Histamine differentially regulates the production of Th1 and Th2 chemokines by keratinocytes through histamine H1 receptor

Histamine is a biological amine that plays an important role in allergic responses. However, the involvement of histamine signaling in late allergic responses in the skin is poorly understood. Therefore, we attempted to investigate the involvement of histamine signaling in late allergic responses, especially in keratinocytes (KCs). HaCaT KCs and normal human KCs (NHKs) predominantly expressed histamine H1 receptor (H1R) and H2 receptor (H2R). Histamine suppressed tumor necrosis factor α (TNF-α)- and interferon-γ (IFN-γ)-induced production of CC chemokine ligand 17(CCL17), a type 2 T-helper (Th2) chemokine, by HaCaT KCs. It suppressed the phosphorylation of p38 mitogen-activated protein (MAP) kinase, but not that of extracellular signal-regulated kinases (ERKs), and TNF-α- and IFN-γ-induced nuclear factor κB (NFκB) activity. In contrast, histamine enhanced the production of CXC chemokine ligand 10 (CXCL10), a Th1 chemokine, by TNF-α- and IFN-γ-stimulated HaCaT KCs and NHKs. TNF-α- and IFN-γ-induced CXCL10 production was upregulated by suppression of p38 MAP kinase or NF-κB activity, which could explain histamine involvement. We concluded that histamine suppresses CCL17 production by KCs by suppressing p38 MAP kinase and NF-κB activity through H1R and may act as a negative-feedback signal for existing Th2-dominant inflammation by suppressing CCL17 and enhancing CXCL10 production.     

Efficient production of a soluble fusion protein containing human beta-defensin-2 in E. coli cell-free system

Human beta-defensin-2 (hBD2), a small cationic peptide, exhibits a broad range of antimicrobial activity and does not cause microbial resistance. In order to produce hBD2 efficiently, an Escherichia coli cell-free biosynthesis system has been developed as an alternative method. A specific plasmid pIVEX2.4c-trxA-shBD2 was constructed for the cell-free expression of fusion protein (hBD2 linked with His-Tag and Trx-Tag). This allowed enhancement of protein stability and facilitated downstream purification process. Significant amount of target fusion protein was synthesized in the batch-mode bioreactor by optimizing the reaction conditions. About five-fold improvement of productivity (ca. 2.0 mg/ml soluble fusion protein) could be achieved by using a continuous exchange cell-free (CECF) system compared to batch system. One-step affinity chromatographic process was developed to recover high purity fusion protein (95.2%) with overall recovery ratio of about 50%. The fusion protein was cleaved by cyanogens bromide (CNBr), and the mature hBD2 had demonstrated strong inhibition on the growth of E. coli D31 at low concentration.     

Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells

Defensins, a family of antimicrobial peptides, are one of the first lines of host defense. Human beta-defensins (hBD) such as hBD-2 and -3 have anti-HIV activity. Previous studies have shown that HIV-1 virion can induce the expression of hBD, although the exact components of HIV-1 virion that are responsible for hBD expression have not yet been elucidated. In this study, we examined the effect of HIV-1 Tat on the expression of hBD in B cells. Stimulation of B cells with HIV-1 Tat protein significantly increased the mRNA and protein levels of hBD-2. HIV-1 Tat also induced the activation of a reporter gene for hBD-2 in a dose-dependent manner in B cells. Pretreatment of B cells with a JNK inhibitor suppressed HIV-1 Tat-induced hBD-2 expression. Pretreatment of B cells with AP-1 inhibitors or NF-κB inhibitors led to a decrease in HIV-1 Tat-induced protein and mRNA expression of hBD-2. Taken together, our results indicate that HIV-1 Tat can up-regulate the expression of hBD-2 via JNK-NF-κB/AP-1-dependent pathways in human B cells.     

Salmonella enteritidis FliC (flagella filament protein) induces human beta-defensin-2 mRNA production by Caco-2 cells

Antimicrobial peptides are crucial for host defense at mucosal surfaces. Bacterial factors responsible for induction of human beta-defensin-2 (hBD-2) mRNA expression in Caco-2 human carcinoma cells were determined. Salmonella enteritidis, Salmonella typhimurium, Salmonella typhi, Salmonella dublin, and culture supernatants of these strains induced hBD-2 mRNA expression in Caco-2 human carcinoma cells. Using luciferase as a reporter gene for a approximately 2.1-kilobase pair hBD-2 promoter, the hBD-2-inducing factor in culture supernatant of S. enteritidis was isolated. The supernatant factor was heat-stable and proteinase-sensitive. After purification by anion exchange and gel filtration chromatography, the hBD-2-inducing factor was identified as a 53-kDa monomeric protein with the amino-terminal sequence AQVINTNSLSLLTQNNLNK, which is identical to that of the flagella filament structural protein (FliC) of S. enteritidis. Consistent with this finding, the 53-kDa protein reacted with anti-FliC antibody, which prevented its induction of hBD-2 mRNA in Caco-2 cells. In agreement, the hBD-2-inducing activity in culture supernatant was completely neutralized by anti-FliC antibody. In gel retardation analyses, FliC increased binding of NF-kappaB (p65 homodimer) to hBD-2 gene promoter sequences. We conclude that S. enteritidis FliC induces hBD-2 expression in Caco-2 cells via NF-kappaB activation and thus plays an important role in up-regulation of the innate immune response.     

Pyridoxine stimulates filaggrin production in human epidermal keratinocytes

Molecular Biology Reports - Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical...     

Induction of human beta-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines

Defensins are cationic peptides with broad-spectrum antimicrobial activity. They are members of a supergene family consisting of alpha and beta subtypes and each subtype is comprised of a number of different isoforms. For example, human alpha-defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human beta-defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD-1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD-1 or HBD-2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD-1 and HBD-2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 microgram/mL, interleukin-1 beta (IL-1beta) at 1-50 ng/mL, or tumor necrosis factor alpha (TNF-alpha) at the same concentrations. The expression of HBD-1 and HBD-2 mRNAs was monitored by RT-PCR. The cDNA products were sequenced to characterize the gene product. HBD-2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD-1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD-2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL-1beta or TNF-alpha. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD-2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.     

Cloning and expression of human beta-defensin-2 gene in Escherichia coli

Human beta-defensin-2 (hBD-2), a small cationic peptide, exhibits a broad range of antimicrobial activity. It has been found to play important roles in innate and adaptive immune responses against microbial invasion. For the purposes of this study, hBD-2 gene was cloned from the lesions of human condyloma acuminatum. An expression vector was constructed and transformed into E. coli. hBD-2 was expressed as a fusion protein in both the soluble and insoluble forms, which was further confirmed by western blotting analysis.     

Growth factor production from fibrin-encapsulated human keratinocytes

Fibrin has been used extensively in cell encapsulation because it has important biological properties. Keratinocyte encapsulation in fibrin is a widely used technique in skin tissue engineering. The production of growth factors (EGF, TGF-β1 and PDGF-BB) was evaluated when keratinocytes are encapsulated in fibrin. Secretions of TGF-β1 and PDGF-BB increased more than five times compared to monolayer cultures. Encapsulated cells secreted about 80% active form of TGF-β1 (monolayer cells only secreted inactive form). An enhanced secretion of TGF-β1 and PDGF-BB was found in encapsulated cells, showing that fibrin capsules are favourable for the production of these growth factors.     

Toll-like receptor 2-mediated expression of beta-defensin-2 in human corneal epithelial cells

We previously showed that human corneal epithelial cells (HCECs) express Toll-like receptors (TLRs), which recognize gram-positive bacteria and respond to Staphylococcus aureus infection by the expression and secretion of proinflammatory cytokines and beta-defensin-2 (hBD2). In this study, we further elucidated the underlying mechanisms regulating hBD-2 expression and its role in innate defense in HCECs in response to S. aureus challenge. Exposure of HUCL cells, a telomerase-immortalized HCEC line, to S. aureus, its exoproducts (1:10 dilution), or synthetic lipopeptide Pam3Cys (10 microg/ml) resulted in the up-regulation of hBD-2, but not hBD1 and hBD3. Similar to HUCL cells, primary HCECs responded to S. aureus-exoproducts and Pam3Cys challenge by expressing hBD2 mRNA and secreting hBD2 into the culture media. Furthermore, these stimuli induced the expression of TLR2 at both mRNA and protein levels. Consistently with its role as a major pattern-recognizing receptor, TLR2 was located at the cell surface by cell surface biotinylation. The treatment of HUCL cells with TLR2 neutralizing antibody resulted in a significant decrease in Pam3Cys-induced hBD2 production as well as IL-6, IL-8, and TNF-alpha secretion. The Pam3Cys-induced hBD2 expression was completely blocked by NF-kappaB inhibitors and partially inhibited by p38 MAP kinase and the JNK inhibitors. Conditioned media derived from HCECs challenged with S. aureus-exoproducts or Pam3Cys exhibited antibacterial activity against S. aureus, Pseudomonas aeruginosa and Escherichia coli. These findings suggest that S. aureus induces hBD2 production through TLR2-mediated pathways in HCECs and that pathogen-challenged, TLR-activated HCECs possess antimicrobial activity. Thus, the epithelium might play a role in innate defense against bacterial infection by directly killing bacteria in the cornea.     

High-level production of bioactive human beta-defensin-4 in Escherichia coli by soluble fusion expression

Human beta-defensin-4 (hBD4) is a cationic 50-amino acid antimicrobial peptide with three conserved cysteine disulfide bonds. It exhibits a broad antimicrobial spectrum. This study describes the synthesis of hBD4 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released hBD4. A PCR-based gene SOEing (splicing by overlap extension) synthesis method was used in the synthesis of the hBD4 gene with optimized codons. By constructing the expression plasmid (pET32-smhBD4), high concentration of soluble hBD4 fusion protein (1.9 g/l) can be obtained in E. coli. Further optimization studies showed that the expression system was very efficient to produce soluble target protein, and the solubility of the target protein could attain more than 99% even when the culture temperature was as high as 37°C. The highest productivity (2.68 g/l) of the hBD4 fusion protein was achieved by cultivating the E. coli (pET32-smhBD4) in MBL medium at 34°C, inducing the culture at the mid-exponential phase with 0.4-mM isopropyl β-d-galactopyranoside (IPTG), and collecting the broth after 6-h expression. The soluble target protein accounted for 64.6% of the total soluble proteins, and the mature hBD4 expression level was stoichiometrically estimated to be 0.689 g/l. This fusion protein was then purified and cleaved to get the mature hBD4 peptide that showed antimicrobial activity against E. coli and Pseudomonas aeruginosa.     

Histamine modulates the production of interferon-gamma and interleukin-2 by mitogen-activated human mononuclear blood cells

Histamine inhibited the production of interferon-gamma and interleukin 2 (IL-2) induced in human peripheral blood mononuclear cells by Staphylococcal Enterotoxin A (SEA) but had no effect on the expression of IL-2 receptors. The effects on lymphokine production were dose dependent with maximal inhibition occurring at histamine concentrations of 10(-4) to 10(-6) M. The H2-agonist 4-methylhistamine but not the H1-agonist 2-methylhistamine modulated lymphokine production in a similar manner as histamine. Histamine at concentrations of 10(-3) to 10(-8) M had no inhibitory effect directly on the activity of admixed IL-2 containing medium. The inhibitory effects of histamine could be reversed by the H2-antagonist cimetidine but not by the H1-antagonist diphenhydramine. This indicates that the inhibitory effects of histamine on lymphokine production are mediated through H2-receptors on mononuclear cells.     

Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes

The epidermis has developed physical and immunological barriers that prevent infiltration of deleterious chemicals and pathogens. As a first step to understanding the relationship between these barriers, we investigated whether TLR2 activation functionally alters tight junctions (TJs) in cultured human keratinocytes. Stimulation with peptidoglycan, a ligand for TLR2, elevated the TJ-associated barrier in the space of 3 h. The increase in TJ-associated barrier function due to peptidoglycan stimulation was suppressed by the knockdown of TLR adaptor MyD88 or the pretreatment with TLR2-neutralizing Ab, indicating that TLR2 activation enhanced TJ-associated barrier. One and 3 h after peptidoglycan stimulation, expression levels of the TJ proteins occludin, claudin-1, claudin-4, and ZO-1 were unchanged. However, immunoprecipitation studies demonstrated that the association of phospho-atypical protein kinase Cζ/ι, crucial for TJ biogenesis, with occludin was increased. Significantly, inhibition of atypical protein kinase Cζ/ι activity completely blocked the immediate elevation of the TJ-associated barrier. Finally, peptidoglycan was applied to the stratum corneum surface of a human skin equivalent, and the TJ barrier was evaluated. In the space of 3 h after the stimulation, the amount of intercellular tracer in the stratum corneum incubated from the dermal side was reduced, indicating that the TJ barrier is strengthened via TLR2 activation. Taken together, our findings indicated that infiltration of pathogens into the epidermis immediately enhanced TJ function via TLR2 signaling. Furthermore, the dynamically controlled TJs in skin are considered fundamental in preventing further invasion of pathogens and maintaining cutaneous barrier homeostasis.     

NOD2/CARD15 mediates induction of the antimicrobial peptide human beta-defensin-2

Production of inducible antimicrobial peptides offers a first and rapid defense response of epithelial cells against invading microbes. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide induced in various epithelia upon extracellular as well as intracellular bacterial challenge. Nucleotide-binding oligomerization domain protein 2 (NOD2/CARD15) is a cytosolic protein involved in intracellular recognition of microbes by sensing peptidoglycan fragments (e.g. muramyl dipeptide). We used luciferase as a reporter gene for a 2.3-kb hBD-2 promoter to test the hypothesis that NOD2 mediates the induction of hBD-2. Activation of NOD2 in NOD2-overexpressing human embryonic kidney 293 cells through its ligand muramyl dipeptide (MDP) induced hBD-2 expression. In contrast, overexpression of NOD2 containing the 3020insC frame-shift mutation, the most frequent NOD2 variant associated with Crohn disease, resulted in defective induction of hBD-2 through MDP. Luciferase gene reporter analyses and site-directed mutagenesis experiments demonstrated that functional binding sites for NF-kappaB and AP-1 in the hBD-2 promoter are required for NOD2-mediated induction of hBD-2 through MDP. Moreover, the NF-kappaB inhibitor Helenalin as well as a super-repressor form of the NF-kappaB inhibitor IkappaB strongly inhibited NOD2-mediated hBD-2 promoter activation. Expression of NOD2 was detected in primary keratinocytes, and stimulation of these cells with MDP induced hBD-2 peptide release. In contrast, small interference RNA-mediated down-regulation of NOD2 expression in primary keratinocytes resulted in a defective induction of hBD-2 upon MDP treatment. Together, these data suggest that NOD2 serves as an intracellular pattern recognition receptor to enhance host defense by inducing the production of antimicrobial peptides such as hBD-2.     

Corticotropin-releasing hormone affects cytokine production in human HaCaT keratinocytes

CRH cutaneous expression is significantly enhanced after exposure to various stimuli (Physiol Rev 2000, 80;979-1020). We evaluated the effect of CRH on cytokine production in HaCaT keratinocytes, a cell line shown to express CRH receptors coupled to cAMP activation and calcium-dependent transmission pathways. It is demonstrated for the first time that exogenously added CRH stimulates production of IL-6 and IL-11. It also inhibits production of IL-1beta and does not affect TNF-alpha production. Our results indicate that CRH function(s) during cutaneous stress may be mediated by differential effects on cytokine production.     

Preferential codons enhancing the expression level of human beta-defensin-2 in recombinant Escherichia coli

Human beta-defensin-2 (hBD2) is a small antimicrobial peptide with potential as a therapeutic agent. The effect of codon usage on the expression of hBD2 in Escherichia coli was studied. Two coding sequences encoding the same hBD2 precursor were both expressed as fusion protein with thioredoxin in E. coli BL21 (DE3). One is the wild-type human cDNA and the other is a gene synthesized by a PCR-based method in which rare codons were altered to those frequently used in E. coli. The expression level of recombinant hBD2 was over 50% of the total cellular protein when the synthetic gene with preferential codons was employed which was a 9-fold enhancement over the wild-type cDNA. The result shows the codon bias of the host was a major barrier in high-level expression of recombinant hBD2 and suggests a similar approach may be used in the expression of other defensins in E. coli.