Beverly A. Dale

Interleukin 20: discovery, receptor identification, and role in epidermal function.

A structural, profile-based algorithm was used to identify interleukin 20 (IL-20), a novel IL-10 homolog. Chromosomal localization of IL-20 led to the discovery of an IL-10 family cytokine cluster. Overexpression of IL-20 in transgenic (TG) mice causes neonatal lethality with skin abnormalities including aberrant epidermal differentiation. Recombinant IL-20 protein stimulates a signal transduction pathway through STAT3 in a keratinocyte cell line, demonstrating a direct action of this ligand. An IL-20 receptor was identified as a heterodimer of two orphan class II cytokine receptor subunits. Both receptor subunits are expressed in skin and are dramatically upregulated in psoriatic skin. Taken together, these results demonstrate a role in epidermal function and psoriasis for IL-20, a novel cytokine identified solely by bioinformatics analysis.

Inducible expression of human beta-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier.

ABSTRACT Human gingival epithelial cells (HGE) express two antimicrobial peptides of the β-defensin family, human β-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract of Fusobacterium nucleatum, an oral commensal microorganism, but not by that of Porphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated with F. nucleatum cell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-α and F. nucleatum cell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower (∼10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-α as an intermediary in F. nucleatum signaling was ruled out by addition of anti-TNF-α that did not inhibit hBD-2 induction. However, inhibitor studies show that F. nucleatum stimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated from Escherichia coli andF. nucleatum were poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.

Epithelial structural proteins of the skin and oral cavity: function in health and disease.

Epithelial tissues function to protect the organism from physical, chemical, and microbial damage and are essential for survival. To perform this role, epithelial keratinocytes undergo a well-defined differentiation program that results in the expression of structural proteins which maintain the integrity of epithelial tissues and function as a protective barrier. This review focuses on structural proteins of the epidermis and oral mucosa. Keratin proteins comprise the predominant cytoskeletal component of these epithelia. Keratin filaments are attached to the plasma membrane via desmosomes, and together these structural components form a three-dimensional array within the cytoplasm of epithelial cells and tissues. Desmosomes contain two types of transmembrane proteins, the desmogleins and desmocollins, that are members of the cadherin family. The desmosomal cadherins are linked to the keratin cytoskeleton via several cytoplasmic plaque proteins, including desmoplakin and plakoglobin (gamma-catenin). Epidermal and oral keratinocytes express additional differentiation markers, including filaggrin and trichohyalin, that associate with the keratin cytoskeleton during terminal differentiation, and proteins such as loricrin, small proline-rich proteins, and involucrin, that are cross-linked into the cornified envelope by transglutaminase enzymes. The importance of these cellular structures is highlighted by the large numbers of genetic and acquired (autoimmune) human disorders that involve mutations in, or autoantibodies to, keratins and desmosomal and cornified envelope proteins. While much progress has been made in the identification of the structural proteins and enzymes involved in epithelial differentiation, regulation of this process is less clear. Both calcium and retinoids influence epithelial differentiation by altering the transcription of target genes and by regulating activity of enzymes critical in epithelial differentiation, such as transglutaminases, proteinases, and protein kinases. These studies have furthered our understanding of how epithelial tissue and cell integrity is maintained and provide a basis for the future treatment of skin and oral disorders by gene therapy and other novel therapeutics.

Single-Nucleotide Polymorphisms (SNPs) in Human β-Defensin 1: High-Throughput SNP Assays and Association with Candida Carriage in Type I Diabetics and Nondiabetic Controls

ABSTRACT β-Defensins are cationic antimicrobial peptides expressed in epithelia. They exhibit antibacterial, antifungal, and antiviral properties. Defensins are a component of the innate immune response, and it has been proposed that they have a protective role in the oral cavity. Previous studies have shown that human β-defensin 1 (hBD-1) is constitutively expressed in oral epithelial cells but that expression varies between individuals. We tested the hypothesis that genetic variations in defensin peptide expression may be associated with opportunistic infections. This may be critical in the immunocompromised patient population, in which innate immune responses may have a relatively more important role. Oral Candida carriage status and the presence of six single-nucleotide polymorphisms (SNPs) in the DEFB1 gene encoding hBD-1 were evaluated in type I diabetic patients (n = 43) and nondiabetic controls (n = 50). Genomic DNA was obtained from buccal swabs. Portions of the DEFB1 gene were amplified, and each SNP was analyzed by a TaqMan assay, standardized with control DNA of known genotype. Candida carriage status was determined from unstimulated saliva on CHROMagar plating medium. A low level of Candida carriage was defined as ≤350 CFU/ml. A high level of Candida carriage was seen in 44% of the diabetic subjects but only in 28% of the nondiabetic controls (P < 0.05). C. albicans predominated; however, diabetic subjects, especially those with high levels of carriage, showed an increased proportion of Candida glabrata and C. tropicalis. There was a strong association between an SNP in the 5′ untranslated region (C→G at position −44) and Candida carriage in both groups. Among individuals in the diabetic population who had the SNP allele 2 (G), 58% had low CFU, while 6% had high CFU. The C→G SNP at position −44 is associated with low levels of Candida carriage. The resultant odd ratios are statistically significant for a protective effect (odd ratios, 25 for diabetic subjects and 8.5 for nondiabetic subjects). These results indicate that genetic variations in the DEFB1 gene encoding hBD-1 may have a major role in mediating and/or contributing to susceptibility to oral infection.

Regulation of human beta-defensin-2 in gingival epithelial cells: the involvement of mitogen-activated protein kinase pathways, but not the NF-kappaB transcription factor family.

Stratified epithelia of the oral cavity are continually exposed to bacterial challenge that is initially resisted by neutrophils and epithelial factors, including antimicrobial peptides of the β-defensin family. Previous work has shown that multiple signaling pathways are involved in human β-defensin (hBD)-2 mRNA regulation in human gingival epithelial cells stimulated with a periodontal bacterium, Fusobacterium nucleatum, and other stimulants. The goal of this study was to further characterize these pathways. The role of NF-κB in hBD-2 regulation was investigated initially due to its importance in inflammation and infection. Nuclear translocation of p65 and NF-κB activation was seen in human gingival epithelial cells stimulated with F. nucleatum cell wall extract, indicating possible involvement of NF-κB in hBD-2 regulation. However, hBD-2 induction by F. nucleatum was not blocked by pretreatment with two NF-κB inhibitors, pyrrolidine dithiocarbamate and the proteasome inhibitor, MG132. To investigate alternative modes of hBD-2 regulation, we explored involvement of mitogen-activated protein kinase pathways. F. nucleatum activated p38 and c-Jun NH2-terminal kinase (JNK) pathways, whereas it had little effect on p44/42. Furthermore, inhibition of p38 and JNK partially blocked hBD-2 mRNA induction by F. nucleatum, and the combination of two inhibitors completely blocked expression. Our results suggest that NF-κB is neither essential nor sufficient for hBD-2 induction, and that hBD-2 regulation by F. nucleatum is via p38 and JNK, while phorbol ester induces hBD-2 via the p44/42 extracellular signal-regulated kinase pathway. Studies of hBD-2 regulation provide insight into how its expression may be enhanced to control infection locally within the mucosa and thereby reduce microbial invasion into the underlying tissue.

Innate Immune Response of Oral and Foreskin Keratinocytes: Utilization of Different Signaling Pathways by Various Bacterial Species

ABSTRACT The innate immune response is critical for the epithelial antimicrobial barrier. The human β-defensins are small, cationic antimicrobial peptides that are made by epithelial cells and that play a role in mucosal and skin defenses. Human β-defensin 1 (hBD-1) is expressed constitutively in epithelial tissues, whereas hBD-2 and hBD-3 are expressed in response to bacterial stimuli or inflammation. Previous studies showed that hBD-2 was induced by Fusobacterium nucleatum cell wall extract without the involvement of the NF-κB transcription factors, which typically are associated with innate immunity and inflammation. The goal of this study was to characterize signaling pathways involved in hBD-2 induction in response to commensal and pathogenic bacteria. Cultured human oral and foreskin keratinocytes were treated separately with inhibitors of NF-κB, c-Jun N-terminal kinase (JNK), and p38 and then stimulated with oral commensal Streptococcus gordonii, oral pathogens Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans, skin commensal Staphylococcus epidermidis, or skin pathogen Streptococcus pyogenes. Different bacteria induced different levels of hBD-2 and in response to the various inhibitors tested, although certain common patterns were observed for commensal- and pathogen-stimulated cells. hBD-2 induction by all bacteria tested was partially or completely blocked by inhibitors of the JNK and p38 pathways. However, in addition, hBD-2 induction by pathogenic bacteria in both oral and foreskin keratinocytes was blocked by inhibitors of NF-κB. The results indicate that commensal and pathogenic bacteria utilize different pathways in hBD-2 induction and suggest that epithelial cells from different body sites have common signaling mechanisms to distinguish between commensal and pathogenic bacteria.

Salivary Antimicrobial Peptide Expression and Dental Caries Experience in Children

ABSTRACT Dental caries is a major worldwide oral disease problem in children. Although caries are known to be influenced by dietary factors, the disease results from a bacterial infection; thus, caries susceptibility may be affected by host factors such as salivary antimicrobial peptides. This study aimed to determine a possible correlation between caries prevalence in children and salivary concentrations of the antimicrobial peptides human beta-defensin-3 (hBD-3), the cathelicidin LL37, and the alpha-defensins HNP1-3 (a mixture of HNP1, 2, 3). Oral examinations were performed on 149 middle school children, and unstimulated whole saliva was collected for immunoassays of the three peptides and for assay of caries-causing bacteria in saliva. The median salivary levels of hBD-3, LL37, and HNP1-3 were in the microgram/ml range but were highly variable in the population. While levels of LL37 and hBD-3 did not correlate with caries experience, the median HNP1-3 levels were significantly higher in children with no caries than in children with caries. Children with high caries levels did not have high levels of salivary Streptococcus mutans, and the HNP1-3 level was not correlated with salivary S. mutans. By immunohistochemistry we localized HNP1-3 in submandibular salivary duct cells. HNPs are also released by neutrophils into the gingival crevicular fluid. Both sources may account for their presence in saliva. Low salivary levels of HNP1-3 may represent a biological factor that contributes to caries susceptibility. This observation could lead to new ways to screen for caries susceptibility and to new means of assessing the risk for this common oral problem.

The characterization of human epidermal filaggrin. A histidine-rich, keratin filament-aggregating protein

Filaggrin is a histidine-rich, cationic protein that aggregates with keratin filaments in vitro and may function as the keratin matrix protein in the terminally differentiated cells of the epidermis. This protein has been previously isolated from rodent epidermis. In this investigation, a similar protein from human skin was identified, isolated and characterized by biochemical and immunologic techniques. Indirect immunofluorescence of human skin using antiserum to rat filaggrin gave positive immunofluorescence of keratohyalin granules and the stratum corneum. This indicated the presence of a human filaggrin in the epidermis in a localization similar to that of the rodent. The protein was isolated from human epidermis and purified by ion-exchange chromatography and preparative gel electrophoresis. The purified protein crossreacts with antibody to rat filaggrin and migrates as a doublet of molecular weight (Mr) approximately 35 000 on SDS-polyacrylamide gels. It is relatively rich in polar amino acids such as histidine, arginine, serine and glycine, but is poor in nonpolar amino acids. Unlike rodent filaggrin, the human protein contains ornithine. This protein aggregates with human keratin filaments, forming compact macrofibrils in a manner analogous to that of rodent filaggrin. Thus, a human epidermal protein has been isolated which has many of the characteristics of rodent filaggrin and may function as the human keratin matrix protein.

Detection of β-defensins secreted by human oral epithelial cells

Abstract Human β-defensins are antimicrobial peptides that may be critical in the innate immune response to infection. hBD1 and hBD2 are expressed in oral epithelial cells and are detected near the surface of oral tissue, consistent with a role in the epithelial protective barrier function. In this report, we examine secretion of β-defensins in vitro and in biological fluid using ProteinChip® Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry. We show that the 47-amino acid form of hBD1 and the 41-amino acid form of hBD2 are the major secreted forms. These forms are both expressed and secreted under conditions anticipated from previous analysis of β-defensin mRNAs; specifically, hBD1 is detected in culture supernatant from both unstimulated and stimulated cells, and hBD2 is detected only in stimulated cells. Identity of hBD1 and hBD2 was confirmed by immunocapture on the ProteinChip surface. Both peptides are also present in gingival crevicular fluid that accumulates between the tissue and tooth surface, although hBD1 is also found in several smaller forms suggesting extracellular proteolysis. This methodology offers several technical advantages for detection of defensins in biological fluids, including ease and speed of screening, no need for HPLC preliminary processing, and small sample size.

Purification and characterization of a basic protein from the stratum corneum of mammalian epidermis

A basic protein has been isolated and purified from the stratum corneum of newborn rat epidermis. This protein is referred to as stratum corneum basic protein. It was purified by ion-exchange chromatography on DE-52 and CM-52 cellulose. The protein has a molecular weight of 50 000 on sodium dodecyl sulfate-polyacrylamide gels. It is composed of one polypeptide chain and contains no detectable carbohydrate. The protein has an isoelectric point in the range of pH 9-10, but decomposes during isoelectric focusing giving rise to a polypeptide of less than 10 000 daltons. Amino acid analysis reveals high quantities of glutamic acid, glycine, serine, arginine and relatively high levels of histidine, with these five amino acids composing 74% of the total residues. The amino acid analysis is very similar to histidine-containing keratohyalin proteins isolated from the granular layer of epidermis by several investigators. The stratum corneum basic protein differs from fibrous proteins isolated from the same cell layer with respect to net charge, amino acid composition, and molecular weight. The protein does not react with antibody to the fibrous protein. The basic protein has properties which are consistent with its possible function as a stratum corneum interfilamentous matrix protein.