Expression and Modulation of LL-37 in Normal Human Keratinocytes, HaCaT cells, and Inflammatory Skin Diseases

Defensins and cathelicidins (LL-37) are major antimicrobial peptides (AMPs) of the innate immune system of the human skin. In normal non-inflamed skin these peptides are negligible, but their expression can be markedly increased in inflammatory skin disease such as psoriasis. We designed this study to identify the expressions of LL-37 in normal human keratinocyte (NHK) and HaCaT cells after exposure to stimulants and to investigate difference of LL-37 expression accompanied with cell differentiation status, and come to understand difference of susceptibility to infection in atopic dermatitis and psoriasis. Expressions of LL-37 in NHKs and HaCaT cells were evaluated by using RT-PCR, Western blotting, and immunohistochemical (IHC) staining at 6, 12, and 24 hr post stimulation after exposure to Ultraviolet B irradiation and lipopolysaccharide. And expression of LL-37 in skin biopsy specimens from patients with atopic dermatitis and psoriasis was determined by immunohistochemical analysis. In time-sequential analyses of LL-37 expression revealed that LL-37 was expressed in NHKs, but not in HaCaT cells. IHC analysis confirmed the presence of abundant LL-37 in the epidermis of psoriasis. Therefore we deduced that expression of LL-37 is affected by UV irradiation, bacterial infection, and status of cell differentiation.

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title: Human beta-defensin-2 production in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation.
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title: Cathelicidin Antimicrobial Peptides are Expressed in Salivary Glands and Saliva
abstract: The expression of antimicrobial peptides at epithelial surfaces such as skin, lung, and intestine is thought to provide protection against infection. Cathelicidin antimicrobial peptides are essential for the protection of skin against invasive bacterial infection. To determine if cathelicidins are also present in the oral cavity, we examined the expression of both mRNA and protein in mice and human saliva. The murine cathelicidin (CRAMP) was detected in the adult by reverse-transcription/polymerase chain-reaction (RT-PCR), and in embryonic, newborn, and adult tissues by in situ hybridization and immunohistochemistry. CRAMP mRNA and protein were localized to the salivary glands, specifically in acinar cells of the submandibular gland and palatine minor glands, as well as in lingual epithelium and palatal mucosa. In man, the human cathelicidin LL-37 was detected in human saliva by Western blotting. These results indicate that cathelicidins are present in the salivary system, in some oral epithelia, and in saliva, contributing to broad-spectrum defense of the oral cavity.

title: Expression of cathelicidin in human salivary glands.
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To examine the expression of cathelicidin in human salivary glands and to investigate up-regulation of cathelicidin in inflammatory conditions.



Reverse transcriptase-polymerase chain reaction and immunohistochemical staining were performed on 20 salivary gland tissues, 10 from normal salivary glands and 10 from glands with chronic sialadenitis.



Cathelicidin messenger RNA transcripts were detected in the tissues from the normal salivary glands and the glands with chronic sialadenitis. The level of cathelicidin messenger RNA in glands with chronic sialadenitis was significantly increased compared with that in normal salivary glands. Cathelicidin protein was expressed in the glandular epithelium of the normal salivary glands and the glands with chronic sialadenitis.



The results indicate that cathelicidin might play an important role in innate host defense of human salivary glands.

title: Differentiation-regulated expression of Toll-like receptors 2 and 4 in HaCaT keratinocytes
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title: The Expression of the Gene Coding for the Antibacterial Peptide LL-37 Is Induced in Human Keratinocytes during Inflammatory Disorders*
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title: Expressions of beta-defensins in human keratinocyte cell lines.
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Current research was done to investigate the expression and modulation of beta-defensin mRNA in human keratinocyte cell lines.



HaCaT and A431 cell lines were used to all culture experiments. Cultured human keratinocytes were stimulated with ultraviolet (UV) B irradiation or tumor necrosis factor-alpha (TNF-alpha) or lipopolysaccharide (LPS) to determine whether defensin mRNA production occurred. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify defesin cDNA from stimulated keratinocytes, and southern blots were used to verify the specificity of RT-PCR amplication products.



Expression of human beta-defensins was upregulated with UVB irradiation, TNF-alpha and LPS in HaCaT cells and in comparison to the control, significantly higher at 6 h post stimulation with UVB 100 mJ/cm2 and peak at 12 to 18 h post stimulation with UVB 30 mJ/cm2, TNF-alpha and LPS. A431 cells did not show expression of human beta-defensins in unstimulated state, even after irradiation with UVB or TNF-alpha or LPS.



This report demonstrates the presence of defensin in human keratinocytes and capacity of human keratinocytes to produce defensin mRNA in response to UVB irradiation, TNF-alpha and LPS. Release of defensins by keratinocytes in response to cytokines elaborated in inflammation may contribute to the host defense responses.

title: Antimicrobial peptides: an emerging concept in cutaneous biology.
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title: Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.
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title: Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.
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title: ENDOGENOUS ANTIMICROBIAL PEPTIDES AND SKIN INFECTIONS IN ATOPIC DERMATITIS
abstract: PY, Ohtake T, Brandt C, et al.  . 2002;347:1151–1160 

The immune system of human skin contains several antimicrobial peptides, particularly cathelicidins (LL-37) and β-defensins. Although these peptides are negligible in normal skin, they accumulate in skin affected by inflammatory diseases such as psoriasis. The purpose of this study was to compare the levels of expression of LL-37 and human β-defensin 2 (HBD-2) in inflamed skin from patients …

title: Cutaneous injury induces the release of cathelicidin anti-microbial peptides active against group A Streptococcus.
abstract: Cathelicidins are a family of peptides thought to provide an innate defensive barrier against a variety of potential microbial pathogens. The human and mouse cathelicidins (LL-37 and CRAMP, respectively) are expressed at select epithelial interfaces where they have been proposed to kill a number of gram-negative and gram-positive bacteria. To determine if these peptides play a part in the protection of skin against wound infections, the anti-microbial activity of LL-37 and CRAMP was determined against the common wound pathogen group A Streptococcus, and their expression was examined after cutaneous injury. We observed a large increase in the expression of cathelicidins in human and murine skin after sterile incision, or in mouse following infection by group A Streptococcus. The appearance of cathelicidins in skin was due to both synthesis within epidermal keratinocytes and deposition from granulocyctes that migrate to the site of injury. Synthesis and deposition in the wound was accompanied by processing from the inactive prostorage form to the mature C-terminal peptide. Analysis of anti-microbial activity of this C-terminal peptide against group A Streptococcus revealed that both LL-37 and CRAMP potently inhibited bacterial growth. Action against group A Streptococcus occurred in conditions that typically abolish the activity of anti-microbial peptides against other organisms. Thus, cathelicidins are well suited to provide defense against infections due to group A Streptococcus, and represent an important element of cutaneous innate immunity.

title: Mucoid Pseudomonas aeruginosa, TNF-alpha, and IL-1beta, but not IL-6, induce human beta-defensin-2 in respiratory epithelia.
abstract: Cultured lung epithelial cells release antibacterial activity upon contact with Pseudomonas aeruginosa (PA), which is impaired in cystic fibrosis (CF). In order to identify the factors responsible for killing PA by a biochemical approach, we purified antimicrobial activity from supernatants of the A549 lung epithelial cell line, previously stimulated with PA bacteria, by subsequent high performance liquid chromatography. NH(2)-terminal sequencing of a major bactericidal compound revealed it to be identical with human beta-defensin-2 (hBD-2). A mucoid phenotype of PA, but not two nonmucoid PA strains, high concentrations (> 10 microg/ml) of PA lipopolysaccharide, tumor necrosis factor alpha, and interleukin (IL)-1beta, but not IL-6, dose-dependently induced hBD-2 messenger RNA in cultured normal bronchial, tracheal, as well as normal and CF-derived nasal epithelial cells. Genomic analysis of hBD-2 revealed a promoter region containing several putative transcription factor binding sites, including nuclear factor (NF) kappaB, activator protein (AP)-1, AP-2, and NF-IL-6, known to be involved in the regulation of inflammatory responses. Thus, hBD-2 represents a major inducible antimicrobial factor released by airway epithelial cells either on contact with mucoid PA or by endogenously produced primary cytokines. Therefore, it might be important in lung infections caused by mucoid PA, including those seen in patients with CF.

title: Cathelicidin anti-microbial peptide expression in sweat, an innate defense system for the skin.
abstract: The eccrine gland is one of the major cutaneous appendages and secretes sweat. Its principal function is thermoregulation during exposure to a hot environment or physical exercise. In addition to this function, we show that LL-37, a member of cathelicidin family of anti-microbial peptides, is expressed in sweat. LL-37 protein and mRNA was seen in the eccrine structures of normal human skin by immunohistochemistry and in situ hybridization. LL-37 was localized to both the eccrine gland and sweat ductal epithelial cells, whereas dermcidin, a previously described natural antibiotic in sweat, was expressed only in the gland itself. The anti-microbial activity of LL-37 and dermcidin against various bacteria in the sweat ionic environment was demonstrated by solution colony forming assay using synthetic peptides, and in sweat obtained from normal volunteers. These results indicate that cathelicidin is secreted in human sweat, has potent anti-microbial activity against both gram-positive and gram-negative bacteria, and can, after processing from the preproform, provide a barrier for protection against infection. Thus, sweat represents a unique mode of delivery for potent innate immune effector molecules in the absence of inflammation.

title: An angiogenic role for the human peptide antibiotic LL-37/hCAP-18.
abstract: Antimicrobial peptides are effector molecules of the innate immune system and contribute to host defense and regulation of inflammation. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. Here we show that LL-37 induces angiogenesis mediated by formyl peptide receptor-like 1 expressed on endothelial cells. Application of LL-37 resulted in neovascularization in the chorioallantoic membrane assay and in a rabbit model of hind-limb ischemia. The peptide directly activates endothelial cells, resulting in increased proliferation and formation of vessel-like structures in cultivated endothelial cells. Decreased vascularization during wound repair in mice deficient for CRAMP, the murine homologue of LL-37/hCAP-18, shows that cathelicidin-mediated angiogenesis is important for cutaneous wound neovascularization in vivo. Taken together, these findings demonstrate that LL-37/hCAP-18 is a multifunctional antimicrobial peptide with a central role in innate immunity by linking host defense and inflammation with angiogenesis and arteriogenesis.

title: Ll-37, the Neutrophil Granule–And Epithelial Cell–Derived Cathelicidin, Utilizes Formyl Peptide Receptor–Like 1 (Fprl1) as a Receptor to Chemoattract Human Peripheral Blood Neutrophils, Monocytes, and T Cells
abstract: We have previously shown that antimicrobial peptides like defensins have the capacity to mobilize leukocytes in host defense. LL-37 is the cleaved antimicrobial 37-residue, COOH-terminal peptide of hCAP18 (human cationic antimicrobial protein with a molecular size of 18 kD), the only identified member in humans of a family of proteins called cathelicidins. LL-37/hCAP18 is produced by neutrophils and various epithelial cells. Here we report that LL-37 is chemotactic for, and can induce Ca2+ mobilization in, human monocytes and formyl peptide receptor–like 1 (FPRL1)-transfected human embryonic kidney 293 cells. LL-37–induced Ca2+ mobilization in monocytes can also be cross-desensitized by an FPRL1-specific agonist. Furthermore, LL-37 is also chemotactic for human neutrophils and T lymphocytes that are known to express FPRL1. Our results suggest that, in addition to its microbicidal activity, LL-37 may contribute to innate and adaptive immunity by recruiting neutrophils, monocytes, and T cells to sites of microbial invasion by interacting with FPRL1.

title: Neonatal Skin in Mice and Humans Expresses Increased Levels of Antimicrobial Peptides: Innate Immunity During Development of the Adaptive Response
abstract: The expression of antimicrobial peptides and proteins is an important innate immune defense mechanism that has recently been shown to be essential for cutaneous defense against invasive bacterial disease. Newborns have an immature cellular immune defense system that leads to increased susceptibility to infections. Here we show that skin from embryonic and newborn mice, as well as human newborn foreskin, express antimicrobial peptides of the cathelicidin and β-defensin gene families. Immunohistochemistry and in situ hybridization demonstrated abundant cathelicidin protein and mRNA is present in normal skin during the perinatal period. Quantitative real-time PCR showed mouse cathelicidin expression (CRAMP) is 10- to 100-fold greater in the perinatal period than adult. Murine β-defensins-1 and -4 and human β-defensin-2 were also present in newborn skin. Combined, human cathelicidin (LL-37/hCAP/18) and β-defensin-2 demonstrated synergistic antimicrobial activity and efficiently killed group B Streptococcus, an important neonatal pathogen. Antimicrobial peptides may therefore provide a compensatory innate defense mechanism during development of cellular immune response mechanisms in the newborn period.

title: A peptide antibiotic from human skin
abstract: To avoid opportunistic infections, plants and animals have developed antimicrobial peptides in their epithelia that can form pores in the cytoplasmic membrane of microorganisms. After contact with microorganisms, vertebrate skin, trachea and tongue epithelia are rich sources of peptide antibiotics, which may explain the unexpected resistance of these tissues to infection. Here we report that human skin is protected in a similar way by an inducible, transcriptionally regulated, antibiotic peptide, which resembles those in other mammals.