Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

By IL-1 signaling,monocyte-derived cells dramatically enhance the epidermal antimicrobial response to lipopolysaccharide

Epithelia react to microbial pathogens by mounting a defensive response that includes the production of antimicrobial peptides. In this study, we show that, in human epidermal cultures, Escherichia coli LPS was a very weak direct inducer of human beta-defensin (HBD)-2 mRNA and peptide, but the induction was greatly amplified when monocyte-derived cells (MoDeC) acted as intermediaries between LPS and the epidermis. IL-1R antagonist largely reversed the effect of MoDeC on epidermal HBD-2, indicating that, from among the many products of MoDeC, IL-1 was the dominant inducer of HBD-2 synthesis. In normal fresh human skin, which contains Langerhans cells and other myeloid cell types, in addition to keratinocytes, LPS also induced HBD-2 in an IL-1-dependent manner. In DNA microarray expression studies, HBD-2 was one of the most abundant mRNAs induced in epidermis by LPS-treated MoDeC, and its induction was reversed by IL-1Ra. Thus, epidermal response to LPS is potently amplified by MoDeC through IL-1-mediated signaling, leading to a selective increase in the synthesis of the antimicrobial peptide HBD-2. This pattern of responses establishes a key role for both IL-1 and HBD-2 in the host defense reaction of the epidermis.     

Secretion of interleukin-1beta by astrocytes mediates endothelin-1 and tumour necrosis factor-alpha effects on human brain microvascular endothelial cell permeability

Evidence suggests that endothelin-1 (ET-1) plays an essential role in brain inflammation. However, whether ET-1 contributes directly to blood-brain barrier (BBB) breakdown remains to be elucidated. Using an in vitro BBB model consisting of co-cultures of human primary astrocytes and brain microvascular endothelial cells (BMVECs), we first investigated the expression of ET-1 by BMVECs upon stimulation with tumour necrosis factor (TNF)-alpha, which plays an essential role in the induction and synthesis of ET-1 during systemic inflammatory responses. Increased ET-1 mRNA was detected in the human BMVECs 24 h after TNF-alpha treatment. This was correlated with an increase in ET-1 levels in the culture medium, as determined by sandwich immunoassay. Both TNF-alpha and ET-1 increased the permeability of human BMVECs to a paracellular tracer, sucrose, but only in the presence of astrocytes. The increase in BMVEC permeability by TNF-alpha was partially prevented by antibody neutralization of ET-1 and completely by monoclonal antibody against IL-1beta. Concomitantly, TNF-alpha induced IL-1beta mRNA expression by astrocytes in co-culture and this effect was partially prevented by ET-1 antibody neutralization. In parallel experiments, treatment of human primary astrocytes in single cultures with ET-1 for 24 h induced IL-1beta mRNA synthesis and IL-1beta protein secretion in the cell culture supernatant. Taken together, these results provide evidence for paracrine actions involving ET-1, TNF-alpha and IL-1beta between human astrocytes and BMVECs, which may play a central role in BBB breakdown during CNS inflammation.     

CXC chemokine receptor 4 expression and function in human astroglioma cells

Chemokines constitute a superfamily of proteins that function as chemoattractants and activators of leukocytes. Astrocytes, the major glial cell type in the CNS, are a source of chemokines within the diseased brain. Specifically, we have shown that primary human astrocytes and human astroglioma cell lines produce the CXC chemokines IFN-gamma-inducible protein-10 and IL-8 and the CC chemokines monocyte chemoattractant protein-1 and RANTES in response to stimuli such as TNF-alpha, IL-1beta, and IFN-gamma. In this study, we investigated chemokine receptor expression and function on human astroglioma cells. Enhancement of CXC chemokine receptor 4 (CXCR4) mRNA expression was observed upon treatment with the cytokines TNF-alpha and IL-1beta. The peak of CXCR4 expression in response to TNF-alpha and IL-1beta was 8 and 4 h, respectively. CXCR4 protein expression was also enhanced upon treatment with TNF-alpha and IL-1beta (2- to 3-fold). To study the functional relevance of CXCR4 expression, stable astroglioma transfectants expressing high levels of CXCR4 were generated. Stimulation of cells with the ligand for CXCR4, stromal cell-derived factor-1alpha (SDF-1alpha), resulted in an elevation in intracellular Ca(2+) concentration and activation of the mitogen-activated protein kinase cascade, specifically, extracellular signal-regulated kinase 2 (ERK2) mitogen-activated protein kinase. Of most interest, SDF-1alpha treatment induced expression of the chemokines monocyte chemoattractant protein-1, IL-8, and IFN-gamma-inducible protein-10. SDF-1alpha-induced chemokine expression was abrogated upon inclusion of U0126, a pharmacological inhibitor of ERK1/2, indicating that the ERK signaling cascade is involved in this response. Collectively, these data suggest that CXCR4-mediated signaling pathways in astroglioma cells may be another mechanism for these cells to express chemokines involved in angiogenesis and inflammation.     

Expression and activity of beta-defensins and LL-37 in the developing human lung

Immaturity of innate immunity contributes to the increased susceptibility of human neonates to infection. The lung is a major portal of entry for potential pathogens in the neonate, and human beta-defensins (HBDs) and LL-37 participate in pulmonary innate immunity. We hypothesized that these antimicrobial factors would be developmentally regulated, expressed by neonatal pulmonary tissues, and participate in neonatal innate immunity. We found HBD-2 to be the predominant beta-defensin in human neonatal lung. HBD-2 mRNA expression was developmentally regulated, induced by the proinflammatory factor IL-1beta, and decreased by dexamethasone. Additionally, HBD-2 abundance in neonatal tracheal aspirates increased as a function of gestational age. HBD-1 had a lower level of expression compared with HBD-2 and was induced by dexamethasone. HBD-3 and LL-37 messages were not detected in airway epithelial cultures. Additionally, each antimicrobial peptide exhibited a unique spectrum of antimicrobial activity and salt sensitivity against bacteria commonly causing sepsis in the neonate. Lower levels of HBD-2 may be one factor contributing to the increased susceptibility of premature infants to pulmonary infections.     

Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-32alpha induction in human pancreatic periacinar myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. IL-32alpha protein expression was evaluated by Western blot analyses, and IL-32alpha mRNA expression was analyzed by Northern blot and real-time PCR analyses. IL-32alpha mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1beta, IFN-gamma, and TNF-alpha. IL-1beta, IFN-gamma, and TNF-alpha enhanced intracellular accumulation of IL-32alpha protein, but IL-32alpha was not detected in supernatants. Each cytokine dose and time dependently induced IL-32alpha mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1beta-, IFN-gamma-, and TNF-alpha-induced IL-32alpha mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1beta- and TNF-alpha-induced NF-kappaB activation and IL-1beta-, TNF-alpha-, and IFN-gamma-induced activated protein-1 (AP-1) activation. Blockade of NF-kappaB and AP-1 activation by an adenovirus expressing a stable mutant form of IkappaBalpha and a dominant negative mutant of c-Jun markedly suppressed IL-1beta-, IFN-gamma-, and/or TNF-alpha-induced IL-32alpha mRNA expression. Human pancreatic periacinar myofibroblasts expressed IL-32alpha in response to IL-1beta, TNF-alpha, and IFN-gamma. IL-32alpha mRNA expression is dependent on interactions between the phosphatidylinositol 3-kinase/Akt-pathway and the NF-kappaB/AP-1 system.     

Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2

The expression of inducible antimicrobial peptides, such as human beta-defensin-2 (HBD-2) by epithelia, comprises a component of innate pulmonary defenses. We hypothesized that HBD-2 induction in airway epithelia is linked to pattern recognition receptors such as the Toll-like receptors (TLRs). We found that primary cultures of well-differentiated human airway epithelia express the mRNA for TLR-4, but little or no MD-2 mRNA, and display little HBD-2 expression in response to treatment with purified endotoxin +/- LPS binding protein (LBP) and soluble CD14. Expression of endogenous MD-2 by transduction of airway epithelial cells with an adenoviral vector encoding MD-2 or extracellular addition of recombinant MD-2 both increased the responses of airway epithelia to endotoxin + LBP and sCD14 by >100-fold, as measured by NF-kappaB-luciferase activity and HBD-2 mRNA expression. MD-2 mRNA could be induced in airway epithelia by exposure of these cells to specific bacterial or host products (e.g., killed Haemophilus influenzae, the P6 outer membrane protein from H. influenzae, or TNF-alpha + IFN-gamma). These findings suggest that MD-2, either coexpressed with TLR-4 or secreted when produced in excess of TLR-4 from neighboring cells, is required for airway epithelia to respond sensitively to endotoxin. The regulation of MD-2 expression in airway epithelia and pulmonary macrophages may serve as a means to modify endotoxin responsiveness in the airway.     

Cytokine regulation of IL-1 beta gene expression in the human polymorphonuclear leukocyte

Although recently polymorphonuclear leukocytes (PMN) have been identified as producers of IL-1 beta in response to LPS and granulocyte/monocyte colony stimulating factor, little is known regarding the ability of other cytokines to induce the production of IL-1 beta in the PMN. Inasmuch as IL-1 and TNF have been shown to be important priming agents, as well as agents that induce migration of PMN, we investigated their effect on IL-1 beta gene expression in human peripheral blood PMN. In the present study, we demonstrate that human peripheral blood PMN produce IL-1 beta in response to IL-1 alpha, IL-1 beta, and TNF-alpha. Control (unstimulated) human PMN had virtually undetectable levels of IL-1 beta mRNA. Either IL-1 beta or TNF, induced PMN to transiently express IL-1 beta mRNA with peak expression at 1 h, returning to untreated levels by 2 h. A dose response indicated that as little as 0.05 ng/ml of IL-1 beta or TNF resulted in IL-1 beta induction, with maximal effects at 1 ng/ml of IL-1 beta and 5 ng/ml of TNF. IL-1 alpha or IL-1 beta exhibited similar dose responses in IL-1 beta mRNA induction. Inasmuch as cytokines have been shown to have synergistic effects in cell function studies, we induced PMN with a combination of maximally effective doses of TNF plus IL-1 beta. They demonstrated a cooperative effect on IL-1 beta gene expression, in that mRNA levels were sustained for three hours. IL-1 beta Ag expression, as measured by ELISA, paralleled IL-1 beta mRNA expression with cell associated peak levels at 2 to 4 h. IL-1 beta Ag levels in PMN lysates and supernatants correlated with IL-1 beta mRNA levels, i.e., TNF + IL-1 greater than TNF greater than IL-1. Thus, these studies represent the first demonstration of IL-1 and TNF induction of IL-1 beta gene expression in the PMN. Furthermore, the time course of induction is unique to the PMN, with peak induction of mRNA at 1 h, which is consistent with the short lived nature of these cells in inflammatory lesions.     

IL-4 and IL-13 negatively regulate TNF-alpha- and IFN-gamma-induced beta-defensin expression through STAT-6, suppressor of cytokine signaling (SOCS)-1, and SOCS-3

Human beta-defensins (HBDs) are a major class of antimicrobial peptides that play an important role in the innate immune response, however, the induction and regulation of these antimicrobial peptides is not well understood. We demonstrate here that stimulation of keratinocytes with TNF-alpha/IFN-gamma induces HBD-2 and HBD-3 by activating STAT-1 and NF-kappaB signaling. We further demonstrate that IL-4 and IL-13 activate STAT-6 and induce the suppressors of cytokine signaling (SOCS)-1 and -3. This interferes with STAT-1 and NF-kappaB signaling, thereby inhibiting TNF-alpha/IFN-gamma-mediated induction of HBD-2 and HBD-3. These data suggest that targeting the STAT-1-signaling pathway or suppressor of cytokine signaling expression enhances beta-defensin expression and represents a new therapeutic strategy for reduction of infection in human diseases associated with beta-defensin deficiency.     

The transcriptional control of TGF-beta in human osteoblast-like cells is distinct from that of IL-1 beta.

Transforming growth factor beta (TGF-beta) and interleukin 1 (IL-1) are among the most potent osteotropic cytokines. The expression of mRNA for both TGF-beta and IL-1 beta was studied in human osteoblast-like cells in vitro. These cells constitutively expressed TGF-beta but not IL-1 beta mRNA. Treatment of the cells with the systemic hormones 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (10(-8) M) and parathyroid hormone (10(-7) M) induced an increase in TGF-beta mRNA but failed to stimulate the production of IL-1-beta mRNA. Retinoic acid (10(-8) M) had no effect on either mRNA species. The cytokines IL-1 alpha (200 pg/ml), tumour necrosis factor alpha (TNF-alpha) (17 ng/ml) and bacterial lipopolysaccharide (LPS) (500 ng/ml) stimulated the production of IL-1 beta mRNA after 6-8 hours. This was followed by an increase in protein production after 24 hours. In contrast, the production of TGF-beta mRNA remained constant after treatment with these agents. Treatment of the cells with hydrocortisone (10(-8) M) resulted in the suppression of both TGF-beta and IL-1 beta mRNA. However, when the stimulating agent 1,25-(OH)2D3 was added in conjunction with hydrocortisone the mRNA expression of TGF-beta mRNA returned to 70% of the stimulated level. In contrast, the addition of the stimulatory agent IL-1 alpha to hydrocortisone-treated cells resulted in no increase in IL-1 beta mRNA. In-situ hybridization demonstrated both TGF-beta and IL-1 beta mRNA at the cellular level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential inhibition of IL-1 and TNF-alpha mRNA expression by agents which block second messenger pathways in murine macrophages

IL-1 and TNF-alpha are induced in macrophages by LPS; however, it is unclear whether similar mechanisms control the expression of both genes. Here, we report on the detection of differential regulation of LPS induced IL-1 and TNF-alpha mRNA expression and protein production in murine macrophages based on the use of inhibitors of second messenger pathways. Northern blot analysis was performed with total RNA obtained from murine (C57Bl/6) peritoneal macrophages stimulated in vitro with LPS with or without an inhibitor of protein kinase C (PKc)(1-(5-isoquinolinesulfonyl)-2-methylpiperazine hydrochloride; H7) or an inhibitor of calmodulin (CaM)-dependent kinase (N-(6-amino-hexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride; W7). Northerns were analyzed with probes for IL-1 alpha and IL-1 beta and TNF-alpha. The expression of the three cytokine mRNA by LPS was inhibited in a dose response manner by H7. In contrast, the expression of IL-1 mRNA, but not TNF-alpha mRNA, was blocked by treatment with W7. Parallel studies monitoring biologic activities of these two cytokines confirm the mRNA data. PKc inhibitors, H7 and retinal, block both IL-1 and TNF-alpha protein production and inhibitors of CaM kinase, W7, N-(6-aminobutyl)-5-chloro-2-naphthalenesulfonamide, calmidazolum, and trifluoperazine dichloride inhibit only IL-1 production. These data suggest that both PKc and CaM kinase dependent pathways are involved in the induction of IL-1 mRNA by LPS. In contrast, TNF-alpha expression appears to be PKc dependent but not CaM kinase dependent.     

Expression of IL-8 gene in human monocytes and lymphocytes: differential regulation by TNF and IL-1

TNF-alpha and IL-1 were reported to be the most powerful inducers of IL-8 in a multitude of cells, including leukocytes. In this study, we investigated TNF-alpha- and IL-1-mediated regulation of IL-8 gene expression in non-fractionated PBMC, and purified monocyte (MO) and lymphocyte (LY) fractions. Our analysis revealed that purified human MO did not respond to exogenous TNF-alpha with the induction of IL-8 mRNA or protein, nor require endogenous TNF-alpha for IL-8 expression. In contrast, in the presence of exogenous IL-1alpha and IL-1beta a substantial enhancement of IL-8 mRNA and protein expression in MO was observed. Nevertheless, antibodies to IL-1alpha and IL-1beta were unable to downregulate the expression of IL-8 in resting adherent or Staphylococcus aureus Cowan 1 (SAC)-stimulated MO. In contrast with MO, purified LY and non-fractionated PBMC expressed IL-8 in response to exogenous TNF-alpha, similar to exogenous IL-1alpha and IL-1beta. As was seen with MO, antibodies to TNF-alpha, IL-1alpha and IL-1beta did not inhibit the expression of IL-8 in purified LY and non-fractionated PBMC stimulated with SAC and LPS. Taken together, our data demonstrate major differences in responsiveness of MO and LY to exogenous TNF-alpha and IL-1, and suggest relative autonomy of IL-8 gene expression in these cells that does not require accessory cytokines but can be induced directly by exogenous stimuli.     

Endotoxin and Staphylococcus aureus enterotoxin A induce different patterns of cytokines.

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-gamma and TNF-beta. LPS exhibited marked production of IL-1 alpha, IL-1 beta, TNF-alpha, IL-6 and IL-8. After LPS stimulation IL-1 alpha, IL-1 beta, TNF-alpha and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-beta, IL-2, IFN-gamma and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours. In contrast, peak production of IL-1 alpha, IL-1 beta and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-beta, TNF-alpha, IFN-gamma and IL-2 was found with peak production 12-48 hours after initiation. Only low amounts of IL-6 were evident. The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections.(ABSTRACT TRUNCATED AT 250 WORDS)