Pr39

tryptophan: ILPWKWPWWPWRRNH2

Indolicidin asparlic acid: DDDDDDD, GDDDDDD

Anionic Peptides

Figure 1. Chemical structural classification of some well-studied mammalian antimicrobial peptides (modified from [3] with permission).

cursor molecules are prepropeptides containing a signalling pre- sequence, a neutralizing propeptide and the carboxy-terminal defensin that is liberated following multiple processing steps. Nearly all mammalian defensins contain six cysteine molecules participating in three disulfide bonds. They can be sorted broadly into three groups based on cysteine spacing and disulfide-bond alignment (Fig. 1).

The a-defensins are generally shorter in length and contain disulfide bridges in a {C1-C6, C2-C4, C3-C5} pattern, while the (3-defensins are more variable in length at both termini and contain disulfide bridges in a {C1-C5, C2-C4, C3-C6} pattern. The core tertiary structure of a- and P-defensins is considered to be a triple-stranded antiparallel [3-sheet [7], The third group of defensins, 6-defensins, has been identified only in rhesus macaques [8], and is unique both in structure (circular) and in mechanism of post-translational assembly (intermolecular disulfide bridging).

As an interesting aside, it was recognised recently that the CXC motif present in the N-ter-minal of defensins is analogous to the CXC motif present in a class of chemokines [9], Specific CXC chemokines demonstrate a pattern of broad-spectrum antimicrobial activity similar to that of the defensins [9], perhaps blurring the distinction between antimicrobial and signalling molecules.

The second main class of mammalian antimicrobial peptides is the cathelicidins, all of which are cleaved from precursor proteins containing the highly conserved propeptide sequence termed cathelin [6]. Interestingly, the cathelin propeptide was discovered independently as a porcine inhibitor of cathepsin L [10,11]. Cathelicidin genes consist of 4 exons, with exons 1-3 encoding the tissue-targeting cathelin portion of the molecule, and exon 4 encoding the active peptide. The active cathelicidin peptides vary widely in their primary sequence, and the cathelicidin gene family varies markedly in size among species. To date, only a single human cathelicidin gene has been identified, hCAP18. hCAP18 refers to the 18 kd cathelicidin prepropeptide, while the processed, active moiety is termed LL-37 (N-terminal leu-leu with length 37 a.a.) (Fig. 1).

Peptides of the defensin and cathelicidin families follow the dual paradigm of being processed from a precursor propeptide and having an antimicrobial activity linked to their net cationic charge. However, nature in its abundant diversity is likely to have evolved multiple paradigms for providing molecular protection of the epithelial barrier. The search for additional, novel classes of antimicrobial peptides has required novel insights and novel laboratory approaches. Among the unique peptide classes under investigation are anionic peptides and histone fragments.

The former are zinc-binding small molecules detected in pulmonary tissues of both sheep and humans [12], In both species, these molecules and their precursors are constitutively expressed in airway tissues [13]. It is interesting to speculate whether they contribute to the proven effectiveness of zinc supplements in ameliorating pulmonary infections.

Many investigators have reported the HPLC purification of histone fragments with antimicrobial activity, and for years this was thought to be of no real consequence. However, it is now better appreciated that cellular apoptosis may be a defence response to microbial invasion. Therefore, it is a reasonable hypothesis that the release of histone fragments from the apoptotic cell chromatin is a highly effective strategy for augmenting host defence [14,15],

1.2. Cellular origin

Each species examined has a unique pattern of antimicrobial peptide expression, perhaps reflective of its unique life cycle and environment. However, a feature common to all species is the presence of abundant antimicrobial peptides in circulating granulocytes (e.g., neutrophils [5] and eosinophils [16]). It has been estimated that the antimicrobial a-defensins stored in human neutrophil granules (termed HNP's) may constitute up to 50% of the total neutrophil protein [5], A subset of these granule antimicrobial peptides can be detected in epithelial tissues, with the most prominent examples being the epidermal expression of human cathelicidin LL-37 and the orthologous mouse cathelicidin CRAMP [11,17], Additionally, cells of both the T and B cell lineages, capable of infiltrating epithelial tissues, demonstrate transcription, translation and release of both defensins and cathelicidins [15].

Defensins and cathelicidins are the principle antimicrobial peptides expressed in the epithelium of every major organ system examined. In human tissues this includes the skin, ear canal, salivary glands, conjunctiva, breast tissue, GI tract from oesophagus to colon, pulmonary tract, and genitourinary tract [6,11,18-20], In addition to their expression in the epithelial lining cells, antimicrobial peptides may be released locally from tissue monocytes encountering microbes or their products [21], Their presence in circulating phagocytic cells as well as at sites of host-environment interface has lead investigators to hypothesise that they are the microbicidal effector molecules of the innate immune system. Recent evidence described below (section 1.8-1.10, and section 2) not only is supportive of this role in innate immunity, but also is suggestive of a more central role in regulating host immunity.

1.3. Mechanisms of microbicidal action

In general, defensins and cathelicidins are bactericidal in vitro against both Gram-positive and Gram-negative bacteria in assays using simplified, low ionic strength media. Subsets of these peptides demonstrate antiviral, antifungal and antiprotozoal activity as well. A working model for their mechanism of activity includes an initial interaction of the cationic moiety with the anionic bacterial membrane phospholipids, subsequent weakening of the microbial membrane integrity and a terminal disruption of membrane function [4,22,23]. This model is consistent with the repeated observations that: (1) elevation of salt or divalent cation concentration in the media may shield the charges on the microbial surface and reduce antimicrobial peptide activity; and (2) bacteria may acquire resistance to microbial activity through alterations in their surface lipid properties.

As we attempt to develop a unifying model for the antimicrobial peptide mechanism of action, we should recognise that a number of independent observations suggest a significant diversity to the microbicidal activity. For example, a novel approach to this question is the use of transcriptional profiling to investigate the bacterial response to treatment with an antimicrobial peptide. E. coli treated with a sublethal dose of an insect antimicrobial peptide, Cecropin A, demonstrated significant changes in transcript levels for 26 different bacterial genes, half of which are yet to be assigned a function [24], These changes differed from those seen in response to thermal, osmotic or nutritional stress.

Other data that need to be considered are the following: (1) several cationic peptides demonstrate cidal activity independent of local salt-concentration, most notably the cathelicidins [7]; (2) transmission EM of S. aureus killed by human (3-defensin-3 (hBD-3) demonstrated a cellular morphology similar to that produced by penicillin, suggesting that primary disruption of cell wall integrity had occurred [25]; (3) neutrophil a-defensins HNP1-3 may bind to viral co-receptors inhibiting HIV replication [26]; and (4) HNP-1 added to rat or mouse lung explants augmented killing of E. coli and P. aeruginosa via enhanced production of reactive oxygen intermediates [27], The take-home message is that in vivo, antimicrobial activity may be a synergy of independent innate immune mechanisms not easily demonstrated in simplified culture conditions.

1.4. Constitutive expression at epithelial surfaces

Constitutive epithelial expression is a feature common to the a-defensins expressed in Paneth cells, but in general is rare for fS-defensins with the exception of hBD-l/mBD-1 [3], The Paneth cell defensins of mouse (cryptdins) and humans (HD5 and HD6) are stored as propeptides in intracellular granules. Their release, either spontaneous or induced, is accompanied by co-release of a processing enzyme that cleaves the propeptide internally, releasing the active moiety. In the mouse, the processing enzyme is a matrix metalloproteinase MMP-7 [matrilysin] [28], and in humans, the analogous activity has been ascribed to an intestinal-specific trypsin isoform [29]. Paneth cell propeptide synthesis and storage appears to be a constitutive process, although release can be regulated at least partially by bacterial exposure through stimulation of Ca2+-activated K+ channels [30],

While most studies of adult animals demonstrate a constitutive level of Paneth cell a-defensin expression, during development there is a well-documented progression in expression levels [31,32] as well as a progression from diffuse epithelial expression to Paneth cell localisation [33], The discovery of this maturation process may shed light on the increased risk of premature individuals for infectious enterocolitis [32]. While the abundance of evidence supports the constitutive nature of Paneth cell a-defensin expression, a study in rats has demonstrated that following haemorrhagic shock, mRNA for rat a-defensin RD-5 was up-regulated 10-fold [34]. While this result may be unique to the rat defensins, our current use of the term "constitutive" may reflect more the lack of an inducible response to traditional proinflammatory mediators. Modulation of high level basal activity in the case of Paneth cell a-defensins may be a characteristic of infectious, inflammatory and hormonal conditions.

Expression of the cathelicidin LL-37 is considered to be constitutive in the human gastrointestinal tract, with highest levels detected in epithelial cells at the surface and upper crypts of the colon. Both in vitro and in vivo, LL-37 expression levels parallel the state of differentiation and are not induced by bacterial exposure nor by treatment with proinflammatory cytokines [35], hBD-1 and its mouse ortholog mBD-1 are unique among the p-defensins discovered to date in that they are expressed constitutively in epithelia and detected generally at low levels in surface lining fluids. The lack of inducibility correlates with an absence of NF-kB and AP-1 binding sites in their promoter regions [7], In the best studied examples, hBD-1 and mBD-1 mRNAs were detected at their highest levels in the lung and in the kidney (both proximal and distal tubular epithelial cells) [36], and hBD-1 peptide isoforms were detectable in human urine [37], While the usual mediators of inflammation do not induce these gene products, hormonal influences and the state of cellular differentiation may influence their level of transcription/ translation. As examples, concentrations of the hBD-1 peptide increase in urine and breast tissue during pregnancy [7,37], and hBD-1 expression in keratinocytes increased with keratinocytes differentiation [38],

There have been several reports of bacteria or inflammatory mediators down-regulating hBD-1 expression as they induce hBD-2/hBD-3 expression [38], The virulence of organisms causing colonic infections, for example Shigella, appears to be linked to the ability of bacteria or bacterial DNA to down-regulate the "constitutive" expression of both LL-37 and hBD-1 [39], Much remains to be discovered regarding the mechanisms underlying this shift in transcriptional programming and its consequence for host immunity.

The unique pattern of hBD-1 /mBD-1 expression may suggest a unique role for this molecule in innate immunity. In sections to follow, we will highlight reported differences in the properties of P-defensin-1 vs. inducible P-defensins, and hypothesise how those properties might provide insights into the general framework of constitutive vs. inducible host defence. Clearly, whatever framework is proposed, recent advances in this field are likely to lead to future paradigm revisions.

First, and perhaps most importantly, computer analysis of the human and mouse genomes has revealed that the p-defensin gene family is far larger than had been predicted. Sophisticated searches have uncovered 5 chromosomal regions that contain clusters of (3-defensin-related genes and are conserved between species [40]. With the knowledge that at least 30 human p-dcfcnsin family genes and >40 mouse P-dcfcnsin family genes may be contributing to the innate immune system, it may be premature to consider hBD-l/mBD-1 as the sole p-defensin expressed constitutively. Furthermore, with these large gene families, one might surmise that the classic approach to proving gene function via single gene knockout would be unlikely to lead to a detectable change in phenotype. Fortunately, the mBD-1 gene knockout mice were constructed prior to the genomic information being available, and they demonstrate a phenotypic change both in lung [41] and urinary tract immunity [42],

Second, a series of five hBD-1 isoforms have been detected that differ only in their N-termini, and may differ functionally [37]. N-terminal processing may be a regulatory step, and a tissue-specific or individual-specific variation in isoform distribution may underlie a variation in hBD-1 activity. As an example, there are distinct patterns of hBD-1 isoforms present in individual urine samples [37].

Third and perhaps most importantly, most of the investigations into P-defensin regulation have been carried out in culture and may not reproduce in vivo conditions. An example is the detection of both hBD-1 and hBD-2 expression in human renal tubular epithelial cells, while biopsy material showed only hBD-1 expression [36], Included in this category may be the demonstration that H. pylori induces hBD-1 expression in some but not all gastric epithelial cell lines [43,44],

1.5. Mechanisms of induction and secretion at epithelial surfaces

Barrier invasion, tissue injury and inflammation all lead to activation of innate immune defence mechanisms. Prominent among the first molecules to be detected are (3-defensins and catheli-cidins. The most detailed knowledge of this process has come from in vitro and in vivo studies of the bovine tracheal antimicrobial peptide (TAP), a (3-defensin [45,46]. E. coli LPS has been shown to be a potent inducer of TAP in vivo and in tracheal epithelial cells in vitro. The LPS molecule binds to a cell surface CD 14/TLR4 receptor (likely to require MD-2 as well) initiating an intracellular signalling pathway whose end result is NF-kB activation. The TAP gene contains consensus NF-kB binding sites leading to enhanced transcription, translation, post-translational processing and active peptide release.

Further investigation into the inducible expression of hBD-2 suggests that epithelial cells undergo dual regulation of [3-defensin genes [19,21,47], High level bacterial and/or LPS exposure activates epithelial cell NF-kB directly via the TLR (Toll-like receptor) pathway. Alternatively, low level microbial stimulation of tissue monocytes results in proinflammatory cytokine release (IL-1 a, IL-1 [3 and TNF-a). These molecules bind to specific receptors on epithelial cells and at low concentrations are potent activators of NF-kB. In fact, in human epidermis, hBD-2 is among the most highly induced mRNAs following IL-la exposure [47]. Theoretically, having these alternate mechanisms of hBD-2 induction allows for: (1) a direct epithelial cell response to high level bacterial exposure; and (2) in the sub-epithelial space, pathogen-macrophage contact leading to proinflammatory cytokine release that provokes a regional upregulation of antimicrobial peptide synthesis in order to limit bacterial spread.

As more and more defensin genes are sequenced, analysis of their promoter regions has uncovered a marked variation in the presence of consensus response elements for the proinflammatory transcriptional enhancers NF-kB, AP-1, NF-IL-6 and STAT [7], For example, the hBD-2 gene contains response elements for NF-kB, NF-IL-6 and AP-1, while the hBD-3 gene has numerous AP-1 but no NF-kB response elements. hBD-2 and 3 expression can be detected in many of the same tissues, however their differential regulation and differential antimicrobial spectra (hBD-2 active primarily against Gm-negatives while hBD-3 active against Gm-negative and Gm-posi-tive organisms including S. aureus) allows for flexibility in the nature of the epithelial innate response. As a corollary, pharmacologic regulation of defensin gene expression may vary based on the presence or absence of gene-specific response elements. Dexamethasone inhibits hBD-3 but not hBD-2 (nor constitutive hBD-1) expression in airway epithelial cells [7],

As we strive to formulate an induction paradigm that focuses on classical pathways, an unusual set of experiments may have identified a novel pathway to NF-kB activation. In an artificial system utilising transfected bovine kidney cells, investigators reported that micromolar levels of L-isoleucine specifically induced expression of a P-defensin/luciferase reporter construct through NF-kB activation [48], The authors speculate that since this essential amino acid is normally in low abundance, elevated levels of isoleucine or a related compound, possibly secreted by bacteria, could be interpreted by epithelial cells as a sign of infection.

1.6. Mechanisms of recruitment and activation

Recruitment of antimicrobial peptide-containing cells to epithelial sites of injury, infection or inflammation involves both circulating granulocytes and monocytes. The recruiting signals include chemokines released from compromised epithelium at tissue surfaces, from exposed tissue macrophages and from injured vascular endothelium [49]. Many of these same signals are known to promote haemostasis and wound repair [6], hence it is not surprising that the recruited antimicrobial elements would be direct participants in the healing process as well.

Antimicrobial peptides can be cytotoxic to the synthesising eukaryotic cells at high concentrations, and it is a challenge for granulocytes/monocytes to avoid cell injury during peptide packaging into granules and long-term peptide storage. Two alternative strategies have evolved to protect host cells from their own antimicrobial peptides until recruited from the circulation into the tissues. The first is best demonstrated in human neutrophils [4]. Initial processing of neutrophil a-defensins cleaves prepropeptides into propeptides. Within the propeptide, the anionic, N-terminal pro segment balances the charge and toxicity of the cationic, C-terminal defensin peptide. Secondary cleavage separates propiece from mature peptide, but their co-storage in the azurophilic granules leads to trans-inactivation until released into the phagolysosome or the external milieu.

The second strategy is best exemplified by the cathelicidins [50,51]. It is well documented in both pig and human neutrophils that the cathelicidins are stored as unprocessed, propeptides in peroxidase-negative granules, while their processing enzymes are stored in a distinct set of granules in the same cell. No active peptides are present as the cell circulates, however cell activation leads to fusion of both sets of granules with the phagolysosome or release of both sets of granules into the tissues. Either results in activation of this binary antimicrobial weapon.

While the pig neutrophils utilise neutrophil elastase to activate their protegrin cathelicidins, human neutrophils cleave hCAP-18 to LL-37 via proteinase 3, a serine proteinase from azurophilic granules [51]. This activation pathway provides an opportunity for the host to dampen an exuberant inflammatory response through proteinase inhibitors present in tissues, e.g., elafin in human skin [52], or co-released by the neutrophil, e.g., alpha 1-antiprotease [53], In addition, pharmacologic modulation of host inflammation may be accomplished through the use of small molecule proteinase inhibitors, e.g., the elastase inhibitor NX21909 [54], Finally, it is likely that among the diversity of mechanisms used by microbial pathogens to escape host defences will be a molecular mimic of host proteinase inhibitors.

1.7. Mechanisms of peptide inactivation

Three mechanisms have been proposed for antimicrobial peptide inactivation: protein binding, proteinase degradation and post-translational modification. As examples of the first mechanism, defensin binding to serum albumin, a-1-antiprotease (also known as a-1-antitrypsin) and a-1-chymotrypsin inhibits its proinflammatory and cytotoxic effects [55], As further evidence for the importance of this protective mechanism, a selected oligonucleotide inhibitor of elastase, NX21909, protects against lung inflammatory injury in an in vivo model of acute disease [54].

Bacterial proteinases from human pathogens including P. aeruginosa and S. pyogenes have been shown to inactivate the human cathelicidin LL-37, possibly revealing a common microbial virulence mechanism [56]. Molecules that block this degradation, e.g., the metalloproteinase inhibitor GM6001, may have significant therapeutic potential in combination with traditional antibiotic treatment.

Finally, ADP ribosyltransferase, present on or secreted from epithelial cells, lymphocytes and neutrophils, can ribosylate the arg-14 of HNP-1 [57]. Ribosylated-HNP-1 is detected at increased levels in the BAL from smokers, and the ribosylated product has decreased antimicrobial and cytotoxic effects. Interestingly, arg-14 HNP-1 ribosylation did not affect T cell chemotactic and IL-8-releasing activities, suggesting that this mode of peptide modification may provide new insights into HNP-1 structure-function relationships.

1.8. Non-antimicrobial effects

A current area of great interest in the study of antimicrobial peptides is the search for dual roles in host immunity. Most if not all antimicrobial peptides are multi-functional, potentiating the innate immune response via mechanisms as diverse as promotion of wound healing, chemokine activity and initiating an adaptive immune response [6]. Indeed, it remains an open question whether antimicrobial activity is their most prominent role in vivo. A note of caution here relates to the fact that molecules like defensins and cathelicidins are membrane active, may have nonspecific effects on membrane functions such as maintenance of ion gradients, and hence may demonstrate activities in specialised assay systems that are of unclear significance to in vivo conditions.

That caveat aside, antimicrobial peptides demonstrate a remarkable spectrum of host defence-related, non-antimicrobial functions including modulation of: bacterial adherence [55,58]; complement activation [55]; fibrinolysis [55]; steroid synthesis [59]; mast cell activation [60]; monocyte, neutrophil, mast cell, T cell and immature dendritic cell chemoattraction [11,61,62]; cytokine expression [63]; cytotoxicity [64]; cell proliferation [7,65]; angiogenesis [66]; protease inhibitor synthesis [67]; keratinocyte differentiation [38]; proteoglycan synthesis [11]; phagocytosis [68] and Ca+2 mobilisation [59]. The breadth of potential functions has led some to consider these molecules as the key regulators of innate immunity. While many recent reviews can be found containing complete descriptions of these activities, here we will highlight four unique aspects.

1 A variety of activities ascribed to these peptides involve binding to specific, high-affinity cell surface receptors. Among the best characterised examples are: [3-defensin binding to the chemokine receptor CCR6 inducing chemotaxis of T-cells and immature dendritic cells [69]; LL-37 binding to the formyl peptide receptor-like 1 (FPRL1) inducing monocyte chemotaxis and Ca+2 mobilisation [17]; and HNP1-3 binding to the chemoreceptor CXCR4 on CD8 T lymphocytes, effectively blocking the replication of HIV-1 virus type X4 and stabilising patients' immunologic status [26], In general, this type of receptor-peptide ligand interaction has enormous potential for drug targeting through development of receptor antagonists, antireceptor antibodies or soluble receptors [70],

2 Constitutive and inducible antimicrobial peptides differ significantly in their activities. The inducible hBD-2 and LL-37 peptides degranulate mast cells via a G protein -phospholipase C-dependent mechanism, while the constitutive hBD-1 lacks this activity [60], Additionally, hBD-2 is a mast cell chemotaxin while hBD-1 is not [17]. As a third example, in immortalised human keratinocytes the state of differentiation regulates hBD-1 mRNA levels while hBD-2 mRNA levels are unchanged [38],

3 Dose-response curves for non-antimicrobial activities often are bell-shaped, with peak activity near the concentration at which peak antimicrobial activity is reached. HNP1-3 enhance proliferation of human lung tumour epithelial cell lines at 4-10 (ig/ml, but are cytotoxic at concentrations > 20 |J.g/ml [65]. LL-37 is chemotactic for mast cells at 5 (ig/ml but not at 1 or 20 |J.g/ml [17]. Finally, pig protegrins, members of the cathelicidin family, trigger the cleavage and export of IL-1 (3 from LPS-primed monocytes with a peak at 12.5 |ig/ml but are inactive at 1 and 100 (J.g/ml [63]. Possible implications of this consistent dose-response pattern are discussed in Section 3.2.

4 The non-antimicrobial activities of these peptides can amplify the innate response via positive feedback loops. IL-8, a CXC chemokine, is one of several signals that attract neutrophils to the lungs in patients with chronic inflammatory diseases, e.g., chronic bronchitis or COPD

[71]. Subsequent neutrophil degranulation then releases a-defensins that induce airway epithelial cells to synthesise additional IL-8.

1.9. Interactions with components of the innate immune system

One should not underestimate the challenge faced by the innate immune system in protecting the delicate epithelial tissues, e.g., those of the pulmonary and gastrointestinal tracts that provide vital physiologic functions in the presence of recurrent and/or continuous pathogen exposure. In many situations, a brisk immune and inflammatory response to low level pathogen exposure is less than ideal, as it may damage and disrupt key tissue elements. Mechanisms to promote a prompt and appropriate response to microbial challenge must be in place, it is our challenge to delineate them.

Evidence is accumulating that polarisation of epithelial cells is a key element in regulation of epithelial innate immunity. The apical cell surface is the primary site of microbial exposure, and analysis of its surface molecules as well as the composition of its secreted surface fluid will continue to provide insights into how host tissues control their adjacent milieu. As a baseline, the apical secretory components include (but are not limited to) organics (e.g., HC1), lysozyme, lactoferrin, SLPI, PLA2 and constitutive antimicrobial peptides (e.g., hBD-1) [72,73], In airway surface fluid there is ample evidence for synergistic interactions between these components, although the (3-defensin component, hBD-1 is more additive in its effects. An analogous system is present in the crypts of the small intestine, where the primary antimicrobial peptides are a-defensins [74], and in vaginal fluid [75]. It is a recurring theme in these studies that individual and synergistic activities are reduced as ionic strength increases.

On the apical surface of epithelial cells can be found bactericidal-permeability inducing protein (BPI), a molecule with both antibacterial and LPS-binding properties [76]. It is hypothesised that surface BPI interacts with constitutive p-defensins in promoting antimicrobial activity against bacteria adhering to the host cell surface, p-defensin and BPI-binding of released Gm-negative bacteria LPS would prevent the initiation of potentially injurious inflammatory responses.

A more dramatic and complcx host response is activated in the setting of barrier compromise and epithelial injury [21]. Microorganisms and their cellular constituents now will come into contact with the epithelial cell basolateral surface where the Toll-like receptors (TLR) are localised and can be activated. TLR activation leads to NF-kB stimulation of P-defensin synthesis, predominantly the hBD-2 and hBD-3 molecules in the human pulmonary and gastrointestinal tissues. Additionally, tissue monocytes/macrophages will be activated by the microbial stimuli leading to proinflammatory cytokine (TNF-a , IL-la, IL-lp) and antimicrobial peptide release. These molecules will then recruit circulating granulocytes and monocytes to an area at risk for significant infection spread.

Surprisingly, while degranulation of the recruited neutrophils leads to release of a-defensins that enhance monocyte TNF-a and IL-lp expression in response to S. aureus or phorbol myr-istate acetate (positive feedback), the same molecules down-regulate endothelial cell VCAM-1 expression reducing cellular recruitment from the circulation (negative feedback) [5], The released a-defensins also bind C1Q and may activate the classical complement pathway [77], another component of the innate immune system preserved in organisms from Drosophila to humans.

1.10. Liaison with the adaptive immune system

It has been a long time coming, but finally a model is evolving that includes the innate immune system in alerting, stimulating and regulating the adaptive immune response. This can be viewed not merely as a step-wise escalation of the host response, but in reality a commitment of the host to a long term expenditure of resources for combating what is perceived as a significant pathogenic organism.

Following this line of reasoning, the most convincing evidence for regulation of adaptive immunity revolves around chemoattraction. While both defensin and cathelicidin peptides are antimicrobial at micromolar concentrations, they are chemotactic for dendritic and memory T cells at nanomolar concentrations [5], hBD-1 and 2 act through binding to the CCR6 chemokine receptor, LL-37 via formyl peptide receptor-like 1 (FPRL1) and the a-defensins through a receptor not yet identified.

The primary adaptive immune response plays out in draining regional lymph nodes somewhat distant from the infected/injured tissue. Initially, immature dendritic cells are recruited to the site of inflammation, they process foreign antigens for presentation, migrate to the regional nodes and finally activate unprimed T and B cells. These activated cells then migrate to the original site of infection/injury to deliver the first effective response of the adaptive immune system. These are key steps where the antimicrobial peptides function in co-ordination with cytokine and chemokine elements of the innate immune system. Four examples are outlined below.

First, hBD-1, hBD-2 and a-defensins are chemotactic for immature dendritic cells (iDC) eliciting their migration to the site of antimicrobial peptide synthesis (epithelial cells) or release (from recruited granulocytes) [49], Second, upon iDC arrival, both mBD-2 and human a-defensins have been shown to induce iDC maturation including the upregulation of surface costimulatory molecules and the enhanced expression of proinflammatory cytokines including IL-12, IL-la and IL-ip [62], Third, antimicrobial peptides recruit memory T cells to sites of inflammation [78], And fourth, a-defensins have been shown to increase the mouse IgG response in a model of immunoglobulin stimulation [79],

2. IN VIVO MODELS OF ANTIMICROBIAL PEPTIDE FUNCTION 2.1. Skin

The ready availability of epidermal tissues for in vivo and in vitro studies has lead to rapid advances in our knowledge of skin antimicrobial peptide biology. Intact and uninflammed, keratinocytes do not express significant amounts of either cathelicidin or defensin peptides. However, both in human and mouse tissues, injured/inflamed skin expresses high levels of one or more antimicrobial peptides [6]. Two lines of evidence support the relevance of antimicrobial peptide expression to skin defence.

First, it is well known from clinical experience that psoriatic lesions are less prone to infection than atopic dermatitis lesions. In a recently published study, immunoblotting and Western blot analysis were used to analyse epidermal expression of both the human cathelicidin peptide LL-37 and the human (3-defensin hBD-2 [80]. These peptides have been shown to be synergistic in activity against S. aureus, a major skin pathogen. In psoriatic surface epidermis, there is abundant expression of both peptides correlating with a decreased risk for bacterial infection [80]. In contrast, in atopic dermatitis lesions there is a significant suppression of expression for both peptides correlating with an increased risk of bacterial infection. Additionally, IL-4 and IL-13, cytokines known to be increased in atopic dermatitis lesions but not in psoriatic lesions, block antimicrobial peptide induction by TNF-a in cultured keratinocytes.

Direct evidence for the role of antimicrobial peptides in skin was provided through use of mouse knockout technology. Mice in which the LL-37 ortholog, CRAMP, was inactivated (CR-/-), demonstrated increased susceptibility to necrotic skin infection by group A Streptococcus (GAS) compared to their wild type littermates (CR+/+) [81]. Heterozygous animals (CR+/-) were intermediate in susceptibility supporting a gene dosage effect. In complementary studies, mutating the GAS bacterium to make it resistant to killing by CRAMP, lead to equal infectivity in CR-/- and CR +/+ animals. This combination of mouse and bacterial genetics provides the most convincing evidence to date that a single antimicrobial peptide provides a critical role in host defence.

2.2. Pulmonary tract

An area of great interest, and of significant controversy, is the role of antimicrobial peptides in lung protection in general, and specifically in patients with cystic fibrosis [82], The leap into studying antimicrobial peptides in cystic fibrosis derives from observations that: (1) CF patients are prone to airway colonisation by bacterial pathogens; (2) CF patients are prone to recurrent lung infections as a source of morbidity and mortality; (3) the airway secretions of CF patients have been reported to contain an increased salt concentration; and (4) airway antimicrobial peptides are salt-sensitive, losing protective activity in the presence of salt concentrations seen in CF airway fluid.

Several well-publicised research reports used both in vitro assays and bronchial xenografts to demonstrate that the activity of the constitutively-expressed lung hBD-1 against common CF pathogens was reduced in the presence of CF airway fluid. Additionally, antisense suppression of xenograft hBD-1 expression abolished airway surface fluid microbicidal activity, and overexpression of the less salt-sensitive LL-37 restored antimicrobial activity in CF xenograft tissue [83-85],

As tantalising as it is to have CF lung disease explained by salt-sensitive antimicrobial activity, this hypothesis is highly controversial, based on the following observations. First, recent data suggests that the purported high salt concentration in CF airway surface fluid may not exist in vivo [86], Second, multiple [3-defensin peptides are expressed in airway epithelial cells [7] so that the hypothesis that hBD-1 suppression alone could change the microbicidal activity has not been accepted readily. Third, there is a great deal of phenotypic variation in infection susceptibility within the CF population, even in patients with the same CFTR genotype, with some patients having little in the way of pulmonary complications and others infected frequently from their early childhood [87],

As an alternative approach to address some of the questions, two laboratories derived mouse lines in which the mBD-1 gene was inactivated. In one report, there was no change in pulmonary susceptibility to a nebulized S. aureus challenge [42], while in the other there was a reproducible, delayed clearance of H. influenza inoculated intranasally [41]. Differences in strain background, pathogen choice and CFU level of inoculation make it difficult to resolve this discrepancy. Additionally, it is a challenge to relate this to CF in humans, as the mouse CF model does not mimic the human condition in pulmonary pathology.

2.3. Gastrointestinal tract

Multiple antimicrobial peptides are expressed in mammalian gastrointestinal tissues, with the greatest abundance in Paneth cells of the distal ileal crypts [74], Initial thoughts were that use of single gene knockouts would be unlikely to produce a detectable phenotype. A successful alternative approach was to inactivate processing pathways common to all mouse intestinal a-defensins. The matrix metalloproteinase matrilysin (MMP-7) co-localises with a-defensins in Paneth cell granules, and its inactivation leads to a deficiency of mature defensins in the Paneth cells, an accumulation of the inactive precursors (implying a block in processing) and an increased susceptibility to an oral challenge with the bacterial pathogen Salmonella [28], While this is very convincing evidence for the role of a-defensins in intestinal immunity, it remains a possibility that the loss of other intestinal activities regulated by MMP-7 contributed to the change in host susceptibility.

In a complementary series of experiments, Bevins CL, Huttner KM, et al. (submitted for publication [96]) have produced and analysed transgenic mouse strains carrying the human Paneth cell defensin gene HD-5. In two independently-derived lines, Paneth cell expression of HD-5 is co-ordinated with that of the endogenous mouse defensins both in spatial orientation and in developmental time-frame. More surprisingly, the addition of this single peptide to the mouse Paneth cell expression of up to 20 different a-defensins led to a reproducible augmentation of host defence.

There are a growing number of published studies related to antimicrobial peptide function in the tongue, ocular mucosa, genitourinary tract and in breast milk. The reader is directed to recent reviews for specific papers in these areas [4,5,7], Clearly this area of research is poised to grow in the next decade.

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