Characterization of the divergent eosinophil ribonuclease, mEar 6, and its expression in response to Schistosoma mansoni infection in vivo.

The eosinophil-associated ribonucleases (Ears) are rapidly evolving proteins found in multigene clusters that are unique to each rodent species. Of the 15 independent genes in the Mus musculus cluster, only mEars 1 and 2 are expressed at significant levels at homeostasis. Here we characterize the expression of mEar 6 in the liver and spleen in mice in response to infection with the helminthic parasite, Schistosoma mansoni. Interestingly, expression of mEar 6 is not directly related to the elevated levels of serum IL-5 or tissue eosinophilia characteristic of this disease, as no mEar 6 transcripts were detected in the liver or the spleen from uninfected IL-5-transgenic mice. The coding sequence of mEar 6 has diverged under positive selection pressure (K(a)/K(s) > 1.0) and has a unique unpaired cysteine near the carboxy-terminus of the protein. The high catalytic efficiency of recombinant mEar 6 (k(cat)/K(m) = 0.9 x 10(6)/M/s) is similar to that of the cluster's closest human ortholog, eosinophil-derived neurotoxin (EDN/RNase 2). In summary, we have identified mEar 6 as one of only two RNase A superfamily ribonucleases known to be expressed specifically in response to pathophysiologic stress in vivo.

Gene expression in epithelial cells in response to pneumovirus infection.

Respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM) are viruses of the family Paramyxoviridae, subfamily pneumovirus, which cause clinically important respiratory infections in humans and rodents, respectively. The respiratory epithelial target cells respond to viral infection with specific alterations in gene expression, including production of chemoattractant cytokines, adhesion molecules, elements that are related to the apoptosis response, and others that remain incompletely understood. Here we review our current understanding of these mucosal responses and discuss several genomic approaches, including differential display reverse transcription-polymerase chain reaction (PCR) and gene array strategies, that will permit us to unravel the nature of these responses in a more complete and systematic manner.

Transcriptional regulation of galectin-10 (eosinophil Charcot-Leyden crystal protein): a GC box (-44 to -50) controls butyric acid induction of gene expression.

Galectin-10 (gal-10, also known as Charcot-Leyden crystal protein) is a member of the galectin family of beta-galactoside binding proteins that is expressed uniquely in eosinophilic and basophilic leukocytes. To gain a better understanding of galectin gene expression, we present an analysis of the transcriptional regulation of the gene encoding gal-10. Analysis of the minimal promoter revealed nine consensus-binding sites for transcription factors, including several that are also found in the minimal promoters of galectins -1, -2, and -3. The decrease in gal-10 promoter activity after disruption of either the GC box (-44 to -50) or the Oct site (-255 to -261) suggests that these sites, along with the previously characterized GATA and EoTF sites, are necessary for full promoter activity. By supershift analysis, we demonstrate binding of the transcription factors Sp1 and Oct1 to the consensus GC box and the Oct site, respectively. Similar to gal-1, gal-10 expression is induced by butyric acid, an effect that is lost upon ablation of the GC box. Additionally, we demonstrate AML3 binding to the consensus AML site and YY1 binding to the Inr sequence, both elements functioning as silencers in the gal-10 promoter.

Epithelial cells infected with respiratory syncytial virus are resistant to the anti-inflammatory effects of hydrocortisone.

In this work we continue our study of the biochemical responses of respiratory epithelial cells to infection with human paramyxovirus pathogens. In our earlier studies, we detected elevated concentrations of the proinflammatory chemokines MIP-1alpha and IL-8 in upper and lower respiratory tract secretions from patients infected with respiratory syncytial virus (RSV). Here we demonstrate the same trend for individuals infected with parainfluenza virus (PIV), with elevated concentrations of MIP-1alpha and IL-8 (means of 309 +/- 51 and 2280 +/- 440 pg/ml/mg protein, respectively) detected in nasal wash samples from 17 patients with culture-positive PIV. Similar to our findings with RSV, cells of the HEp-2 epithelial line and primary cultures of human bronchial epithelial cells respond to PIV infection with production and release of both MIP-1alpha and IL-8. Addition of the glucocorticoid anti-inflammatory agent hydrocortisone (200-1000 ng/ml) attenuated the production of MIP-1alpha and IL-8 in PIV-infected cells while having minimal to no effect on the production of these mediators from cells infected with RSV. Neither virus infection resulted in a change in the total cellular concentration of glucocorticoid receptors, nor did hydrocortisone exert any differential effect on viral replication. As repression of chemokine production by epithelial cells is likely to result in diminished recruitment of proinflammatory leukocytes, these results may explain in part why glucocorticoid therapy reduces the symptoms associated with acute PIV infection, but have little to no effect in the overall outcome in the case of RSV.

Cytokeratin 17 is expressed in cells infected with respiratory syncytial virus via NF-kappaB activation and is associated with the formation of cytopathic syncytia.

We used differential display to detect enhanced expression of an mRNA fragment encoding cytokeratin 17 (Ck-17) in respiratory syncytial virus (RSV)-infected epithelial cells. Expression increased 12-fold by 96 h after infection but remained unchanged in cells challenged with virus in the presence of neutralizing anti-RSV fusion protein antibody. Immunoblots of RSV-infected cell lysates probed with an anti-keratin antibody demonstrated stable expression of total cytokeratins over time. When probed with an anti-Ck-17 monoclonal antibody, Ck-17 was first detected at 4 days after infection. In situ staining demonstrated that Ck-17 expression localized to regions of syncytia formation. Expression of Ck-17 mRNA also increased in response to intracellular RSV-F protein in the absence of active RSV infection. No increase in Ck-17 mRNA expression and no syncytia were observed in RSV-infected cells grown in the presence of the NF-kappaB inhibitor gliotoxin. These results suggest that RSV-induced transcriptional activation of the Ck-17 gene is dependent on an NF-kappaB-associated signaling pathway.

The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators.

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designated MEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase-polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) gamma, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor beta inhibited such expression. Induction by IFN-gamma occurred rapidly and was resistant to cycloheximide. IFN-alpha also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-gamma and the combination of IFN-alpha and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-gamma immediate early gene.

Shared features of transcription: mutational analysis of the eosinophil/basophil Charcot-Leyden crystal protein gene promoter.

The lineage-specific Charcot-Leyden crystal (CLC) protein is found in human eosinophils and basophils where it comprises 7-10% of the cellular protein content. Previous work from our laboratory has identified the motif GGAGA[A/G] as a powerful enhancer of gene transcription ill two eosinophil ribonuclease genes. To evaluate a potentially larger role for this motif in the transcriptional regulation of eosinophil genes, we have isolated 1504 nucleotides 5' to the transcriptional start site of the gene encoding CLC protein and identified a functionally active promoter that includes three distinct copies of the GGAGAA motif. Destruction of only one of the three motifs by site-directed mutagenesis resulted in loss of promoter activity (73 +/- 6% reduction), suggesting that this core motif is necessary but not sufficient to support enhanced transcriptional activity. Sequence comparisons and site-specific mutagenesis has permitted further delineation of this enhancer element which, as a result of this work, is now defined as GGAGA[A/G]NNNA. Electromobility shift assays demonstrated specific binding of nuclear protein(s) from an eosinophilic clone-15 nuclear extract to this extended motif. Similar analysis of a GATA-1 binding site demonstrated enhancer activity, with mutagenesis resulting in a 94 +/- 1.4% reduction in activity, whereas the AML1 site functioned as a gene silencer.

Respiratory syncytial virus infection induces expression of the anti-apoptosis gene IEX-1L in human respiratory epithelial cells.

By means of differential display reverse-transcriptase polymerase chain reaction, increased expression of the mRNA encoding the anti-apoptosis gene IEX-1L was found in respiratory epithelial cells infected with respiratory syncytial virus (RSV). IEX-1L mRNA expression increased 5-7-fold in RSV-infected cells at 72 h after infection but remained unchanged in cells exposed to irradiated, replication-incompetent RSV. Because IEX-1L is reported to protect cells from apoptosis induced by tumor necrosis factor (TNF)-alpha, the effect of TNF-alpha on epithelial cell apoptosis in the context of RSV infection was determined. Epithelial cells were exposed to vehicle, RSV, or irradiated RSV for 72 h, and then TNF-alpha was added to appropriate cultures. Cytochemical staining of cellular DNA with 4,6-diamidino-2-phenylindole demonstrated TNF-alpha-induced apoptosis in 23.4% of control cells but only 5% of RSV-infected cells. These data show that RSV infection protects epithelial cells from TNF-alpha-induced apoptosis and that this effect is temporally associated with IEX-1L gene expression.

Macrophage inflammatory protein-1alpha and RANTES are present in nasal secretions during ongoing upper respiratory tract infection.

Macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated upon activation, normal T-cell expressed and secreted) were measured by enzyme-linked immunosorbent assay (ELISA) from virus-infected respiratory cell culture supernatants and from 100 nasal wash samples obtained from patients aged 8 d to 10 yr. The results of the nasal wash samples were analyzed in relation to the etiology of the viral infection. In vitro, respiratory syncytial virus (RSV) induced the production of MIP-1alpha, while both RSV and adenovirus were associated with the production of RANTES. Both MIP-1alpha and RANTES were detected in nasopharyngeal secretions from pediatric patients with acute upper respiratory tract RSV, adenovirus, influenza, and parainfluenza virus infection (p<0.001 by Fisher's exact test). As both of these chemokines have potent effects on the recruitment and degranulation of eosinophils and basophils, further understanding of their role in upper respiratory tract infections may provide valuable insights into the immunopathogenesis of respiratory viral infections.

Respiratory syncytical virus-induced chemokine expression in the lower airways: eosinophil recruitment and degranulation.

Characterization of chemokine expression patterns in virus-infected epithelial cells provides important clues to the pathophysiology of such infections. The aim of this study was to determine the chemokine response pattern of respiratory epithelium when infected with respiratory syncytial virus (RSV). Macrophage inflammatory protein-1-alpha (MIP-1-alpha), interleukin-8 (IL-8), and RANTES concentrations were measured from RSV-infected HEp-2, MRC-5, and WI-38 cell culture supernatants daily following infection. Additionally, MIP-1-alpha, IL-8, and RANTES concentrations were measured from lower respiratory secretions obtained from 10 intubated infants (0-24 mo) with RSV bronchiolitis, and from 10 control subjects. Our results indicate that respiratory epithelial cells respond to RSV infection by producing MIP-1-alpha, IL-8, and RANTES. Production of MIP-1-alpha required ongoing viral replication, whereas RANTES and IL-8 could be elicited by inactivated forms of the virus. MIP-1-alpha, RANTES, and IL-8 were also present in lower airway secretions obtained from patients with RSV bronchiolitis. Eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN), the eosinophil secretory ribonucleases, were detected in lower airway secretions from RSV-infected patients; ECP concentrations correlated with MIP-1-alpha concentrations (r = 0.93). We conclude that MIP-1-alpha is present in the lower airways during severe RSV disease. The correlation between MIP-1-alpha and ECP concentrations suggests a role for eosinophil degranulation products in the pathogenesis of RSV bronchiolitis.

Ribonuclease k6: chromosomal mapping and divergent rates of evolution within the RNase A gene superfamily.

We have localized the gene encoding human RNase k6 to within approximately 120 kb on the long (q) arm of chromosome 14 by HAPPY mapping. With this information, the relative positions of the six human RNase A ribonucleases that have been mapped to this locus can be inferred. To further our understanding of the individual lineages comprising the RNase A superfamily, we have isolated and characterized 10 novel genes orthologous to that encoding human RNase k6 from Great Ape, Old World, and New World monkey genomes. Each gene encodes a complete ORF with no less than 86% amino acid sequence identity to human RNase k6 with the eight cysteines and catalytic histidines (H15 and H123) and lysine (K38) typically observed among members of the RNase A superfamily. Interesting trends include an unusually low number of synonymous substitutions (Ks) observed among the New World monkey RNase k6 genes. When considering nonsilent mutations, RNase k6 is a relatively stable lineage, with a nonsynonymous substitution rate of 0.40 x 10(-9) nonsynonymous substitutions/nonsynonymous site/year (ns/ns/yr). These results stand in contrast to those determined for the primate orthologs of the two closely related ribonucleases, the eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), which have incorporated nonsilent mutations at very rapid rates (1.9 x 10(-9) and 2.0 x 10(-9) ns/ns/yr, respectively). The uneventful trends observed for RNase k6 serve to spotlight the unique nature of EDN and ECP and the unusual evolutionary constraints to which these two ribonuclease genes must be responding. [The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF037081-AF037090.]

Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus.

A dose-dependent decrease in infectivity was observed on introduction of eosinophils into suspensions of respiratory syncytial virus group B (RSV-B). This antiviral effect was reversed by ribonuclease inhibitor, suggesting a role for the eosinophil secretory ribonucleases. Recombinant eosinophil-derived neurotoxin (rhEDN), the major eosinophil ribonuclease, promoted a dose-dependent decrease in RSV-B infectivity, with a 40-fold reduction observed in response to 50 nM rhEDN. Ribonucleolytically inactivated rhEDN (rhEDNdK38) had no antiviral activity. Semiquantitative reverse transcriptase-polymerase chain reaction demonstrated loss of viral genomic RNA in response to rhEDN, suggesting that this protein promotes the direct ribonucleolytic destruction of extracellular virions. Ribonuclease A had no antiviral activity even at approximately 1000-fold higher concentrations, suggesting that rhEDN has unique features other than ribonuclease activity that are crucial to its effectiveness. These results suggest that rhEDN may have potential as a therapeutic agent for prevention or treatment of disease caused by RSV.

Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism.

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene beta-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for beta-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 x 10[6]/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an approximately 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.

Molecular cloning of four novel murine ribonuclease genes: unusual expansion within the ribonuclease A gene family.

We have characterized four novel murine ribonuclease genes that, together with the murine eosinophil-associated ribonucleases 1 and 2, form a distinct and unusual cluster within the RNase A gene superfamily. Three of these genes (mR-3, mR-4, mR-5) include complete open reading frames, encoding ribonucleases with eight cysteines and appropriately spaced histidines (His11 and His124) and lysine (Lys35) that are characteristic of this enlarging protein family; the fourth sequence encodes a non-functional pseudogene (mR-6P). Although the amino acid sequence similarities among these murine ribonucleases varies from 60 to 94%, they form a unique cluster, as each sequence is found to be more closely related to another of this group than to either murine angiogenin or to murine pancreatic ribonuclease. Interestingly, the relationship between the six genes in this 'mR cluster' and the defined lineages of the RNase A gene family could not be determined by amino acid sequence homology, suggesting the possibility that there are one or more additional ribonuclease lineages that have yet to be defined. Although the nature of the evolutionary constraints promoting this unusual expansion and diversification remain unclear, the implications with respect to function are intriguing.

Suppression of HIV-1 transcription by beta-chemokines RANTES, MIP1-alpha, and MIP-1beta is not mediated by the NFAT-1 enhancer element.

Soluble factors derived from human CD8+ T-lymphocytes inhibit HIV-1 replication by suppressing transcription from the viral long terminal repeat (LTR), an effect shown to be mediated in part by an NFAT-1 enhancer sequence. We show here that the CD8+ derived beta-chemokines, RANTES, MIP1-alpha, and MIP-1beta, known suppressors of HIV-1 replication in human peripheral blood mononuclear cells, can suppress transcription from the HIV-1 LTR in transient transfection assays in cells of the Jurkat (acute T leukemia) line. Surprisingly, the suppression mediated by these beta-chemokines persisted in the absence of an intact NFAT-1 element, suggesting that there are at least two classes of HIV-1 suppressor factors--NFAT-1-dependent and NFAT-1-independent factors--produced by CD8+ T-lymphocytes.

The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes.

The Charcot-Leyden crystal (CLC) protein, or eosinophil lysophospholipase, is a characteristic protein of human eosinophils and basophils; recent work has demonstrated that the CLC protein is both structurally and functionally related to the galectin family of beta-galactoside binding proteins. The galectins as a group share a number of features in common, including a linear ligand binding site encoded on a single exon. In this work, we demonstrate that the intron-exon structure of the gene encoding CLC is analogous to those encoding the galectins. The coding sequence of the CLC gene is divided into four exons, with the entire beta-galactoside binding site encoded by exon III. We have isolated CLC beta-galactoside binding sites from both orangutan (Pongo pygmaeus) and murine (Mus musculus) genomic DNAs, both encoded on single exons, and noted conservation of the amino acids shown to interact directly with the beta-galactoside ligand. The most likely interpretation of these results suggest the occurrence of one or more exon duplication and insertion events, resulting in the distribution of this lectin domain to CLC as well as to the multiple galectin genes.

Intronic enhancer activity of the eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3) genes is mediated by an NFAT-1 consensus binding sequence.

The eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) are both small, cationic ribonuclease toxins that are stored in and secreted by activated human eosinophilic leukocytes. We have previously shown that optimal expression of the EDN gene is dependent on an interaction between an intronic enhancer element or elements and the 5' promoter region. Here we present evidence demonstrating that the gene encoding ECP is regulated in an analogous fashion and that an intronic enhancer element functioning in both genes is a consensus binding sequence for the transcription factor NFAT-1. Our initial results demonstrate that one or more nuclear proteins isolated from human promyelocytic leukemia (HL-60) cells bind specifically at this consensus site (5'-GGAGAA-3') within the intron of the EDN gene and that disruption of this sequence reduced the characteristic 20-30-fold increase in reporter gene activity observed with the tandem EDN promoter/exon 1/intron construct to background levels. The NFAT-1 consensus site in the ECP gene differs from that found in the EDN gene by a single nucleotide (5'-GGAGAG-3'); the conversion of the 3' G to an A resulted in a further enhancement of the reporter gene activity supported by the ECP promoter/exon 1/intron construct. Interestingly, no "supershift" was observed in gel shift assays performed in the presence of anti-NFAT-1 antiserum, suggesting that a nuclear protein other than NFAT-1 may be acting at this consensus site.

Molecular cloning and characterization of a novel human ribonuclease (RNase k6): increasing diversity in the enlarging ribonuclease gene family.

The discovery of Ribonuclease k6 (RNase k6) was an unexpected result of our ongoing efforts to trace the evolutionary history of the ribonuclease gene family. The open reading frame of RNase k6, amplified from human genomic DNA, encodes a 150 amino acid polypeptide with eight cysteines and histidine and lysine residues corresponding to those found in the active site of the prototype, ribonuclease A. The single-copy gene encoding RNase k6 maps to human chromosome 14 and orthologous sequences were detected in both primate and non-primate mammalian species. A single mRNA transcript (1.5 kb) was detected in all human tissues tested, with lung representing the most abundant source. At the cellular level, transcripts encoding RNase k6 were detected in normal human monocytes and neutrophils (but not in eosinophils) suggesting a role for this ribonuclease in host defense. Of the five previously identified human ribonucleases of this group, RNase k6 is most closely related to eosinophil-derived neurotoxin (EDN), with 47% amino acid sequence identity; slight cross-reactivity between RNase k6 and EDN was observed on Western blots probed with polyclonal anti-EDN antiserum. The catalytic constants determined, Km = 5.0 microM and Kcat = 0.13 s-1, indicate that recombinant RNase k6 has approximately 40-fold less ribonuclease activity than recombinant EDN. The identification and characterization of RNase k6 has extended the ribonuclease gene family and suggests the possibility that there are others awaiting discovery.

Characterization of eosinophils generated in vitro from CD34+ peripheral blood progenitor cells.

Methods for isolation and cultivation of CD34+ peripheral blood progenitor cells (PBPCs) have facilitated their use in autologous transplantation and as potential targets for gene therapy. In this work, we present the possibility of using these isolated cells to study lineage-specific hematopoietic differentiation. We have shown that differentiating PBPCs faithfully replicate transcriptional events that occur during maturation of the eosinophil lineage; messenger RNAs encoding the five eosinophil granule proteins were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) after 2-3 days of cytokine-stimulated growth. Only three of the five proteins were detected by immunofluorescence staining after 14 days of cytokine-stimulated growth; the percentage of Charcot-Leyden crystal protein (CLC)-containing cells (16-18%) exceeded that of eosinophil peroxidase (EPO)-containing cells (7-8%), which in turn exceeded that of eosinophil-derived neurotoxin (EDN)-containing cells (2-4%). While the electrophoretic mobilities of both CLC and EPO synthesized by differentiating PBPCs were similar to those of their normal counterparts, immunoreactive EDN was found to be heterogeneous and of higher molecular weight that EDN found in mature eosinophils. It is not clear whether our results, which show progressive, but incomplete, differentiation of PBPCs into eosinophils, reflect a lack of knowledge as to what factors are essential for complete differentiation in vitro or relate to the inherent capacity of PBPCs to differentiate along this lineage.