Masamoto Murakami

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.

Neonatal Skin in Mice and Humans Expresses Increased Levels of Antimicrobial Peptides: Innate Immunity During Development of the Adaptive Response

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.

Cathelicidin antimicrobial peptide LL-37 in psoriasis enables keratinocyte reactivity against TLR9 ligands

Here we show that keratinocytes in psoriatic lesional skin express increased Toll-like receptor (TLR) 9 that similarly localizes with elevated expression of the cathelicidin antimicrobial peptide LL-37. In culture, normal human keratinocytes exposed to LL-37 increased TLR9 expression. Furthermore, when keratinocytes were exposed to LL-37 and subsequently treated with TLR9 ligands such as CpG or genomic DNA, keratinocytes greatly increased production of type I interferons. This response mimicked observations in the epidermis of psoriatic lesional skin as keratinocytes in psoriatic lesions produce greater amounts of interferon-β than normal skin lacking LL-37. The mechanism for induction of type I interferons in keratinocytes was dependent on TLR9 expression but not on a DNA-LL-37 complex. These findings suggest that keratinocytes recognize and respond to DNA and can actively participate in contributing to the immunological environment that characterizes psoriasis.

Expression and secretion of cathelicidin antimicrobial peptides in murine mammary glands and human milk

Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.

Tight junctions in the stratum corneum explain spatial differences in corneodesmosome degradation.

Abstract:u2002 To maintain stratum corneum integrity while simultaneously desquamating at a steady rate, degradation of corneodesmosomes must proceed in a controlled manner. It is unknown why corneodesmosomes are present only at the cell periphery in the upper stratum corneum. To explore this, we studied distributions of three major corneodesmosomal components, corneodesmosin, desmoglein 1 and desmocollin 1 in normal adult human epidermis. Immunofluorescent microscopy studies of skin surface corneocytes detected all three components only at the cell edges. Immunoelectron microscopy revealed selective loss of these components at the central areas starting from the deep cornified layers. We hypothesized that tight junctions (TJs) formed in the superficial granular layer may prevent protease access by functioning as a barrier between the peripheral and the central intercellular spaces in the stratum corneum. Ultrastructural examination demonstrated TJs up to the junctions between the seventh and the eighth deepest cornified layers. Immunoelectron microscopy also detected clusters of occludin and claudin‐1 immunolabels at the cell periphery, and kallikrein 7 immunolabels outside of TJs in the lower cornified layers. With colloidal lanthanum nitrate perfusion assay of stripped stratum corneum, the tracer was excluded from TJ domains. Taken together, we propose that TJs inhibit access of proteases to the peripheral corneodesmosomes forming the structural basis for the basket‐weave‐like appearance of the stratum corneum.

Patients with palmoplantar pustulosis have increased IL-17 and IL-22 levels both in the lesion and serum

Abstract:u2002 Recent findings about the pathogenesis of pustulosis palmaris et plantaris (PPP), also known as palmoplantar pustulosis, suggest that IL‐17 expression in the acrosyringium as well as infiltration of IL‐17 positive cells, e.g. Langerhans cells may play important roles. However, to date, it has not been established whether circulating IL‐17 related cytokines are involved in PPP. We studied the circulating IL‐17 related cytokines as well as the mRNA levels in lesional skin. IL‐17 related cytokine mRNAs were increased in the PPP lesions compared with the control tissues (five patients vs five controls). The serum levels of TNF‐alpha, IL‐17, IL‐22 and IFN‐gamma also were significantly increased in PPP, but not IL‐23 and IL‐8 (48 patients vs 20 controls). Our findings document that not only the serum IL‐17 but also tissue IL‐17 are elevated in PPP and may be in the pathogenesis of this disorder.

Acrosyringium Is the Main Site of the Vesicle/Pustule Formation in Palmoplantar Pustulosis

Pustulosis palmaris et plantaris or palmoplantar pustulosis (PPP) is a refractory pustular eruption on the palms and soles with unknown etiology. Numerous eccrine sweat pores exist on the palms and soles, suggesting the involvement of eccrine sweating in the pathogenesis of PPP. To the best of our knowledge, however, no definite abnormality in sweating has been documented in PPP. Accordingly, we analyzed the eccrine sweat duct involvement in the mechanism of vesicle formation in PPP. Dermatoscopy showed that PPP vesicles are located on the top of the ridges but not in the furrows. The sweat secretion in the lesional area was much lower than that in the nonlesional area, with or without pain stimulation to induce sweating. Immunostaining of horizontal sections of the lesions using antibodies against gross cystic disease fluid protein-15 (GCDFP-15) and epithelial membrane antigen (EMA) showed that these markers were localized in the cells lining the intraepidermal vesicles. Although the sweat antimicrobial peptides, dermcidin and human cathelicidin antimicrobial peptide 18 (hCAP-18)/LL-37, were detected in the fluid of the vesicles/pustules, neither dermcidin nor hCAP-18/LL-37 were overexpressed by neighboring keratinocytes. These findings suggest that the acrosyringium may be involved as the main site of the vesicle formation in the pathomechanism of PPP.

Lamellar Granule Secretion Starts before the Establishment of Tight Junction Barrier for Paracellular Tracers in Mammalian Epidermis

Defects in epidermal barrier function and/or vesicular transport underlie severe skin diseases including ichthyosis and atopic dermatitis. Tight junctions (TJs) form a single layered network in simple epithelia. TJs are important for both barrier functions and vesicular transport. Epidermis is stratified epithelia and lamellar granules (LGs) are secreted from the stratum granulosum (SG) in a sequential manner. Previously, continuous TJs and paracellular permeability barriers were found in the second layer (SG2) of SG in mice, but their fate and correlation with LG secretion have been poorly understood. We studied epidermal TJ-related structures in humans and in mice and found occludin/ZO-1 immunoreactive multilayered networks spanning the first layer of SG (SG1) and SG2. Paracellular penetration tracer passed through some TJs in SG2, but not in SG1. LG secretion into the paracellular tracer positive spaces started below the level of TJs of SG1. Our study suggests that LG-secretion starts before the establishment of TJ barrier in the mammalian epidermis.

Characterization of the expression and function of N-methyl-D-aspartate receptor in keratinocytes

Abstract:u2002 The N‐methyl‐D‐aspartate (NMDA) receptor is expressed on neural tissue where it gates calcium ion entry upon stimulation. Using immunohistochemistry, it has been demonstrated in this study that the NMDAR1 receptor is also expressed on keratinocytes (KCs) in normal human skin and inflamed psoriatic skin in vivo. Furthermore, the NMDA receptor was functional as demonstrated by the ability of this receptor to trigger Ca++ influx in KCs. Incubation of cultured, human KCs with MK‐801 decreases the cell growth and induces an increase in apoptosis. These findings demonstrate that the KC expression of NMDA receptor is a mechanism through which the influx of Ca++ into the cell can be regulated and suggest that the expression of this receptor may play a role in the regulation of KC growth and differentiation.

Recognition of prostate and melanoma tumor cells by six-transmembrane epithelial antigen of prostate-specific helper T lymphocytes in a human leukocyte antigen class II-restricted manner.

The six-transmembrane epithelial antigen of prostate (STEAP) protein is an attractive candidate for T cell-based immunotherapy because it is overexpressed in prostate cancer and various other tumor types. Several peptide epitopes capable of stimulating CTLs that killed STEAP-expressing tumor cells have been described. Our goal was the identification of helper T lymphocyte (HTL) epitopes of STEAP for the optimization of T cell-based immunotherapies against STEAP-expressing malignancies. Candidate HTL epitopes for STEAP were predicted using in silico algorithms for HLA class II-binding peptides and were tested for their ability to elicit HTL responses by in vitro peptide vaccination of CD4 T lymphocytes from healthy individuals and prostate cancer patients. Two peptides (STEAP(102-116) and STEAP(192-206)) were effective in stimulating in vitro antitumor HTL responses in both normal individuals and prostate cancer patients. Notably, both STEAP HTL peptides behaved as promiscuous T-cell epitopes because they stimulated T cells in the context of more than one MHC class II allele. These newly described STEAP HTL epitopes could be of value for the design and optimization of T cell-based immunotherapy against STEAP-expressing tumors.