Topic Article
The Human Antimicrobial Peptide LL-37 Suppresses Apoptosis in Keratinocytes
Clara I Chamorro, Günther Weber, Alvar Grönberg, Andor Pivarcsi and Mona Ståhle
Journal of Investigative Dermatology (2009) 129, 937–944; doi:10.1038/jid.2008.321

Defense of the Skin with LL-37
Navid Bouzari ¹, Nancy Kim ¹ and Robert S. Kirsner ¹
Journal of Investigative Dermatology (2009), 129, 814. doi:10.1038/jid.2009.37

As the first line of defense against invading organisms, the skin uses a multiplicity of processes to ensure host survival, including physical, immunological, and innate systems of defense. The innate immune system, which defends skin in a nonspecific fashion, includes the cathelicidins and the single currently known human cathelicidin, hCAP18. This 8-kDa cationic antimicrobial protein is produced by leukocytes, as well as epithelial and mucosal cells (Agerberth et al., 1995), as part of a broader defense system. The active region is its cationic C-terminal 37-amino-acid domain, LL-37, which mediates broad antimicrobial activity (Dorschner et al., 2001) by disruption of the microbial cell membrane (Oren et al., 1999). Beyond this function, LL-37 defends the organism by stimulating inflammation (Koczulla et al., 2003) and speeding wound healing (Dorschner et al., 2001). LL-37’s effect on host cells appears to be concentration dependent, but its specific effect on keratinocytes has not been previously studied.
Chammorro et al. (2009) studied whether LL-37 induced or suppressed apoptosis in keratinocytes and by which mechanisms this occurred. Through the use of camptothecin (CAM, a widely used experimental apoptotic inducing agent) and the study of capsase-3 (a protein in the apoptotic pathway), cycloxygenase-2, and other genes involved in regulating apoptosis, these investigators were able to better understand the role of LL-37 in keratinocyte apoptosis. LL-37 appears to protect keratinocytes from apoptosis by inhibiting CAM-induced capsase-3 activation. This is thought to occur by upregulation of several antiapoptotic genes, including cyclooxygenase-2 and inhibitor apoptosis protein-2. This action is mediated, at least in part, via prostaglandin E-2; LL-37 ensures keratinocyte survival while its antimicrobial action takes place.

Reprinted from Dürr et al., Biochim Biophys Acta (2006) 1758:1408–25, with permission from Elsevier.

Through the following questions, we examine this paper in greater detail.

QUESTIONS
1. What are the roles of the epidermis in immunity?
2. What is LL-37, and what is the authors’ rationale for studying it?
3. What methods can be employed to study apoptosis?
4. What were the major findings of the study?
5. What may be the clinical implications of the study?
6. What further studies could be performed to confirm or further the observations?

ANSWERS

1. Human skin is the interface between the body’s internal environment and an external environment that entails assault by microbial pathogens. At one time it was considered an inactive protective barrier that participated in host defense merely by blocking entry of microbial pathogens, but it is now apparent that the skin is able to defend the body by mounting both innate and adaptive immunities. The innate immune system includes hundreds of peptides and proteins that engage in potent microbicidal activities. An increasing body of research suggests that these microbicidal agents include a vast array of antimicrobial peptides composed of histatins, granulysin, lactoferricin, defensins, and cathelicidins. Among these antibacterial peptides, two major classes of human defensins and cathelicidins have the capacity to kill a broad spectrum of pathogens, including Gram-positive and -negative bacteria, fungi, and viruses (Braff and Gallo, 2006; Lehrer and Ganz, 1999). To date, six human β-defensins (hBD-1 through -6) have been identified in human tissues. Some (e.g., hBD-1) are constitutively produced, whereas others (e.g., hBD-2) are induced by bacterial lipopolysaccharides and cytokines such as tumor necrosis factor (TNF)- α, IFN-γ, and IL-1 (Harder et al., 2001). Nearly 30 cathelicidin members have been found in mammals; however, only one cathelicidin—human cationic antibacterial protein, 18 kDa (hCAP-18)—has been identified in humans. Its mature antibacterial peptide is termed LL-37 because it begins with two leucine residues and is 37 amino acid residues long. LL-37 was first identified in neutrophils and was later shown to be expressed in various epithelia, lymphocytes, monocytes, keratinocytes, and glands (Niyonsaba and Ogawa, 2006). Figure 1 shows circumstances in which LL-37 has been found. In addition, LL-37 is secreted in wounds and seminal fluid, providing a sterile environment during wound healing and fertilization, respectively (Andersson et al., 2002).

Figure 1. Influences and effects of LL-37. Cytokines such as IL-6 increase the level of LL-37 whereas others, such as IL-4, decrease its level. These changes result in augmenting or diminishing antimicrobial and immunomodulatory activity, respectively. The diagram shows a variety of site-specific antimicrobial and immunomodulatory activities of LL-37. G+, Gram-positive; G -, Gram negative.

Adaptive immunity is a powerful, flexible, and “learning” surveillance system that complements and extends epidermal barrier function. The members most commonly sent to effect responses are T cells, often accompanied by neutrophils and sometimes by monocytes. Adaptive immunity has the advantage not only of flexibility but also memory. Its response is slower than that of innate immunity, but it is able to combat infections that have evolved to evade innate responses. Adaptive immunity can recognize and respond to virtually any protein or carbohydrate imaginable, yet it is dependent on elements of the innate immune system to initiate and direct its response. The combination of innate and adaptive responses leads to highly efficient recognition and clearance of pathogens. On the other hand, maladaptive responses of these two systems can lead to injury, as one observes in allergy, autoimmunity, and allograft rejection.

2. LL-37, the cationic C-terminal of hCAP-18, was first identified in neutrophils (Sørensen et al., 1997) and was later shown to be expressed in various squamous epithelia (e.g., airways), serous and mucous cells of the submucosal glands, and specific lymphocyte and monocyte populations, as well as in keratinocytes in inflamed skin. LL-37 displays antimicrobial activity against a broad spectrum of pathogenic microorganisms and neutralizes lipopolysaccharide bioactivity. Besides its killing properties, a wide range of biological activities have been attributed to LL-37, such as inhibiting biofilm formation, causing chemotaxis and mast cell degranulation, inducting chemokines, enhancing keratinocyte migration and proliferation, and enhancing vascularization. LL-37 shows contrasting effects on apoptosis in different cell types; for example, it suppresses apoptosis in neutrophils but promotes apoptosis in some epithelial cells (Nijnik and Hancock, 2009). Taking into consideration the in vitro effects of LL-37 on the migration and proliferation of keratinocytes, its role in wound healing, and its contrasting effects on apoptosis of different cell types, Chamorro et al. (2009) investigated the role of LL-37 in apoptosis by keratinocytes.

The promoter of the LL-37 gene contains potential regulatory motifs involved in inflammation. These motifs include NF-κB, γ-IFN response element (γ-IRE), NF-IL-6, and the acute-phase response factor STAT3. Regulation of the LL-37 gene has not been fully clarified. For example, cytokines such as IL-6 and IFN-γ may increase LL-37 expression in epithelial cells while decreasing its expression in monocytes, T cells, and natural killer cells. In addition, LL-37 regulation seems to be site-specific. For example, LL-37 is highly expressed in skin inflammation whereas its expression is low in colon inflammation (Braff and Gallo, 2006; Niyonsaba et al., 2006). Figure 1 shows some of the factors involved in the regulation and expression of LL-37. In summary, it appears that LL-37 is differentially regulated among various cell types.

3. Only 33 years have passed since Olsen and Everett (1975) first described apoptosis in normal skin. Since then, studies in mammalian cells have characterized multiple, interconnected apoptotic pathways and regulatory factors in its control. Cells respond to environmental, extracellular, and internal death signals (Figure 2). The extrinsic pathway is stimulated by binding of Fas ligand, TNF, or related cytokines to “death receptors.” The intrinsic pathway is triggered by cytotoxic drugs and DNA damage (e.g., UV radiation). In addition, several regulatory factors play roles in both intrinsic and extrinsic pathways. One is inhibitor of apoptosis (IAP); in intrinsic apoptosis, mitochondria release Smac/DIABLO to block IAP and promote apoptosis. The Bcl-2 family of proteins can both promote and suppress apoptosis. The p53 tumor suppressor promotes apoptosis. Ultimately, however, the end products of both intrinsic and extrinsic pathways of apoptosis are caspase-3 (discussed in the article by Chamorro et al., 2009) and caspase-7. To study apoptotic pathways and their regulation, several methods have been developed. Most methods include single-cell analyses of the events that occur during apoptosis. These include visualizing cytochrome c release, assessing mitochondrial transmembrane potentials, evaluating translocation of Bcl-2-family proteins, assessing caspase activation and phosphatidylserine flip, and measuring plasma membrane integrity (Bouchier-Hayes et al., 2008). Although these methods provide valuable data, in vitro studies are not ideal because cultured cells may behave differently in the cellular and tissue context of in vivo apoptosis. In order to validate in vitro studies, animal models have been developed in which apoptotic regulatory molecules are deleted or overexpressed. For example, p53-deficient mice were used to show that p53 is essential for UVB-induced apoptosis. Transgenic mice for Bcl-x or K5-PTEN-flox/flox are available for assessing chemically induced tumors (Raj et al., 2006).

Figure 2. Apoptotic pathways. The extrinsic pathway is stimulated by binding of Fas ligand or related cytokines to “death receptors.” The intrinsic pathway is triggered by cytotoxic drugs and DNA damage, both of which lead to a cascade of programmed cell death. IAP, inhibitor of apoptosis.

4. On the basis of previously reported contrasting effects of LL-37 on apoptosis in different mammalian cells, and in view of the role of LL-37 in wound healing, Chamorro et al. (2009) studied the role of LL-37 in apoptosis by keratinocytes. They first showed that LL-37 suppresses camptothecin (CAM)-induced apoptosis. Two methods were used to evaluate membrane integrity. YO-PRO-1 and propidium iodine staining showed that LL-37 is effective in protecting keratinocytes from CAM-induced cell death. These findings were validated by DNA content analysis. Another confirmatory finding was that LL-37 decreases caspase-3, which is one of the key effector proteins for apoptosis. Chamorro et al. then applied the Significance Analysis of Microarrays algorithm, looking for the differences in expressed genes between LL-37-treated and untreated keratinocytes. Among the genes expressed, the authors focused on the apoptosis-related gene COX-2, finding an increase in its expression. They further confirmed this finding using real-time PCR and showed an increase in IAP-2, another anti-apoptotic gene. Accordingly, they demonstrated a COX-2-dependent increase in prostaglandin E-2 as a result of the addition of LL-37 to keratinocytes. Finally, they showed that inhibition of COX-2 repressed the anti-apoptotic effects of LL-37, strengthening the hypothesis that the anti-apoptotic role of LL-37 is—at least partly—via COX-2.

5. In no other organ system does apoptosis play as many vital roles as it does in skin. It is critical in balancing keratinocyte proliferation with formation of stratum corneum. An increase in apoptotic keratinocytes is a feature of a variety of cutaneous diseases, including toxic epidermal necrolysis, sunburn, graft-versus-host disease, alopecia areata, Kindler’s syndrome, lichen planus, incontinentia pigmenti, and cutaneous viral infections. Decreased apoptosis, on the other hand, can be seen in skin conditions characterized by epidermal hyperplasia and hyperkeratosis, e.g., skin cancer and psoriasis. Although apoptosis-based therapies have had limited use in patients, many have been suggested by animal models or are already in clinical development (Raj et al., 2006). LL-37 peptide could represent a way to treat skin conditions in which the apoptosis rate is elevated, such as toxic epidermal necrolysis. Adenoviral transfer of LL-37, currently used to treat cystic fibrosis (Bals et al., 1999), could be used in skin diseases with high apoptotic rates. Moreover, conditions that increase the level of LL-37 (as vitamin D3 does in skin) might be of indirect use in treating skin diseases with high rates of apoptosis. By contrast, suppressing LL-37 production could potentially be useful in therapy of skin diseases such as psoriasis and skin cancer. Several studies have shown the efficacy of COX-2 inhibitors in several types of cancer. A recent case report showed remission of an advanced melanoma during treatment with the COX-2 inhibitor rofecoxib (Lejeune et al., 2006). In psoriasis, IL-17 and TNF- α increase the level of COX-2, making COX-2 inhibition (and/or LL-37 inhibition) a potential therapeutic option for this disease. Nonetheless, many challenges must first be overcome, including the expense of peptide production, assurance of peptide stability, full elucidation of pharmacokinetics, and an assessment of potential toxicities.

6. Chamorro et al. (2009) assessed the role of LL-37 in CAM-induced apoptosis of keratinocytes. They looked at caspase-3, a key protein in the apoptosis pathway, and two important anti-apoptotic genes, COX2 and IAP-2. It is important to note that apoptotic stimuli may activate multiple pathways simultaneously. Therefore, it would be important to evaluate the effects of LL-37 on different pathways involved in apoptosis by keratinocytes. One of the important signaling pathways is p38 mitogen-activated protein kinase (MAPK), which is promoted by p53 and Bax. The MAPK pathway is modulated by epidermal growth factor (EGF) (Raj et al., 2006), which is known to protect keratinocytes against apoptosis. Given the role of EGF in wound healing and keratinocyte apoptosis, assessment of the function of LL-37 in this pathway would also be valuable. Keratinocytes, like most other cell types, are more susceptible to apoptosis in vitro than in vivo. Multiple survival signals present in tissues are likely to account for this increased keratinocyte resistance to apoptosis in vivo. Growth factors other than EGF, such as nerve growth factor and hepatocyte growth factor, protect keratinocytes against apoptosis by maintaining the level of Bcl-2 and Bcl-x. Whether they have any association with LL-37 would be important to know. Unlike other known caspases, which are expressed ubiquitously, caspase-14 is found only in skin (Raj et al., 2006). The effects of LL-37 on this caspase might help to explain the contrasting effects of LL-37 on apoptosis in different mammalian cell types. As mentioned above (question 3), animal models of apoptosis are available to validate in vitro findings. Future challenges include the examination of LL-37 in these animal models.

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¹ Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA