins serve important immunoregulatory functions [138
]. In many instances, the potency of defensins with respect to immunomodulatory activities greatly exceeds that for direct antimicrobial activity. For example, at nM concentrations, human -defensins are selectively chemotactic for resting CD4/CD45RA and CD8 T cells [139
]. Human ß-defensins are chemotactic for immature DC and memory T cells via the human CCR6 [140
].
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EOSINOPHILS |
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Major basic protein (MBP) is 10–15 kDa cationic polypeptide aggregates, which form the crystalline core characteristic of eosinophil secondary granules. MBP is highly abundant, accounting for 50% of eosinophil granule protein content, and demonstrates antihelminthic (Brugia spp., Schistosoma mansoni, Trichinella spiralis), antiprotozoal (Trypanosoma cruzi), and antibacterial (e.g., E. coli, S. aureus) activities [23
, 141
].
Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) are 17–21 kDa proteins with homology to RNases. The highly cationic ECP demonstrates direct bactericidal activity, independent of its RNase activity [142 ], forms voltage-gated ion channels in target membranes [143 ], and possesses antiparasitic activity toward schistosomula of S. mansoni [144 ]. Rosenberg and colleagues [145 , 146 ] have found that eosinophils can mediate a direct, RNase-dependent reduction in infectivity of respiratory syncytial virus (RSV) in vitro and that EDN can function alone as an independent antiviral agent against RSV, a single-stranded RNA virus. EDN also demonstrates chemotactic activity toward DC [147 ].
Human eosinophils also express BPI, although its subcellular localization (specific granules) and amounts (25% of PMN BPI content on a cellular basis) are distinct from its expression in PMN [148
]. Lz is also found in eosinophils, as is the bOBP, a PGRP ortholog [78
].
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MONOCYTES AND MACROPHAGES |
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- and ß-defensin peptides, respectively [10
, 149
]. Murine macrophage-like RAW cells as well as resident peritoneal macrophages express arginine- and lysine-rich murine microbicidal proteins (MUMPs) with high primary homology to histones [150
]. MUMP-1, MUMP-2, and MUMP-3 demonstrated broad, in vitro microbicidal activity against S. typhimurium, E. coli, S. aureus, L. monocytogenes, Mycobacterium fortuitum, and Cryptococcus neoformans. Monocytes also express the cytosolic calprotectin [151
]. Recent studies suggest that monocytes can express -defensins [130
, 152
], but the mass amounts per cell remain to be defined. Chemokines are low molecular weight, cationic, disulfide-linked peptides secreted by monocytes, macrophages, and other leukocytes [153 ] with some general, structural similarities to defensins (i.e., cationicity and a high disulfide content) [154 ]. Whereas it has been demonstrated that defensins possesses chemoattractant activity (see above), Cole and co-workers [155 ] demonstrated the obverse, that chemokines have salt-sensitive, antimicrobial activity when tested in vitro against E. coli and L. monocytogenes.
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MAST CELLS |
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-defensin peptides (70–2500 nM) induce rapid degranulation of mast cells by a rapid G protein-dependent mechanism [158
]. Defensins and cathelicidins induce mast cell histamine release and prostaglandin D2 synthesis [159
]. |
LYMPHOCYTES |
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T cells and NK cells, express and secrete -defensins (HNP 1–3), the cathelicidin peptide LL-37, Lz, and a fragment of histone H2B. |
NONLEUKOCYTE SOURCES OF APP |
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APP MODULATE THE INFLAMMATORY ACTIVITY OF MICROBIAL PRODUCTS |
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1000-fold greater than that of the most potent (cationic) rabbit -defensins and the rabbit cathelicidin peptide p15 [188
]. This study also revealed that different cationic APP may bind different regions of the LPS molecule in that unlike BPI, -defensins and p15s did not inhibit long-chain ("smooth-type") LPS effectively. Remarkably, proCAP18 demonstrates extremely potent endotoxin-neutralizing activity at subnanomolar concentrations, showing a molar potency more than 100-fold greater than that of the CAP18 peptide (20 nM) or BPI (50 nM) [184
]. The human cathelicidin-derived peptide LL-37 inhibits macrophage stimulation by LPS, LTA, and noncapped mycobacterial lipoarabinomannan [120
]. Oral administration of Lf to germ-free piglets reduces mortality induced by intravenous (i.v.) LPS, apparently by translocation of Lf to the systemic circulation, binding of systemic LPS, and reducing its interaction with porcine monocytes [190
].
It is interesting that Lf has been reported to enhance LPS-induced release of IL-6 [55
], a cytokine with anti-inflammatory effects. Remarkably, Azu alone (10 µg/ml) stimulated the production of TNF- from isolated monocytes and has an additive effect on LPS-induced production of TNF- [179
]. Monocytes bind and internalize Azu via an as-yet undefined mechanism.
The ability of serprocidins to modulate the inflammatory potential of microbial products is increasingly appreciated. NE, at relatively low concentrations, selectively degrades virulence factors of Shigella flexneri, S. typhimurium, and Yersinia enterocolitica [178 ], as well as bacterial flagellin [191 ]. Lf may enhance degradation of microbial products by altering the conformation of bacterial virulence proteins secreted by S. flexneri, thereby rendering them more susceptible to proteolytic degradation [176 ].
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APP INTERACT WITH THE TLR PATHWAY |
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Calprotectin release from neutrophils in response to P. gingivalis LPS occurs via the CD14-TLR2-nuclear factor-
B pathway [192
]. LPS-induced expression of ß-defensin-2 expression in human tracheobronchial epithelium occurs via a CD14-dependent pathway [193
]. Upon exposure to LPS, monocyte-derived cells secrete IL-1, a cytokine that signals via a TLR-related receptor/pathway system, thereby greatly amplifying human epidermal expression of ß-defensin [194
]. Bacterial lipopeptide acts via TLR-2 to induce ß-defensin-2 expression in human airway epithelial cells [195
]. Intestinal infections with Shigella spp. are associated with down-regulation of epithelial defensin and cathelicidin peptide expression [196
]. Shigella-derived plasmid DNA was able to replicate this inhibitory effect, raising the possibility that DNA of this intestinal pathogen is able to engage the innate-immune recognition system (via TLR9?) to reduce expression of APP.
Some APP apparently activate TLRs and cytosolic pathways downstream of TLRs. Study of the potential inflammatory role of serprocidins in human disease has prompted evaluation of the effect of these protease homologues on cytokine synthesis. NE induces IL-8 synthesis in bronchial epithelial cells via a MyD88/IL-1 receptor-associated kinse/TNF receptor-associated factor-6 pathway [197 ]. Human ß-defensin-2 apparently directly activates immature DC via TLR4, inducing up-regulation of costimulatory molecules and DC maturation and thereby type 1-polarized adaptive-immune responses [198 ].
Much remains to be learned about the interactions of APP and TLRs, including evaluation of the possibility that particular patterns of TLR activation may serve to tailor the innate-immune response to specific pathogens.
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DEFINING THE ROLE OF APP IN VIVO |
|---|
-defensins and cathelicidins act in synergy with BPI to inhibit growth and TNF- induction by encapsulated E. coli K1/r [89
]. Human and guinea pig defensins are synergistic with CAP18/LL-37 in permeabilizing the outer and inner membranes of E. coli ML-35p [200
]. In addition to the complex, granule-based arsenal PMN deploy against microbes at sites of infection, soluble components of plasma that may bind the microbe prior to ingestion, such as complement components and PLA2, can impact subsequent intracellular killing and digestion [201
, 202
]. One means of determining the contribution of individual APP to the activity of complex biologic fluids involves depletion of activity with neutralizing antibodies or selective inhibitors. For example, addition of a neutralizing anti-BPI serum substantially reduces activity of rabbit ascitic fluid against E. coli [70 ], a neutralizing anti-PLA2 serum blocks microbicidal activity of a sterile rabbit peritoneal exudate against S. aureus [126 ], inhibiting NE with the active site inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone blocks proteolytic activation of cathelicidin-based protegrin peptides and reduces microbicidal activity in neutrophil secretions [203 ], and addition of zinc, an antagonist of calprotectin, reduces the anti-Candidal activity of empyema fluid [204 ]. Although these well-designed studies are highly suggestive of a role for APP in host defense, they fall short of proving their importance in vivo.
More definitive answers may be obtained in murine models that are amenable to genetic manipulation. Whereas mice deficient in NE demonstrate enhanced susceptibility to Gram-negative bacterial sepsis [80 , 205 ], those deficient in PLA2 are more susceptible to infection by S. aureus [206 ]. Reeves and co-workers [82 ] evaluated the susceptibility of NE- and/or CatG-deficient mice to microbial infection and found that the former demonstrated increased susceptibility to infection by C. albicans, and the latter were more susceptible to S. aureus. In both instances, increased susceptibility to infection-induced mortality correlated with reduced activity of NE- or CatG-deficient PMN tested against these pathogens in vitro. Mice rendered deficient in the cathelicidin-related antimicrobial peptide demonstrate increased susceptibility to epithelial infection with Group A Streptococcus [207 ].
There are some human conditions and diseases characterized by susceptibility to infection in which the expression of APP is reduced. Humans with defects in the gene encoding NE suffer cyclic or severe congenital neutropenia (Kostmann syndrome) as a result of maturational arrest of promyelocytic development in the bone marrow, suggesting that NE regulates myelopoiesis [208
]. PMN of patients with Kostmann syndrome (under treatment with G-CSF) are deficient in cathelin-LL-37 and -defensins HNP 1–3 yet have normal content of Lf and normal oxidative burst, suggesting that deficiency of antimicrobial peptides in such patients may contribute to their risk of infection [209
]. Specific granule deficiency is a rare genetic disorder characterized by recurrent bacterial infections, which are caused by mutations in the gene encoding the transcription factor CCAAT/enhancer-binding protein (
) [210
]. Neutrophils derived from patients with specific granule deficiency lack defensins [211
] but also lack other PMN proteins and exhibit alteration in the properties of monocytes [212
]. PMN of human newborns, whose immunologic, functional immaturity renders them at high risk of invasive microbial infection [213
], are selectively deficient in BPI but not in other granule components such as defensins or myeloperoxidase, and BPI levels are three- to fourfold lower than those of adults [214
, 215
]. Although diminished expression of APP in these disorders may well contribute to susceptibility to infection, the pleiotropic nature of these disorders precludes firm conclusions at this time.
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ROLES OF APP IN HUMAN DISEASE |
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Clp levels are elevated in meconium passed by preterm and low birth weight neonates, as well as those with some degree of perinatal asphyxia, reflecting immaturity of and ischemic damage to the intestinal mucosa [220 ]. Accordingly, Clp is significantly elevated in stool derived from neonates with necrotizing enterocolitis, raising the possibility that it may have use as a clinical marker for this life-threatening disease process [221 ].
There is evidence in that in some disease states, APP may be normally expressed but functionally inhibited. For example, the elevated osmolarity of the fluid overlying the respiratory tract epithelium in patients with cystic fibrosis (CF) may block activity of local, salt-sensitive APP (e.g., ß-defensins) and thereby contribute to the susceptibility of these patients to chronic pulmonary infections [222 ]. However, the tonicity of airway surface fluid is a matter of contention, and this interesting hypothesis remains to be proven [223 ]. APP, in particular, BPI and the serprocidins, are targets of autoantibodies in a variety of diseases. Azu and PR3 are targets of antineutrophil cytoplasmic antibodies (ANCA) in patients with systemic vasculitides, including Wegner’s granulomatosis [224 , 225 ]. Patients with CF, inflammatory bowel disease, vasculitis, and primary sclerosing cholangitis often develop ANCA against BPI [226 ]. The presence of BPI-ANCA in such patients is associated with higher inflammatory disease activity and organ damage. Sera of CF patients, which contain anti-BPI ANCA, inhibit neutrophil killing of P. aeruginosain vitro [227 ], raising the possibility that the development of anti-BPI ANCA may contribute to susceptibility of CF patients to bacterial infection.
Zhang and co-workers [228
] reported that human -defensins HNP 1–3, from leukocyte cultures enriched in CD8+ T cells, may play important roles in preventing clinical progression of HIV-positive patients to AIDS. Although they later found that neutrophils from allogenic feeder cells accounted for the defensin peptide content, the anti-HIV activity of defensins was confirmed [229
].
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BIOPHARMACEUTICAL DEVELOPMENT OF APP |
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|
Activity against microorganisms with high degrees of resistance to conventional antibiotics is an attractive feature of APP [126 , 238 ]. A topical formulation of a congener of the porcine leukocyte antimicrobial peptide protegrin (IBD-367, IntraBiotics) is currently in clinical development [239 ]. A phase III trial of IBD-367 for the prevention and treatment of mucositis in patients undergoing myeloablative chemotherapy demonstrated trends toward improvement (P=0.1) but did not achieve statistical significance. An aerosolized formulation of IBD-367 is currently being evaluated as an agent to prevent ventilator-associated pneumonia in an international, multicenter phase III clinical trial. A topical formulation of a congener of the cathelicidin peptide indolicidin (omiganan/MBI 226, Micrologix) recently completed a phase-III trial toreduce catheter-related infections with some evidence of benefit with respect to reduction of catheter colonization and local catheter site infections [240 ]. Based on its activity against Propionibacterium acnes and other skin flora, a topically applied BPI-derived peptide, XMP.629, is currently in phase II clinical trial as a novel treatment for acne.
Systemic administration
The endotoxin-neutralizing activity of BPI, based on its high-affinity binding to LPS (Kd, nM), is manifest in biologic fluids, including whole human blood, thereby rendering it an attractive target for biopharmaceutical development [241
]. Recombinant N-terminal fragments of BPI, including a 21-kDa fragment (rBPI21 or Neuprex, XOMA U.S. LLC), have demonstrated antiendotoxic and antibacterial activity in animal models of bacteremia [242
] and safety, nonimmunogenicity, and efficacy with respect to endotoxin neutralization in phase I clinical studies of human volunteers [243
, 245
]. rBPI21 has been evaluated in a number of clinical indications in which Gram-negative bactermia and/or endotoxemia are believed to play a role. Perhaps the most compelling indication thus far has been as an adjunctive therapy for patients with fulminant sepsis as a result of Neisseria meningitides (
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ABSTRACT
INTRODUCTION