Skin 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. Cutaneous defense mechanisms by antimicrobial peptides

The following review discusses the biology and clinical relevance of antimicrobial peptides expressed in the skin. The importance of the epithelial contribution to host immunity is evident, as alterations in antimicrobial peptide expression have been associated with various pathologic processes.

Antimicrobial peptides are normally small cationic polypeptides that are classified together due to their faculty to obstruct the development of microbes.

As effectors of natural immunity, antimicrobial peptides directly kill a wide spectrum of bacteria, fungi, and viruses. In addition, these peptides modify the local inflammatory reaction and stimulate mechanisms of adaptive and cellular immunity. Cathelicidins and defensins constitute the most important families of antimicrobial peptides in the dermis, although other cutaneous peptides, such as proteinase inhibitors, chemokines, and neuropeptides also indicate antimicrobial activity.

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

 

Antimicrobial Peptides in the Skin: Biological Relevance

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

In fact, a recent study has discovered that mammalian dermis includes a fundamental antimicrobial-enhancing factor that renders bacteria susceptible to cathelicidin in vitro, despite the presence of physiological salt and serum (Dorschner et al, 2004). The in vivo relevance of antimicrobial peptides in the physiological environment is further highlighted by the laboratory animal models and human skin diseases that are exposed below.

Cathelicidins are characterized by an N-terminal signal peptide, a highly conserved cathelin domain and a structurally irregular 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 rapid, potent, and broad-spectrum antimicrobial activity and have been implicated in various immunomodulatory faculties(Koczulla et al, 2003).

Human cathelicidin, LL-37, assumes an a-helical structure in solutions with ion compositions similar to human plasma, interstitial fluid, 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 controlled in particular inflammatory contexts (Frohm et al, 1997; Dorschner et al, 2001). LL-37 is generated in eccrine structures, where it is yielded and processed in sweat, indicating a further barrier role against topical skin inflammation (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 production is upregulated in neonatal skin, where it may adjust for the empirical immaturity of adaptive immune responses (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 have six cysteine residues that form particular disulfide bridges. Disulfide bridge alignment and molecular structure separate this major antimicrobial peptide group into a-, b-, and y-defensins. Mammalian defensins exhibit antimicrobial activity against fungi,bacteria and enveloped viruses. á-Defensins have 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 stored in azurophil granules of neutrophils as fully processed, mature peptides. Like cathelicidins, á -defensins alter 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 reduce the expression of the adhesion molecule VCAM-1 in endothelial cells activated by TNF-a (Chaly et al, 2000).

b-Defensins have 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 different cell types, including 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 sterilized from human psoriatic scales and calluses (Harder et al, 2001), is inducible in a range of tissues. b-Defensins have broad-spectrum antimicrobial activity 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 stimulates histamine release and prostaglandin D2 production in mast cells, indicating a possible immunotherapeutic role as a vaccine adjuvant to enhance antibody production (Befus et al, 1999). hBD-2 is practically 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 illustrates the important role that defensins play in host defense against cutaneous pathogens.

Interestingly, the stability of the antimicrobial activity of these peptides on their originally described function transforms, and no clear trend is noted. For example, the antimicrobial activity of ECP/RNase 3 does not need 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 inhibits bacterial proteinase activity as a mechanism of microbial growth inhibition (Takahashi et al, 1994), whereas cystatin C antimicrobial activity does not depend on its capacity to inhibit bacterial proteinases (Blankenvoorde et al, 1998). The antiviral activity of cystatin C, however, appearsto reside 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 susceptibility of patients with chronic inflammatory skin disorders to infectious complications.

For example, LL-37 is activated 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 increased 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 achieve immunological relevance 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), a leading 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 activated within the epidermis during development of verruca vulgaris and condyloma accuminata, indicating that it represents a component 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 plenteous in lesions of superficial folliculitis, a typical skin disease distinguished by inflammation of the hair follicle and infection with S. aureus (Oono et al, 2003). These investigations demonstrate 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, induce 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 capacity of LL-37 to induce angiogenesis further highlights the relevance 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 infection and sepsis.

After injury, antimicrobial peptide levels in the skin rise quickly due to enlarged synthesis by keratinocytes and deposition from degranulation of recruited neutrophils. The chemoattractant properties of cathelicidins and defensins may further amplify this process through their functional interactions with leukocyte surface receptors. The growing number of multifunctional peptides found to frustrate microbial growth further expands the mammalian antimicrobial arsenal, demonstrating that the host antimicrobial peptide defense system acts both directly and indirectly to avoid skin infection.

Disruption of the skin by acne inflammation, invading micro-organisms, sun damage, disease, injury due to trauma, surgery, burns, accidents, or by chemical, dermabrasion or laser procedures used for skin renewal, generates a signal to the natural immune system and initiates responses that may or may not be effective in a) preventing an impending invasion from microbes by a secretion of antimicrobial peptides on the surface of the skin and b) in triggering the regeneration of new healthy cells to replace those damaged.

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