Abstract: Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1beta and TNFalpha acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface.
Abstract: The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD-/- mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/- littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases.
Abstract: The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.
Abstract: Toll-like receptors (TLRs) are mammalian innate immune recognition receptors that are activated by pathogen associated molecular patterns (PAMPs). TLR4 is the signaling molecule of the lipopolysaccharide (LPS) receptor complex. TLR4 associates with its adapter molecule, MD-2, which is absolutely required for LPS-induced activation of TLR4. MD-2 exists as a cell surface protein in association with TLR4 and as secreted forms consisting of MD-2 monomers and multimers. To facilitate the studies of MD-2 distribution, abundance, and function, we produced monoclonal antibodies (MAbs) to baculovirally expressed soluble MD-2 (sMD-2). Eleven MAbs were characterized by enzyme-linked immunosorbent assay (ELISA) with soluble TLR4/MD-2 complex (sTLR4/MD-2) and sMD-2, Western blotting against sMD-2 monomer and multimers, and inhibition of direct LPS binding to sMD-2. Four MAbs preferentially recognized mainly MD-2 oligomers, not monomers, as judged by Western blotting and ELISA. Anti-MD-2 MAbs useful for indirect immunofluorescent staining of cells expressing TLR4 and MD-2 were identified. One MAb that recognized all forms of MD-2 was used in an ELISA to measure sMD-2 in normal human sera as well as sera from intensive care patients with and without sepsis. Serum levels of sMD-2 were undetectable or very low in normal and in nonsepsis patients but significantly (p < 0.05) increased in sepsis patients. These MAbs should therefore be very useful new tools for studies of MD-2 expression and function in health and disease.
Abstract: Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.
Abstract: This study evaluated the expression and regulation of beta-defensins DEFB-104 and the recently identified DEFB-105-14 in gingival keratinocytes. Keratinocytes from healthy subjects were exposed to cytokines, Escherichia coli lipopolysaccharide or Candida species. Total RNA was extracted and defensin expression analyzed by reverse transcription-polymerase chain reaction. Three patterns of expression were seen: no expression, constitutive expression and inducible expression. Constitutive mRNA expression was evident for DEFB-104, 107, 109, 111, and 112. DEFB-108 and 114 were induced by interleukin (IL)-1beta and Candida species. For DEFB-108 expression, synergism was observed when IL-1beta was combined with tumor necrosis factor-alpha or interferon-gamma. Downregulation of DEFB-109 occurred following treatment with Candida albicans. These findings suggest a role for multiple beta-defensins in response to oral infection. Further investigation is needed to better understand their function, both in terms of antimicrobial activities and contributions to innate and acquired immunity.
Abstract: 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.
Abstract: Chemokine ligand 20 (CCL20) and human beta-defensins (HBDs) share structural and functional properties, including antiparallel beta-pleated sheet core structures, charge distribution, and signaling to adaptive immune cells via the highly selective CCR6 receptor. Because of their similarities, we hypothesized that in addition to its known adaptive immune signaling functions, CCL20 has antimicrobial properties and participates in pulmonary innate immunity. We found that primary cultures of human airway epithelial and cultured fetal lung explants expressed CCL20 mRNA. Expression of CCL20 transcripts were significantly induced by interleukin (IL)-1beta and tumor necrosis factor-alpha, and inhibited by dexamethasone. Primary cultures of airway epithelia secreted CCL20 both apically and basolaterally, and CCL20 abundance was increased over 30-fold with IL-1beta stimulation, achieving an estimated concentration of 167 ng/ml in airway surface liquid. CCL20 abundance in bronchoalveolar lavage fluid from patients with cystic fibrosis was nearly 90-fold higher compared with bronchoalveolar lavage fluid from healthy volunteers. Interestingly, CCL20 exhibited salt-sensitive antimicrobial activity, mainly against Gram-negative bacteria in low mug/ml concentrations. Additionally, apical washings from IL-1beta-stimulated primary cultures of human airway epithelia had significantly more antimicrobial activity than unstimulated controls. CCL20 rapidly permeabilized bacterial membranes with a time course intermediate to HBD-2 and HBD-3. Thus, CCL20 is a bi-functional peptide with both innate and adaptive immune properties that is regulated by inflammatory mediators, expressed by airway epithelia, and increased in cystic fibrosis airway secretions.
Abstract: The severity of periodontal disease is dependent on a combination of host, microbial agent and environmental factors. One strong correlate related to periodontal disease pathogenesis is the immune status of the host. Here we show that human neutrophil peptide (HNP) defensins or human beta-defensins (HBD), co-administered intranasally with the antigen ovalbumin (OVA), induce unique immune responses that if used with microbial antigens may have the potential to hinder the pathogenesis of periodontal disease. C57BL/6 mice were immunized intranasally with phosphate buffered saline (PBS) containing 1 micro g HNP-1, HNP-2, HBD1 or HBD2 with and without 50 microg OVA. At 21 days, isotypes and subclasses of OVA-specific antibodies were determined in saliva, serum, nasal wash, bronchoalveolar lavage fluid, and fecal extracts. OVA-stimulated splenic lymphoid cell cultures from immunized mice were assessed for interferon (IFN)-gamma, Interleukin (IL)-4 and IL-10. In comparison with mice immunized with only OVA, HNP-1 and HBD2 induced significantly higher (P < 0.05) OVA-specific serum IgG, lower, but not significant, serum IgM and significantly lower (P < 0.05) IFN-gamma. In contrast, HNP-2 induced low OVA-specific serum IgG and higher, but not significant, serum IgM. HBD1 induced significantly higher (P < 0.05) OVA-specific serum IgG, higher, but not significant, serum IgM, and significantly higher (P < 0.05) IL-10. The elevated serum IgG subclasses contained IgG1 and IgG2b.
Abstract: The innate immune system includes antimicrobial peptides that protect multicellular organisms from a diverse spectrum of microorganisms. beta-Defensins comprise one important family of mammalian antimicrobial peptides. The annotation of the human genome fails to reveal the expected diversity, and a recent query of the draft sequence with the blast search engine found only one new beta-defensin gene (DEFB3). To define better the beta-defensin gene family, we adopted a genomics approach that uses hmmer, a computational search tool based on hidden Markov models, in combination with blast. This strategy identified 28 new human and 43 new mouse beta-defensin genes in five syntenic chromosomal regions. Within each syntenic cluster, the gene sequences and organization were similar, suggesting each cluster pair arose from a common ancestor and was retained because of conserved functions. Preliminary analysis indicates that at least 26 of the predicted genes are transcribed. These results demonstrate the value of a genomewide search strategy to identify genes with conserved structural motifs. Discovery of these genes represents a new starting point for exploring the role of beta-defensins in innate immunity.
Abstract: SMAP29, an ovine cathelicidin, was systematically altered to create a family of 23 related peptides for MIC and minimum bactericidal concentration determinations. SMAP28, SMAP29, and a derivative of SMAP29 called ovispirin were all antimicrobial. However, many congeners of SMAP29 and ovispirin were not as active as the parent molecules. With immunoelectron microscopy, SMAP29 was seen on membranes and within the cytoplasm of Pseudomonas aeruginosa PAO1.
Abstract: Human beta-defensin-1 (HBD-1) was detected in breast milk in concentrations of approximately 1 to 10 microg/mL. Breast tissue during lactation showed HBD-1 expression in mammary gland epithelia and within luminal secretions. The peptide demonstrated antimicrobial activity against Escherichia coli. HBD-1 may augment neonatal host defenses through antimicrobial effects or prime the adaptive immune system at mucosal surfaces.
Abstract: beta-Defensins are endogenous cationic peptides with broad-spectrum antimicrobial activity that are thought to play a role in the innate immune response. Two human beta-defensins, beta-defensin-1 (HBD-1) and beta-defensin-2 (HBD-2), have been identified. These peptides have recently been characterized in several human tissues. The presence of these peptides in the paranasal sinuses has not been investigated. We examined maxillaary sinus secretions from six patients with sinusitis and 10 patients without signs, symptoms, or radiologic evidence of sinus disease for the presence of beta-defensins. Cationic peptides were extracted from antral lavage specimens and examined for the presence of HBD-1 and HBD-2 by Western blot. Normal maxillary sinus epithelium was obtained from two patients and analyzed by RT-PCR for the presence of HBD-1 and HBD-2 mRNA. Tissue immunostaining for the two peptides was also used. Western blot analysis identified HBD-1 in two of 10 patients in the control group and in three of six patients in the sinusitis group. HBD-2 was identified in one of 10 patients in the control group and in four of six patients in the sinusitis group. RT-PCR revealed HBD-1 mRNA in one of two normal controls tested. Immunostaining localized HBD-1 and HBD-2 to the epithelial cell cytoplasm. This is the first demonstration of HBD-1 and HBD-2 production in the paranasal sinuses. In the present study, HBD-1 and HBD-2 were detected more frequently in the maxillary sinus fluid of patients with inflamed sinuses than in normal controls.
Abstract: Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.
Abstract: Human beta-defensin-2 (HBD-2) is a member of the defensin family of antimicrobial peptides. HBD-2 was first isolated from inflamed skin where it is posited to participate in the killing of invasive bacteria and in the recruitment of cells of the adaptive immune response. Static light scattering and two-dimensional proton nuclear magnetic resonance spectroscopy have been used to assess the physical state and structure of HBD-2 in solution. At concentrations of < or = 2.4 mM, HBD-2 is monomeric. The structure is amphiphilic with a nonuniform surface distribution of positive charge and contains several key structural elements, including a triple-stranded, antiparallel beta-sheet with strands 2 and 3 in a beta-hairpin conformation. A beta-bulge in the second strand occurs at Gly28, a position conserved in the entire defensin family. In solution, HBD-2 exhibits an alpha-helical segment near the N-terminus that has not been previously ascribed to solution structures of alpha-defensins or to the beta-defensin BNBD-12. This novel structural element may be a factor contributing to the specific microbicidal or chemokine-like properties of HBD-2.
Abstract: 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.
Abstract: The presence of the complement-derived anaphylatoxin peptides, C3a and C5a, in the lung can induce respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and pulmonary arteries and aggregation of platelets and leukocytes in pulmonary vessels. C3a and C5a mediate these effects by binding to their specific receptors, C3aR and C5aR, respectively. The cells that express these receptors in the lung have not been thoroughly investigated, nor has their expression been examined during inflammation. Accordingly, C3aR and C5aR expression in normal human and murine lung was determined in this study by immunohistochemistry and in situ hybridization. In addition, the expression of these receptors was delineated in mice subjected to LPS- and OVA-induced models of inflammation. Under noninflamed conditions, C3aR and C5aR protein and mRNA were expressed by bronchial epithelial and smooth muscle cells of both human and mouse lung. C3aR expression increased significantly on both bronchial epithelial and smooth muscle cells in mice treated with LPS; however, in the OVA-challenged animals only the bronchial smooth muscle cells showed increased C3aR expression. C5aR expression also increased significantly on bronchial epithelial cells in mice treated with LPS, but was not elevated in either cell type in the OVA-challenged mice. These results demonstrate the expression of C3aR and C5aR by cells endogenous to the lung, and, given the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for these receptors during lung inflammation.
Abstract: Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.
Abstract: beta-Defensins are broad spectrum antimicrobial peptides expressed at epithelial surfaces. Two human beta-defensins, HBD-1 and HBD-2, have been identified. In the lung, HBD-2 is an inducible product of airway epithelia and may play a role in innate mucosal defenses. We recently characterized rat homologs (RBD-1, RBD-2) of the human genes and used these sequences to identify novel mouse genes. Mouse beta-defensin-4 (MBD-4) was amplified from lung cDNA using polymerase chain reaction primers designed from conserved sequences of RBD-2 and HBD-2. A full-length cDNA was cloned which encodes a putative peptide with the sequence MRIHYLLFTFLLVLLSPLAAFTQIINNPITCMTNGAICWGPCPTAFRQIGNCGHFKVRCCKIR. The peptide shares approximately 40% identity with HBD-2. MBD-4 mRNA was expressed in the esophagus, tongue, and trachea but not in any of 20 other tissues surveyed. Cloning of the genomic sequence of MBD-4 revealed two nearly (>99%) identical sequences encoding MBD-4 and the presence of numerous additional highly similar genomic sequences. Radiation hybrid mapping localized this gene to a region of chromosome 8 near several other defensins, MBD-2, MBD-3, and alpha-defensins (cryptdins)-3 and -17, consistent with a gene cluster. Our genomic cloning and mapping data suggest that there is a large beta-defensin gene family in mice. Identification of murine beta-defensins provides an opportunity to understand further the role of these peptides in host defense through animal model studies and the generation of beta-defensin-deficient animals by gene targeting.
Abstract: beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that may play a role in mucosal defenses of several organs. They have been isolated in several species, and in humans, two beta-defensins have been identified. Here, we report the identification of two genes encoding beta-defensin homologues in the rat. Partial cDNAs were found by searching the expressed-sequence-tag database, and primers were designed to generate full-length mRNA coding sequences. One gene was highly similar to the human beta-defensin-1 (HBD-1) gene and mouse beta-defensin-1 gene at both the nucleic acid and amino acid levels and was termed rat beta-defensin-1 (RBD-1). The other gene, named RBD-2, was homologous to the HBD-2 and bovine tracheal antimicrobial peptide (TAP) genes. The predicted prepropeptides were strongly cationic, were 69 and 63 residues in length for RBD-1 and RBD-2, respectively, and contained the six-cysteine motif characteristic of beta-defensins. The beta-defensin genes mapped closely on rat chromosome 16 and were closely linked to the alpha-defensins genes, suggesting that they are part of a gene cluster, similar to the organization reported for humans. Northern blot analysis showed that both RBD-1 and RBD-2 mRNA transcripts were approximately 0.5 kb in length; RBD-1 mRNA was abundantly transcribed in the rat kidney, while RBD-2 was prevalent in the lung. Reverse transcription-PCR indicated that RBD-1 and RBD-2 mRNAs were distributed in a variety of other tissues. In the lung, RBD-1 mRNA expression localized to the tracheal epithelium while RBD-2 was expressed in alveolar type II cells. In conclusion, we characterized two novel beta-defensin homologues in the rat. The rat may be a useful model to investigate the function and contribution of beta-defensins to host defense in the lung, kidney, and other tissues.
Abstract: Cystic fibrosis mice have been generated by gene targeting but show little lung disease without repeated exposure to bacteria. We asked if murine mucosal defenses and airway surface liquid (ASL) Cl(-) were altered by the DeltaF508 cystic fibrosis transmembrane conductance regulator mutation. Naive DeltaF508 -/- and +/- mice showed no pulmonary inflammation and after inhaled Pseudomonas aeruginosa had similar inflammatory responses and bacterial clearance rates. We therefore investigated components of the innate immune system. Bronchoalveolar lavage fluid from mice killed Escherichia coli, and the microbicidal activity was inhibited by NaCl. Because beta-defensins are salt-sensitive epithelial products, we looked for pulmonary beta-defensin expression. A mouse homolog of human beta-defensin-1 (termed "MBD-1") was identified; the mRNA was expressed in the lung. Using a radiotracer technique, ASL volume and Cl(-) concentration ([Cl(-)]) were measured in cultured tracheal epithelia from normal and DeltaF508 -/- mice. The estimated ASL volume was similar for both groups. There were no differences in ASL [Cl(-)] in DeltaF508 -/- and normal mice (13.8 +/- 2.6 vs. 17.8 +/- 5.6 meq/l). Because ASL [Cl(-)] is low in normal and mutant mice, salt-sensitive antimicrobial factors, including MBD-1, may be normally active.
Abstract: beta-Defensins are cationic peptides with broad-spectrum antimicrobial activity that are produced by epithelia at mucosal surfaces. Two human beta-defensins, HBD-1 and HBD-2, were discovered in 1995 and 1997, respectively. However, little is known about the expression of HBD-1 or HBD-2 in tissues of the oral cavity and whether these proteins are secreted. In this study, we characterized the expression of HBD-1 and HBD-2 mRNAs within the major salivary glands, tongue, gingiva, and buccal mucosa and detected beta-defensin peptides in salivary secretions. Defensin mRNA expression was quantitated by RNase protection assays. HBD-1 mRNA expression was detected in the gingiva, parotid gland, buccal mucosa, and tongue. Expression of HBD-2 mRNA was detected only in the gingival mucosa and was most abundant in tissues with associated inflammation. To test whether beta-defensin expression was inducible, gingival keratinocyte cell cultures were treated with interleukin-1beta (IL-1beta) or bacterial lipopolysaccharide (LPS) for 24 h. HBD-2 expression increased approximately 16-fold with IL-1beta treatment and approximately 5-fold in the presence of LPS. Western immunoblotting, liquid chromatography, and mass spectrometry were used to identify the HBD-1 and HBD-2 peptides in human saliva. Human beta-defensins are expressed in oral tissues, and the proteins are secreted in saliva; HBD-1 expression was constitutive, while HBD-2 expression was induced by IL-1beta and LPS. Human beta-defensins may play an important role in the innate defenses against oral microorganisms.
Abstract: We cloned a second human beta-defensin gene, HBD-2, and determined its gene structure and expression in inflamed tissue sections. The entire gene spanned about 2 kb with two small exons and one intron. Radiation hybrid studies confirmed the location on chromosome 8p, were consistent with the order HNP-1, HBD-1 and HBD-2, and located HBD-2 as the most centromeric of the genes. By three-color fluorescence in situ hybridization on both free chromatin fiber mapping and interphase mapping, HBD-1, HBD-2 and HNP-1 were mapped to chromosome 8p23. HBD-1 was within 40-100kb of HNP-1, while HBD-2 was about 500-600 kb from HBD-1, with the most likely order HNP-1, HBD-1, HBD-2. The expression of HBD-2 was locally regulated by inflammation. HBD-2 mRNA was markedly increased in the epidermis surrounding inflamed regions, but not detectable in adjacent non-inflamed areas, a distribution that was confirmed at the peptide level by immunostaining with HBD-2 antibody. The HBD-2 gene is the first member of the human defensin family that is locally inducible by inflammation.
Abstract: Human beta-defensins (HBDs) are antimicrobial peptides that may play a role in mucosal defense. Diminished activity of these peptides has been implicated in the pathogenesis of cystic fibrosis (CF) lung disease. We show that HBD-1 and HBD-2 mRNAs are expressed in excised surface and submucosal gland epithelia from non-CF and CF patients. The pro-inflammatory cytokine interleukin-1beta stimulated the expression of HBD-2 but not HBD-1 mRNA and peptide in primary cultures of airway epithelia. HBD-1 was found in bronchoalveolar lavage (BAL) fluid from normal volunteers, CF patients, and patients with inflammatory lung diseases, whereas HBD-2 was detected in BAL fluid from patients with CF or inflammatory lung diseases, but not in normal volunteers. Both HBD-1 and HBD-2 were found in BAL fluid in concentrations of several ng/ml, and both recombinant peptides showed salt-sensitive bactericidal activity. These data suggest that in the lung HBD-2 expression is induced by inflammation, whereas HBD-1 may serve as a defense in the absence of inflammation.
Abstract: The effects of a partially purified antihypertensive factor (AHF) from erythrocytes of spontaneously hypertensive rats (SHR) on the blood pressure (BP) and Ca2+ influx of vascular smooth muscle (VSM) in rats were studied. The results indicated that AHF could produce a marked prolonged depressor effect and significantly inhibit the Ca2+ influx dose-dependently on both SHR and renal hypertensive rat (RHR) either in acute or in chronic experiments, but not on normotensive rats. It suggested that the inhibition of Ca2+ influx might be one of the important mechanisms for AHF as an endogenous depressor substance.
