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Department of Biomedical Science and Biotechnology, University of Udine, Italy
Correspondence: Angela Risso, Department of Biomedical Science and Biotechnology, University of Udine, p.le Kolbe, 4, I-33100 Udine, Italy. E-mail: ARisso{at}makek.dstb.uniud.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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Key Words: natural antibiotics • innate and adaptive immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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In mammals, neutrophils, macrophages, and natural killer cells are primarily involved in these mechanisms. They use a variety of proteins and peptides as effector molecules that are able to kill or to inactivate microbial pathogens.
Proteins of complement cascade, a variety of lectins, polypeptides present in neutrophil granules, such as the iron-binding lactoferrin, the bactericidal permeability-increasing protein, secretory phospholipase A2, serprocidins and lysozyme, are endowed with microbicidal activity. Comprehensive reviews on these subjects have recently been published [2 , 3 ].
This review focuses on mammalian leukocyte antimicrobial peptides, molecules with a molecular size lower than 10 kDa, which have aroused interest for their variety in structure and in sequence, and their efficacy in killing microorganisms such as bacteria, fungal cells, or enveloped viruses.
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ANTIMICROBIAL PEPTIDES: GENERAL FEATURES AND SITES OF PRODUCTION |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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These natural antibiotics lack the antigen recognition specificity of the antibody molecules but are rapidly delivered from innate immunity cells after an infection. Then, in innate immune response, the lack of immunological memory and of specific interaction with foreign pathogens, which is typical of adaptive immunity, is counterbalanced by the ability to neutralize a microbial infection in a short time [1 ].
Recent studies have shown that some mammalian defense peptides have regulatory or triggering functions on antigen-driven immune responses, in addition to antimicrobial properties. These observations have contributed to increased interest in such molecules and to shaping the notion of an interplay between innate and acquired immunity [4 , 5 ].
The anatomical sites of production and storage of antimicrobial peptides in mammals are related to the immediate response that they provide to invading microbes. Antibiotic peptides are present in tissues exposed to microbes such as mucosal surfaces and skin [6 7 8 ] and in cytoplasmic granules of professional phagocytes [9 ].
Of the leukocytes, polymorphonuclear cells are the major sources of antimicrobial peptides. This is reflected in the early investigations on these molecules, which were performed by isolating the peptides from cytosolic extracts of neutrophils from a number of species [1 ].
Since then, several reports on antimicrobial peptide synthesis and storage have shown that they are expressed as propeptides in bone marrow polymorphonuclear precursors and are stored in neutrophil granules, from which the mature peptides are released by proteolytic cleavage of the prodomain [10 11 12 ].
All three types of granules, azurophils, specific, and secretory, may be the storage compartments of the propeptides. Storage in a specific type of granule has been reported for some pro-forms [3 ]. On bacterial infection, the mature peptides are released to the microbes engulfed within the phagosome [10 , 13 14 15 ] or can be secreted in extracellular fluids such as the ascitic fluid or the inflammatory exudates [16 , 17 ].
The synthesis and compartmentalization of antimicrobial peptides in macrophages is still a matter of investigation, with the exception of some studies of rabbit defensins. These peptides have been found to be localized in alveolar macrophage phagosomes, where their expression could be up-regulated by immune stimulation with complete Freund’s adjuvant [18 , 19 ].
The reports on natural killer (NK)-lysin [20 ], an antimicrobial and tumorolytic peptide present in porcine cytotoxic T lymphocytes (CTL) and NK cells, and granulysin [21 ], a microbicidal peptide of human CTL and NK, have shown that antibiotic peptides are components of the cytotoxic arsenal of these cells. These molecules are likely to be inducible in vivo during activation and differentiation of mature T lymphocytes and NK, as suggested by the data on NK-lysin. Synthesis of this peptide was greatly increased on in vitro stimulation of pig T and NK cells with interleukin-2 [20 ].
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CLASSIFICATION OF ANTIMICROBIAL PEPTIDES |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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-helices or ß-sheets. In 1995, Boman proposed a classification of animal peptide antibiotics based on structural similarities [1 ]. This study has recently been reviewed by the author on the basis of the new peptides described in the past 5 years [22 ]. The interested reader can find an updated list of sequences of gene-encoded peptides in the database by Tossi [23 ].
Two classes of peptides can be recognized according to the presence or absence of intramolecular disulfide bonds.
One group of peptides without disulfide bridges consists of linear,
mostly -helical peptides, such as human FALL-39, subsequently named
LL-37/hCAP-18 [24
], bovine BMAP-27 and BMAP-28
[25
], porcine PMAP-36 [26
], and PMAP-37
[27
]. Another group, also without disulfide bridges,
includes peptides characterized by a high content of one or two amino
acids such as proline and arginine, overexpressed in bovine Bac 5 and
Bac 7 [28
], and in porcine PR39 [29
], or
tryptophan-rich molecules such as bovine indolicidin
[30
] and porcine PMAP-23 [31
].
The cysteine-containing class has been divided into different groups according to the number of intramolecular disulfide bonds.
There are relatively few peptides with one or two disulfide bonds. Among mammalian species, cyclic dodecapeptide from bovine granulocyte [32 ] and protegrins from porcine neutrophils [33 ] belong to the one-bond group and to the two-bond group, respectively.
Three disulfide bonds are the structural feature characterizing the
defensin family [34
], which includes two subfamilies
designated - and ß-defensins. The two subgroups differ primarily
in their size, -defensins containing 29–35 residues
[34
] and ß-defensins exhibiting 34–42 residues
[35
]. Furthermore, they differ in the position of their
cysteine residues and of the disulfide bridges [35
].
Equine eNAP-1 and eNAP-2 are cysteine-rich peptides belonging to the granulin family [36 ] and to the four-disulfide core peptide family [37 ], respectively. They contain four disulfide bonds and show no amino acid sequence similarities with defensins.
The two peptides found in CTL and NK cells, NK-lysin [20 ] and granulysin [21 ] are also unrelated to the defensin family. They show structural homology in amino acid sequence, in positioning of the cysteines, and in secondary structural motifs with amoebapores, pore-forming peptides of the protozoan parasite Entamoeba hystolytica [38 ] and with saposins, small lipid-associated proteins present in the central nervous system [39 ].
In addition to the structural classification of animal defense peptides, studies of the sequences and expression of mammalian peptides and of their proforms in different species have enabled the identification of two major families: the defensins [34 , 35 ] and the cathelicidins [40 ]. The defensins form the largest group of mammalian peptide antibiotics and, as mentioned above, are characterized by a similar structural conformation and a three-disulfide motif in mature forms. The cathelicidin-derived peptides share a conserved prosequence in the precursor polypeptides. In contrast, the antibacterial domains are highly variable both in sequence and in structure and can be assigned to the structural groups listed above (Table 1 ).
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DEFENSINS AND CATHELICIDINS |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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- and ß-defensins, have been described [34
,
35
].
-Defensins have been found in human [43
], rabbit
[44
], guinea pig [45
], rat
[46
], macacque [47
], and hamster
[48
] neutrophils, in rabbit alveolar macrophages
[49
], and human [50
] and rodent
[51
, 52
] small-intestinal Paneth cells. The
active peptides contain 29–35 amino acid residues, including six
invariant cysteines and five other residues that are highly conserved
among the various members of the family. Nuclear magnetic resonance
(NMR) spectroscopy and X-ray crystallographic analysis conducted on
several human and rabbit defensins have shown that they share a common
conformation, consisting of an amphiphilic triple-stranded ß-sheet
stabilized by three intramolecular disulfide bonds [34
].
The amino- and carboxy-terminal residues lie on the polar face of the
structure and are crucial determinants of microbicidal potency and
target specificity [53
].
In neutrophils, -defensins are stored as processed mature peptides
in the azurophil granules. In human neutrophils, four defensins (HNP-1,
human neutrophil peptide-1, -2 -3, and -4) have been described. HNP-1,
-2, and -3 account for 5% of the total cellular protein and 30–50%
of the azurophil granule protein [34
]. Other human
-defensins are enteric human defensins (HD) -5 and -6 expressed by
small intestinal Paneth cells [50
].
Like most antimicrobial peptides, defensins are synthesized as 93–96 amino acid prepropeptides containing a 19-residue signal sequence and a 40- to 45-residue anionic proregion. The prodomain is followed by the antimicrobial peptide, which is generated from the precursor molecule by proteolytic processing [34 ].
ß-Defensins have been found in bovine tracheal mucosa [54 ], bovine neutrophils [35 ], bovine tongue epithelium [6 ], and human epithelia [55 ]. A novel ß-defensin has been identified in human plasma but its cellular source has not been identified [56 ].
The similarity in structure and amphiphilicity of - and
ß-defensins is reflected in their broad spectrum of activity
[34
, 35
, 57
].
When used in in vitro assays at micromolar concentrations,
defensins kill a wide variety of gram-positive and gram-negative
bacteria and fungi [see refs. 6
34
35
53
] (Table 2
). In addition, some defensins are effective against enveloped
viruses, such as herpes simplex [34
]. In general,
defensins require low salt concentrations for their activity, which is
enhanced in the absence of Ca2+ or Mg2+
[34
]. Studies of antimicrobial mechanisms of defensins
have shown that they permeabilize the outer and inner membranes of
Escherichia coli. After initial electrostatic binding to
negatively charged molecules (head groups of polar membrane lipids) on
the target cell surface, they insert into the energized cell membrane
and seem to form multimeric ion-permeable channels in a
voltage-dependent manner [34
]. This is consistent with
the observation that metabolically active bacterial cells are more
susceptible to human -defensins than resting microbes
[58
].
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-defensin peptides have
revealed that, although the preproregion is highly conserved, there is
some diversity among the primary sequences of antibiotic peptides
[34
].
Differences have been found in the middle portion and in the amino- and
carboxy-terminal residues of -defensins. These have been related to
their different spectra of activity. For example, a difference in a
single amino acid at the amino terminus was reported to strongly
decrease the candidacidal activity of HNP-3 in comparison with HNP-1
and -2. The functional relevance of the amino- and carboxy-terminal
portions of defensins was confirmed by means of synthetic analogs of
human neutrophil defensins [53
].
A remarkable variability in the antimicrobial domains has also been found in the cathelicidin family of antimicrobial peptide precursors, where the highly conserved amino-terminal propieces (99–114 residues long) are followed by carboxy-terminal antimicrobial regions that are highly variable both in sequence, length (12–100 residues), and structural motifs [40 ]. The precursors have been identified in cows [25 ], pigs [26 , 27 ], rabbits [59 ], and humans [24 ]. More recently, they have been identified in mouse [60 , 61 ], rat [62 ], sheep [63 ], and horse [64 ] myeloid cells. The propiece sequence shows similarity with sequences of cystatin superfamily members, proteins known to inhibit cysteine proteinases. It has been suggested that the prodomain may play a role in targeting or in assisting the correct maturation of the peptides [40 ].
Cathelicidins are synthesized during differentiation of bone marrow myeloid cells as prepropeptides, and have been found as proforms in the large granules of bovine neutrophils and in the specific granules of human neutrophils [40 ]. Studies on two bovine cathelicidins, proBac 5 and proBac 7, have clarified the processing and the delivery pathway of the mature peptides [12 , 40 ]. These two propeptides lack antimicrobial activity, probably because the anionic propiece causes inactivation of the cationic carboxy-terminal portion. Upon cleavage of the amino-terminal domain by elastase at a valyl residue, the mature peptides are either discharged into phagocytic vacuoles or released extracellularly. Because most cathelicidins have a valyl residue in a corresponding position, they may undergo a similar cleavage by elastase [40 ].
Sequencing of cathelicidin cDNAs has allowed the chemical synthesis and
biochemical studies of the corresponding antimicrobial peptides whose
sequences were deduced from cDNAs encoding for the precursors. From
analysis by circular dichroism spectroscopy, an amphiphilic -helical
conformation has been suggested for some peptides such as those listed
in Table 1
. A poly(L)-proline type II structure has been
proposed for the proline-arginine-rich peptides PR-39
[65
] and Bac-5 [66
]. This conformation
seem to be biologically active for the candidacidal activity of Bac-5
[67
]. The characterization of the structure and
antimicrobial activities of synthetic peptides has shown that, although
highly variable in sequence and structure, they share a wide spectrum
of action against gram-positive and gram-negative bacteria and fungi
(see Table 2
). Anti-HIV-1 activity by indolicidin, a bovine
cathelicidin-derived peptide, has also been described
[68
].
Like the defensin family, cathelicidins are also membrane-active molecules, as is shown by their ability to permeabilize the bacterial cell wall and inner membrane [40 ]. However, the cytotoxic mechanism of some cathelicidin-derived peptides is different because PR-39 inhibits bacterial DNA synthesis before membrane permeabilization is detectable [22 ] and Bac-5 and Bac-7 cause a rapid decrease in RNA and protein synthesis [40 ].
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INTERACTION OF ANTIMICROBIAL PEPTIDES WITH TARGET CELLS AND RARE OCCURRENCE OF RESISTANT STRAINS |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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Similarly, it has been proposed that effector molecules of innate immunity, such as antimicrobial peptides, can interact with the anionic phospholipids of the bacterial inner membrane or with the LPS of the cell wall of gram-negative bacteria by means of their structure and positive charge. Interaction takes place in a relatively nonspecific manner, which can be rationalized on the basis of biophysical principles [70 , 71 ]. This feature ensures a broad spectrum of action and good efficacy of the peptides in killing infectious agents. In in vitro assays, minimal inhibitory concentrations against a panel of bacteria range between 0.5 to 10 or 100 µM for most peptides (Table 2) [22 ].
The interaction and insertion within the bacterial (or fungal) cell wall and inner membrane have been thoroughly investigated by means of membrane mimetic systems such as artificial lipid bilayers [72 73 74 75 ]. In most cases the initial step of insertion is associated with membrane damage. Such damage has been observed by means of standard assays where peptides were shown to rapidly permeabilize the inner membrane of a susceptible E. coli strain [58 ]. The membrane alteration is followed by the uptake of normally excluded molecules and by leakage of essential ions and metabolites [34 ].
The interaction with target cells, involving recognition of molecular pattern and ruling out any specific receptor, can account for the rare occurrence of resistant strains because resistance implies mutations in outer and inner membrane, namely in structures that are relatively invariant [1 , 76 , 77 ].
Indeed, the reported resistance to antibacterial peptides in Salmonella (gram-negative) and Staphylococcus aureus (gram-positive) strains has been related to variations in chemical composition of LPS (the major cell surface molecule of gram-negative bacteria) and teichoic acids (present in gram-positive cell wall), respectively [78 , 79 ].
On the basis of the above features, antimicrobial peptides have attracted the interest of pharmacological companies as potential novel antibiotics. These observations have also prompted studies by biotechnological companies aimed at synthesizing newly modified molecules that could be more potent than the natural peptides.
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MULTIPLE ROLES OF ANTIMICROBIAL PEPTIDES |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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In intact organisms, peptide concentrations reach significant levels in the sites of their production and release, where they probably synergize with each other in clearing pathogens, as indicated in a recent study by Nagaoka et al. [82 ]. Some peptides can be present in granulocytes in very high quantities, as has been reported for human defensins (5 µg/106 human granulocytes [34 ]) and for Bac-5 and Bac-7 (125 ng/106 bovine granulocytes [83 ]).
It is likely that these concentrations and the possible synergistic action contribute to overcome the above-mentioned inhibitory effects.
The apparent redundancy of antimicrobial peptides can also be related to the pleiotropic activity of some of these molecules which, in addition to microbicidal activities, exert other defense-related functions. Individual antimicrobial peptides appear to be characterized by multiple roles, which may account for the great variety of this class of molecules.
Peptides from both mammalian families (defensins and cathelicidins) are
multifunctional. -Defensins, with their chemotactic functions on
monocytes [84
], are involved in inflammation and may
exert an activation and potentiation role in adaptive immune response.
The human defensin HNP-1 is a chemoattractant for human monocytes
in vitro at concentrations as low as 5 x
10-9 M, supporting the notion that this activity could be
dependent on interaction with a specific receptor [84
].
Human defensins HNP-1 and HNP-2 are also chemotactic for human and murine T cells, playing a recruitment role in the antigen-specific response [34 ]. A recent report has shown that the presence of human neutrophil defensins in mucosal epithelium may enhance the systemic adaptive immune response and increases antigen-specific IgG and IgM levels in serum [85 ]. Moreover, the ability of ß-defensins to induce chemotaxis for dendritic and memory T cells through the specific chemokine receptor CCR6 [86 ] demonstrates the recruitment role played by defensins on adaptive immune effector cells.
The human defensin HNP-2 is able to inhibit fibrinolysis by modulating tissue-type plasminogen activator and plasminogen binding to fibrin and endothelial cells [87 ]. This may have important implications for the pathophysiology of vessel walls, in particular for the thrombotic pathology.
Defensins are released in the circulation, where they may reach high levels in pathological conditions, and they adhere to the endothelium, as shown by their immunohistochemical localization in human coronary vessels [88 ]. Then, defensins present in the vascular tissue may contribute to the pathological consequences of inflammation, in addition to playing the chemotactic and recruitment roles mentioned above.
Among other properties of defensins, the specific interaction of rabbit defensin NP-3a and human HNP-4 with the ACTH receptor of rat adrenal cells in vitro was reported. This effect inhibited ACTH-induced steroidogenesis in vitro [34 ].
-Defensins are mast-cell-agonistic, acting at nanomolar
concentrations as potent secretagogues. This effect is mediated by a G
protein-dependent response, which was shown to be distinct from the
antigen-IgE-mediated activation [89
].
Finally, a report on the inhibition of C1q hemolytic activity by HNP-1 indicates that defensins can control the classical complement pathway [90 ].
Among cathelicidin-derived peptides PR-39, a porcine proline-arginine-rich molecule, has been assigned to a growing list of functions, which together play a modulating role during inflammation.
PR-39 induces cell surface syndecan-1 expression in mammalian mesenchymal cells during wound repair. Because syndecan-1 is a transmembrane proteoglycan involved in cell-to matrix interactions, the peptide can influence cell migration in injured tissues [91 ]. The altered invasiveness and the abnormal actin structure of hepatocarcinoma cells transfected with the PR-39 gene, were related to the interference of PR-39 with induction of syndecan-1 [92 ]. Furthermore, the peptide can inhibit production of superoxide anions mediated by neutrophil NADPH oxidase, thereby influencing the oxidative killing mechanisms of neutrophils [93 ]. These activities were related to the signaling pathway triggered by PR-39. Like other proline-rich peptides possessing a poly(L)-proline type II conformation [94 ], PR-39 is able to bind recombinant proteins containing Src homology 3 (SH3) domains, and the inhibition of NADPH oxidase was attributed to PR-39 interaction with SH3 domains of p47phox [93 ].
Furthermore, an amino-terminal fragment of PR-39 was shown to gain access to the cytosol of endothelial cells and to interact with an SH3-containing protein, p130Cas, causing a redistribution of this molecule from cytosol to cytoskeleton. Because p130Cas is involved in multiple signaling pathways, this observation could explain the cell-surface syndecan expression induced by PR-39 [95 ].
In addition to its regulatory function on oxygen-dependent activities of neutrophils, PR-39 may affect the role played by these cells in inflammation in a more general way because it was shown to act as a chemotactic agent on polymorphonuclear cells. This effect was associated with a transient Ca2+ flux into the cells. The chemotaxis mediated by the peptide was observed at concentrations lower than the minimal bactericidal doses (0,5–2 µM) and was abrogated in the absence of extracellular Ca2+ [96 ].
A modulating role in the immune response has been assigned to other cathelicidin-derived peptides, especially in relation to the LPS binding activity of some members of this group.
CAP-18, a rabbit cathelicidin peptide, is able to inhibit LPS-induced release of cytokines and nitric oxide from macrophages, LPS-induced LAL (Limulus amebocyte lysis assay) coagulation and to protect mice from endotoxin-mediated lethality [97 ]. Like rabbit CAP18, LL37/hCAP18 is also able to inhibit LPS-induced nitric oxide release from macrophages and LPS-mediated lethal effect on mice [98 ].
Recently, cationic antimicrobial peptides with different secondary structures have been reported to block the binding of LPS to LPS-binding protein. This effect may explain their ability to inhibit LPS-induced tumor necrosis factor production by a macrophage cell line [99 ].
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CYTOTOXICITY OF ANTIMICROBIAL PEPTIDES ON MAMMALIAN CELLS |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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NK-lysin has a cytolytic activity against the murine tumor cell line YAC-1 in low serum concentration [20 ].
The defensins HNP-1,-2, and -3 from human neutrophils, the major antimicrobial peptides expressed by human neutrophils, and five defensins from rabbits are toxic for human and murine tumor cell lines at micromolar concentrations [100 ]. There is no target selectivity for transformed cells because human defensins also lyse human lymphocytes, endothelial cells, as well as murine lymphoid cells [34 ]. The cytotoxic mechanisms include an early step of membrane binding, which is inhibited by the presence of serum, low temperature, or addition of heparin [34 ], and is followed by an energy- and cytoskeletal-dependent series of events leading to cytolysis [101 , 102 ]. The toxic activity of defensins probably contributes to the nonoxidative injury of targets by polymorphonuclear cells in antibody-dependent cell cytotoxicity (ADCC) [34 ].
Recently, two members of the cathelicidin family, named BMAP-27 and
BMAP-28, have been reported to be cytotoxic to mammalian cells. They
are two similar bovine peptides of 26 and 27 amino acids with an
amidated carboxy-terminal glycine, which assume an -helix
conformation in a hydrophobic environment [25
]. These
peptides exhibit a toxic activity to several bacterial and fungal
strains. They are also cytotoxic to transformed cell lines, fresh
hematopoietic tumor cells, and normal proliferating, but not resting,
lymphocytes at microbicide-level concentrations (3–6 µM)
[103
]. Cyclic dodecapeptide and indolicidin, which are
also cathelicidins, are cytotoxic to T cell lines and neuronal cells,
but only in the absence of serum [104
,
105
]. In contrast, the activity of BMAP peptides is not
inhibited by the presence of 10% serum. Toxicity is primarily due to
damage of plasma membrane integrity associated with a Ca2+
influx into the cytosol. This effect is followed by apoptosis of the
majority of the cells, both in the transformed U937 cell line and in
in vitro activated human lymphocytes. The toxic activity of
both peptides requires an active metabolism of target cells and is also
dependent on sialyl residues of membrane glycoproteins, as U937 cells
treated with neuraminidase are resistant to the toxic effect
[103
].
Because the sialylation of glycoproteins is increased on activated lymphocyte membrane [106 , 107 ], an initial interaction of the peptides with these negatively-charged residues could account for their selectivity to stimulated versus resting lymphocytes. It is likely that their release by activated granulocytes can provide an easy and safe disposal of aged, unwanted cells present at inflammation sites, such as activated lymphocytes.
By inducing apoptosis of these target cells, the peptides could contribute to regulating late immune response. This has already been shown for apoptosis triggered by the interaction Fas-Fas ligand in the context of antigen-driven immunity [108 ]. Furthermore, BMAP peptides could induce active death of transformed cells during ADCC mediated by neutrophils.
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CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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These investigations have also prompted researchers and pharmaceutical companies to use the natural antibiotics as templates for novel antimicrobial drugs. The use of synthetic peptides identical or similar to those that exist in nature has already provided promising results in clinical trials of therapy against infectious diseases [110 ].
Recent data on antimicrobial peptides in mammals have provided new insights into their roles. It seems that in this vertebrate class, an antigen-specific, clonally based immune system is flanked by multifunctional innate immunity peptides, which have acquired the ability to modulate the inflammatory response or the antigen-specific immune response by mediating recruitment of antigen-presenting cells (dendritic cells, monocytes) and T cells (defensins) or by inhibiting LPS-mediated cytokine release and inducing apoptotic death of activated lymphocytes (cathelicidins).
It is possible that these functions are relevant in vivo in sites of microbial invasion where antimicrobial peptides, while playing a bactericidal or bacteriostatic role, contribute to enhancing the mechanisms of cellular innate resistance and set the stage for the activation and modulation of specific immunity.
Future challenges will be to investigate the possible interaction or cooperation of antimicrobial peptides with cytokines and chemokines in these modulating roles. Furthermore, a physiological approach, involving animal models, will help to better define their profiles of biological activity and will clarify to what extent the antibiotic peptides are endowed in vivo with the multifunctional properties discovered in in vitro experimentation.
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ACKNOWLEDGEMENTS |
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Received August 7, 2000; revised September 24, 2000; accepted September 26, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONANTIMICROBIAL PEPTIDES: GENERAL...CLASSIFICATION OF ANTIMICROBIAL...DEFENSINS AND CATHELICIDINSINTERACTION OF ANTIMICROBIAL...MULTIPLE ROLES OF ANTIMICROBIAL...CYTOTOXICITY OF ANTIMICROBIAL...CONCLUDING REMARKSREFERENCES |
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tossi/search.htlm ,
6N-acetylglucosaminyltransferase up-regulation and sialyl-Le(X) expression during activation of human tonsillar B lymphocytes FEBS Lett 4631,25-28
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