Defense of the Skin with Antimicrobial Peptides

Anti-microbial peptides are mainly minuscule cationic polypeptides that have the capacity to obstruct the multiplication of microbes.

As effectors of natural immunity, antimicrobial peptides instantly kill a broad spectrum of bacteria, fungi, and viruses. In addition, these peptides transform the local inflammatory answer and activate mechanisms of adaptive and cellular immunity. Cathelicidins and defensins constitute the most important families of antimicrobial peptides in the skin, although other cutaneous peptides, such as chemokines, proteinase inhibitors and neuropeptides also demonstrate antimicrobial action.

Together, these multifunctional antimicrobial peptides play a significant role in skin immune defense and disease pathogenesis.

Cutaneous defense mechanisms by antimicrobial peptides Braff MH, Bardan A, Nizet V, Gallo RL. Department of Medicine, University of California San Diego, and VA San Diego Healthcare System, San Diego, California, USA.

Antimicrobial Peptides in the Skin: Biological Relevance

Antimicrobial peptides, which are synthesized in the skin at sites of potential microbial entry, provide a soluble boundary that acts as an obstruction to infection. In the case of infection or trauma, antimicrobial peptide appearance in the skin is upregulated due to enlarged synthesis by keratinocytes and deposition from degranulation of recruited neutrophils. Although antimicrobial peptides evidently demonstrate in vitro antimicrobial activity, studies have revealed that many such peptides, including cathelicidins and defensins, are inactivated by physiological salt concentrations (Goldman et al, 1997).

In fact, a recent study has revealed that mammalian skin contains an essential antimicrobial-enhancing factor that renders bacteria susceptible to cathelicidin in vitro, despite the existence of physiological salt and serum (Dorschner et al, 2004). The in vivo importance of antimicrobial peptides in the physiological milieu is further accentuated by the laboratory animal models and human skin diseases that are exposed below.

Cathelicidins are defined by an N-terminal signal peptide, a highly sustained cathelin domain and a structurally variable cationic antimicrobial peptide at the C-terminus. The cathelin domain functions as both a protease inhibitor and as an antimicrobial peptide in humans (Zaiou et al, 2003). Mature cathelicidin peptides show potent, rapid, and broad-spectrum antimicrobial activity and have been implicated in various immunomodulatory functions(Koczulla et al, 2003).

Human cathelicidin, LL-37, assumes an a-helical structure in solutions with ion compositions similar to intestinal fluid, human plasma, or intracellular fluid. Processing of LL-37 from the cathelicidin precursor is essential for activation of its antimicrobial activity and is accomplished by neutrophil proteases such as proteinase 3 (Sorensen et al, 2001).

LL-37 expression in squamous epithelia is differentially regulated in certain inflammatory circumstances (Frohm et al, 1997; Dorschner et al, 2001). LL-37 is generated in eccrine structures, where it is secreted and processed in sweat, suggesting a further barrier function against topical skin infection (Murakami et al, 2004).

In addition, LL-37 is generated by mast cells and recruits mast cells (Di Nardo et al, 2003), thereby cooperating in innate immunity both by direct antimicrobial activity and by recruitment of cellular human b-defensin defenses. LL-37 generation is upregulated in neonatal skin, where it may adjust for the empirical immaturity of adaptive immune reactions (Dorschner et al, 2003). A true immunomodulatory effector molecule, LL-37 has unequivocal antimicrobial activity, acts synergistically with other antimicrobial peptides, works as a chemoattractant for neutrophils, monocytes and T cells, and activates endothelial cell proliferation by binding to formyl peptide receptor-like 1(FPRL-1) (Koczulla et al, 2003).

The multilayered expression and multifunctionality of cathelicidin in the skin present a formidable innate defense system against infection.

Cathelicidins Cathelicidins are strategically expressed and contribute multiple functions to skin defense. The human cathelicidin precursor protein hCAP18 is expressed by several cell types in the skin including keratinocytes, neutrophils, eccrine ducts, and mast cells. Cathelicidins are processed to active peptides such as LL-37 in neutrophils and more potent peptides in sweat.

These peptides have been best characterized as natural antibiotics, killing a variety of bacterial, fungal, and viral pathogens. Other functions include chemotactic and angiogenic behaviors, and an ability to modify fibroblast proteoglycan synthesis. The N-terminal cathelin-like domain of the hCAP18 precursor protein contains both antimicrobial and proteinase inhibitor activity.

Defensins Defensins contain six cysteine residues that form particular disulfide bridges. Disulfide bridge alignment and molecular structure divide this fundamental antimicrobial peptide group into a-, b-, and y-defensins. Mammalian defensins exhibit antimicrobial activity against bacteria, fungi and enveloped viruses. á-Defensins contain three disulfide bridges in a 1–6, 2–4, 3–5 alignment. Human neutrophils express four á-defensins, which are also referred to as human neutrophil peptides 1 through 4 (HNP-1 to -4) (Harwig et al, 1994). Human defensins 5 and 6 (HD-5 and -6) are abundantly expressed as propeptides in Paneth cells of small intestinal crypts and in epithelial cells of the female urogenital tract.

In humans, defensins are accumulated in azurophil granules of neutrophils as fully processed, mature peptides. Like cathelicidins, á -defensins disturb both microbes and the host. For example, HNP-1, -2, and -3 upregulate tumor necrosis factor alpha (TNF- á) and IL-1 in human monocytes that have been activated by bacteria; these peptides also diminish the expression of the adhesion molecule VCAM-1 in endothelial cells activated by TNF-a (Chaly et al, 2000).

b-Defensins contain three disulfide bridges that are spaced in a 1–5, 2–4, 3–6 pattern. The four best-known human b-defensins, hBD-1 to -4, have been identified in various cell types, along with epithelial and peripheral blood mononuclear cells (Harder et al, 2001; Fang et al, 2003; Liu et al, 2003). hBD-1 is constitutively expressed in epithelia, whereas hBD-2 is highly upregulated in inflamed skin. hBD- 3, which was purified from human psoriatic scales and calluses (Harder et al, 2001), is inducible in a variety of tissues. b-Defensins have broad-spectrum antimicrobial action and additional immune-related cellular functions.

For example, hBD-2 binds to CCR6 and is chemotactic for immature dendritic cells and memory T cells (Yang et al, 1999). hBD-2 also promotes histamine release and prostaglandin D2 production in mast cells, suggesting a potential immunotherapeutic role as a vaccine adjuvant to enhance antibody production (Befus et al, 1999). hBD-2 is virtually absent in normal skin and its expression in human keratinocytes requires stimulation by cytokines or bacteria (Liu et al, 2003). The upregulation of hBD-2 by keratinocytes exhibits the crucial role that defensins play in host defense against cutaneous pathogens.

Interestingly, the dependence of the antimicrobial activity of these peptides on their originally described function modifies, and no clear trend is observed. For example, the antimicrobial activity of ECP/RNase 3 does not require ribonuclease activity, which is essential for the antiviral activity of both ECP/RNase 3 and EDN/RNase 2 (Domachowske et al, 1998a, b). P-cystatin a impedes bacterial proteinase activity as a mechanism of microbial growth inhibition (Takahashi et al, 1994), whereas cystatin C antimicrobial activity does not depend on its ability to inhibit bacterial proteinases (Blankenvoorde et al, 1998). The antiviral activity of cystatin C, however, appearsto remain in the proteinase-binding domain. Calprotectin contains zinc-binding sites and inhibits microbial growth through competition for metals (Sohnle et al, 2000), whereas NGAL interferes with bacterial iron acquisition (Goetz et al, 2002).

Antimicrobial Peptides in the Skin: Clinical Relevance

Differential expression of antimicrobial peptides appears to play a role in the vulnerability of patients with constant inflammatory skin diseases to infectious complications.

For example, LL-37 is stimulated in human keratinocytes during psoriasis, lupus erythematosus and contact dermatitis (Frohm et al, 1997). hBD-2 and hBD-3 are also upregulated in keratinocytes of inflamed psoriatic lesions (Harder et al, 2001; Nomura et al, 2003).

The magnified expression of antimicrobial peptides in psoriasis correlates with a low rate of secondary infection. In contrast, the expression of LL-37 and hBD-2 is not upregulated in individuals with atopic dermatitis, who are highly susceptible to bacterial and viral infections (Ong et al, 2002). The differences in antimicrobial peptide expression between these two disorders gain immunological importance in light of the antimicrobial activity of LL-37 against S. pyogenes (Dorschner et al, 2001) and its synergistic activity with b-defensins against S. aureus (Ong et al, 2002), an important agent of human skin infections.

LL-37 expression is upregulated in inflammatory skin lesions of erythema toxicum neonatorum and immunolocalizes within CD15-expressing neutrophils, EG-2-expressing eosinophils, and CD1a-expressing dendritic cells (Marchini et al, 2002). LL-37 is also stimulated within the epidermis during development of verruca vulgaris and condyloma accuminata, suggesting that it represents a element of the innate immune response to papillomavirus infection (Conner et al, 2002).

Both hBD-1 and hBD-2 are upregulated in the lesions of acne vulgaris and may therefore be included in the pathogenesis or resolution of this condition (Philpott, 2003).

In addition, hBD-2 and the HNP are abundant in lesions of superficial folliculitis, a common skin disease distinguished by inflammation of the hair follicle and infection with S. aureus (Oono et al, 2003). These studies show potential roles for antimicrobial peptides in host immune defense against skin infection.

Cathelicidin is generated at high levels in the skin after wounding (Dorschner et al, 2001) and is strongly expressed in healing skin epithelium (Heilborn et al, 2003). After cutaneous wounding, growth factors activate tissue regeneration until the physical barrier protecting the skin from microbial infections has been re-established.

Growth factors crucial in skin wound healing, such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-a (TGF-a, produce the expression of cathelicidins and defensins in human keratinocytes (Sorensen et al, 2003). LL-37 antibodies impede post-wounding re-epithelialization in a concentration-dependent manner and cathelicidin expression is low or absent in chronic ulcers (Heilborn et al, 2003).

The ability of LL-37 to produce angiogenesis further highlights the importance of cathelicidin in wound healing and tissue repair (Koczulla et al, 2003). In addition, the expression of hBD-2 is dramatically diminished in burn wounds and blister fluid from partial thickness burns (Ortega et al,2000), providing evidence that innate immune defects may contribute to the risk of burn wound sepsis and infection.

Even under resting conditions, low levels of antimicrobial peptides are synthesized at sites of potential microbial entry into the skin and provide a further impediment to infection.