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.

Glucocorticoid administration accelerates mortality of pneumovirus-infected mice.

The use of glucocorticoids for the treatment of symptoms associated with respiratory syncytial virus (RSV) infection has been questioned. To evaluate the sequelae of glucocorticoid administration in the setting of pneumovirus infection in vivo, hydrocortisone was administered to mice infected with pneumonia virus of mice (PVM), a pneumovirus and natural rodent pathogen that is closely related to RSV and replicates the signs and symptoms of severe human RSV infection. Results showed that hydrocortisone spared the pulmonary neutrophilia but resulted in ablation of the pulmonary eosinophilia, despite continued production of the relevant chemoattractant, macrophage inflammatory protein-1alpha. Hydrocortisone also led to diminished production of inducible nitric oxide synthase and accumulation of reactive nitrogen species in lung tissue and bronchoalveolar lavage fluid and diminished lymphocyte recruitment. PVM-infected mice responded to hydrocortisone with enhanced viral replication and accelerated mortality. These results suggest several mechanisms to explain why glucocorticoid therapy may be of limited benefit in the overall picture of pneumovirus infection.

Rapid diversification of RNase A superfamily ribonucleases from the bullfrog, Rana catesbeiana.

We present sequences of five novel RNase A superfamily ribonuclease genes of the bullfrog, Rana catesbeiana. All five genes encode ribonucleases that are similar to Onconase, a cytotoxic ribonuclease isolated from oocytes of R. pipiens. With amino acid sequence data from 14 ribonucleases from three Rana species (R. catesbeiana, R. japonica, and R. pipiens), we have constructed bootstrap-supported phylogenetic trees that reorganize these ribonucleases into five distinct lineages--the pancreatic ribonucleases (RNases 1), the eosinophil-associated ribonucleases (RNases 2, 3, and 6), the ribonucleases 4, the angiogenins (RNases 5) and the Rana ribonucleases--with the Rana ribonucleases no more closely related to the angiogenins than they are to any of the other ribonuclease lineages shown. Further phylogenetic analysis suggests the division of the Rana ribonucleases into two subclusters (A and B), with positive (Darwinian) selection (dN/dS > 1.0) and an elevated rate of radical nonsynonymous substitution (dR) contributing to the rapid diversification of ribonucleases within each cluster. This pattern of evolution-rapid diversification via positive selection among sequences of a multigene cluster-bears striking resemblance to what we have described for the eosinophil-associated ribonuclease genes of the rodent Mus musculus, a finding that may have implications with respect the physiologic function of this unique family of proteins.

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.

Gene structure and enzymatic activity of mouse eosinophil-associated ribonuclease 2.

Mouse eosinophil-associated ribonuclease-2 (mEAR-2) is one of a cluster of genes identified in the genome of the mouse Mus musculus that are highly divergent orthologs of the primate ribonucleases, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP). Northern analysis revealed expression of genes hybridizing to mEAR-2 in mouse lung, liver and spleen tissues. We obtained full-length cDNA by hybridization screening of mouse eosinophil and lung cDNA libraries and by rapid amplification of cDNA ends (RACE) from liver, spleen and lung RNA. Using these methods we have isolated the 195 base pair (bp) 3' untranslated region (UTR) that includes a typical polyadenylation signal preceding a poly A tail and the 5' UTR which includes 63-71 bp and three distinct transcriptional start sites. Using unidirectional PCR we isolated a 361-bp 5' promoter region and delineated the intronic / exonic boundaries which include a non-coding exon 1, a single intron, and a coding exon 2, a structure that is typical of genes of the RNase A superfamily. Consensus sites for PU.1 and EoTF, both active as intronic enhancer elements of the gene encoding EDN, are also present in the intron of the gene encoding mEAR-2. The catalytic activity of recombinant baculovirus-derived mEAR-2 is similar to that of rhEDN from this source, with catalytic constants k(cat)/K(m)=5.6x10(6) M(-1) s(-1) and 10.5x10(6) M(-1) s(-1), respectively, against a standard yeast tRNA substrate. Sequence analysis of the non-coding regions and enzymatic characterization of the gene product provide further evidence indicating that mEAR-2 is a structural and functional ortholog of primate EDNs and ECPs.

The expression of GAD67 isoforms in zebrafish retinal tissue changes over the light/dark cycle.

We show the levels of glutamic acid decarboxylase (GAD), the enzyme catalyzing the conversion of glutamic acid to GABA, changes in zebrafish retinal tissue during the light/dark cycle. Further, we identify two transcripts of the GAD67 gene, full-length GAD67 and the truncated 25 kDa alternative splice variant (ES), as the major GAD isoforms in this tissue. GAD-positive neurons were identified immunocytochemically by probing retinal sections with K2, an antibody to the GAD67 isoform, and with an antibody specific for the 25 kDa splice variant. For both antibodies, GAD-immunoreactivity was observed in horizontal cells in the distal retina and amacrine cells in the proximal retina, with both cell bodies and processes labeled. No apparent difference in K2 labeling pattern was observed in tissue harvested 8 hrs after light offset or onset, whereas ES label was identified in more structures in dark tissue. Quantification of GAD levels was determined by densitometry of Western Blots. The protein content of GAD67 and ES varied between tissue harvested during the light and the dark. ES expression was up-regulated in dark tissue; whereas, full-length GAD67 expression increased in light tissue. In vivo GABA content, measured with high performance liquid chromatography (HPLC), was found to increase in light tissue, paralleling the expression of full-length GAD67 transcripts. Expression of ES did not correlate with measured GABA levels, suggesting this isoform, which lacks the catalytic domain necessary for enzymatic activity, may have a different physiological role in retinal tissue. The inverse expression patterns of full-length GAD67 and ES suggest that alternative splicing of GAD67 may be triggered by the light and/or dark cycle, resulting in a change in inhibitory neurotransmitter content in retinal tissue.

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.

Pulmonary eosinophilia and production of MIP-1alpha are prominent responses to infection with pneumonia virus of mice.

Human eosinophils secrete two distinct ribonucleases that have antiviral activity against pathogens of the family Paramyxoviridae. To examine the role of eosinophils and their ribonucleases in host defense against paramyxovirus pathogens in vivo, we have developed a mouse model involving a viral pathogen that naturally targets a rodent host. In this work we describe infection of Balb/c mice with pneumonia virus of mice (PVM, strain J3666), a paramyxovirus pathogen found frequently among rodent populations. We show here that pulmonary eosinophilia is an immediate response to infection with PVM, with bronchoalveolar lavage fluid containing 12-14% eosinophils obtained as early as day 3 postinoculation. Infection is accompanied by the production of macrophage inflammatory protein-1-alpha (MIP-1alpha), a chemokine that has been associated with the pulmonary eosinophilia observed in response to respiratory syncytial virus infection in humans and with enhanced clearance of influenza virus in mice. Interestingly, we observed no changes in expression of the chemoattractants eotaxin and RANTES in response to PVM infection, and interleukin-5 remained undetectable throughout. These responses-clinical pathology, viral recovery, pulmonary eosinophilia, and production of MIP-1alpha-will provide a means for exploring the role of eosinophils, eosinophil secretory ribonucleases, and eosinophil chemoattractants in host defense against PVM and related paramyxovirus pathogens in vivo.

Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection.

The mammalian RNase A superfamily comprises a diverse array of ribonucleolytic proteins that have a variety of biochemical activities and physiological functions. Two rapidly evolving RNases of higher primates are of particular interest as they are major secretory proteins of eosinophilic leukocytes and have been found to possess anti-pathogen activities in vitro. To understand how these RNases acquired this function during evolution and to develop animal models for the study of their functions in vivo, it is necessary to investigate these genes in many species. Here, we report the sequences of 38 functional genes and 23 pseudogenes of the eosinophil-associated RNase (EAR) family from 5 rodent species. Our phylogenetic analysis of these genes showed a clear pattern of evolution by a rapid birth-and-death process and gene sorting, a process characterized by rapid gene duplication and deactivation occurring differentially among lineages. This process ultimately generates distinct or only partially overlapping inventories of the genes, even in closely related species. Positive Darwinian selection also contributed to the diversification of these EAR genes. The striking similarity between the evolutionary patterns of the EAR genes and those of the major histocompatibility complex, immunoglobulin, and T cell receptor genes stands in strong support of the hypothesis that host-defense and generation of diversity are among the primary physiological function of the rodent EARs. The discovery of a large number of divergent EARs suggests the intriguing possibility that these proteins have been specifically tailored to fight against distinct rodent pathogens.

Rapid evolution of the ribonuclease A superfamily: adaptive expansion of independent gene clusters in rats and mice.

The two eosinophil ribonucleases, eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3), are among the most rapidly evolving coding sequences known among primates. The eight mouse genes identified as orthologs of EDN and ECP form a highly divergent, species-limited cluster. We present here the rat ribonuclease cluster, a group of eight distinct ribonuclease A superfamily genes that are more closely related to one another than they are to their murine counterparts. The existence of independent gene clusters suggests that numerous duplications and diversification events have occurred at these loci recently, sometime after the divergence of these two rodent species ( approximately 10-15 million years ago). Nonsynonymous substitutions per site (d(N)) calculated for the 64 mouse/rat gene pairs indicate that these ribonucleases are incorporating nonsilent mutations at accelerated rates, and comparisons of nonsynonymous to synonymous substitution (d(N) / d(S)) suggest that diversity in the mouse ribonuclease cluster is promoted by positive (Darwinian) selection. Although the pressures promoting similar but clearly independent styles of rapid diversification among these primate and rodent genes remain uncertain, our recent findings regarding the function of human EDN suggest a role for these ribonucleases in antiviral host defense.

Evolution of antiviral activity in the ribonuclease A gene superfamily: evidence for a specific interaction between eosinophil-derived neurotoxin (EDN/RNase 2) and respiratory syncytial virus.

We have demonstrated that the human eosinophil-derived neurotoxin (EDN, RNase 2), a rapidly evolving secretory protein derived from eosinophilic leukocytes, mediates the ribonucleolytic destruction of extracellular virions of the single-stranded RNA virus respiratory syncytial virus (RSV). While RNase activity is crucial to antiviral activity, it is clearly not sufficient, as our results suggest that EDN has unique structural features apart from RNase activity that are necessary to promote antiviral activity. We demonstrate here that the interaction between EDN and extracellular virions of RSV is both saturatable and specific. Increasing concentrations of the antivirally inactivated, ribonucleolytically inactivated point mutant form of recombinant human EDN, rhEDNdK38, inhibits rhEDN's antiviral activity, while increasing concentrations of the related RNase, recombinant human RNase k6, have no effect whatsoever. Interestingly, acquisition of antiviral activity parallels the evolutionary development of the primate EDN lineage, having emerged some time after the divergence of the Old World from the New World monkeys. Using this information, we created ribonucleolytically active chimeras of human and New World monkey orthologs of EDN and, by evaluating their antiviral activity, we have identified an N-terminal segment of human EDN that contains one or more of the sequence elements that mediate its specific interaction with RSV.

Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity.

Eosinophil cationic protein (ECP) is one of two RNase A-superfamily ribonucleases found in secretory granules of human eosinophilic leukocytes. Although the physiologic function of eosinophils [and thus of the two eosinophil ribonucleases, ECP and eosinophil-derived neurotoxin (EDN)] remains controversial, we have recently shown that isolated human eosinophils promote ribonuclease-dependent toxicity toward extracellular virions of the single-stranded RNA virus, respiratory syncytial virus, group B (RSV-B). We have also shown that recombinant human EDN (rhEDN) can act alone as a ribonuclease-dependent antiviral agent. In this work, we provide a biochemical characterization of recombinant human ECP (rhECP) prepared in baculovirus, and demonstrate that rhECP also promotes ribonuclease-dependent antiviral activity. The rhECP described here is N-glycosylated, as is native ECP, and has approximately 100-fold more ribonuclease activity than non-glycosylated rhECP prepared in bacteria. The enzymatic activity of rhECP was sensitive to inhibition by placental ribonuclease inhibitor (RI). Although rhECP was not as effective as rhEDN at reducing viral infectivity (500 nM rhECP reduced infectivity of RSV-B approximately 6 fold; 500 nM rhEDN, >50 fold), the antiviral activity appears to be unique to the eosinophil ribonucleases; no reduction in infectivity was promoted by bovine RNase A, by the amphibian ribonuclease, onconase, nor by the closely-related human ribonuclease, RNase k6. Interestingly, combinations of rhEDN and rhECP did not result in either a synergistic or even an additive antiviral effect. Taken together, these results suggest that that the interaction between the eosinophil ribonucleases and the extracellular virions of RSV-B may be specific and saturable.

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.

Ribonucleases and host defense: identification, localization and gene expression in adherent monocytes in vitro.

Several ribonucleases of the RNase A family function as antibacterial, anti-parasitic and anti-viral agents. In this work, we have shown that mRNAs encoding five of the six known human ribonucleases of the RNase A family are expressed in cultured human monocytes, and that ribonucleases are released by adherent monocytes in culture. Using a polyclonal antiserum prepared against recombinant protein, we have detected one of these ribonucleases, RNase 4, in lysates of normal human peripheral blood monocytes, but not granulocytes or lymphocytes, by Western blotting. Subcellular localization by immunoelectron microscopy demonstrated the presence of RNase 4 in the cytoplasmic granules of isolated monocytes. Interestingly, mRNA encoding RNase 4 could not be detected in freshly isolated monocytes, emerging only after 16 h in culture, suggesting the possibility of de novo protein synthesis in association with monocyte differentiation.

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.

Diversity among the primate eosinophil-derived neurotoxin genes: a specific C-terminal sequence is necessary for enhanced ribonuclease activity.

The human eosinophil-derived neurotoxin (hEDN) is a secretory effector protein from eosinophilic leukocytes that is a member of the ribonuclease A (RNase A) family of ribonucleases. EDN is a rapidly evolving protein, accumulating non-silent mutations at a rate exceeding those of most other functional coding sequences studied in primates. Although all primate EDNs retain the structural and functional residues known to be prerequisites for ribonuclease activity, we have shown previously that recombinant EDN derived from a New World monkey sequence ( Saguinus oedipus ) had significantly less catalytic activity than the human (hEDN) ortholog.In this work, we have prepared recombinant proteins from EDN from sequences derived from orangutan (Pongo pygmaeus, oEDN) and Old World monkey (Macaca fascicularis, mcEDN) genomic DNAs, and from a second New World monkey sequence (Aotus trivirgatus, omEDN) as well. The catalytic efficiencies [ k cat/ K m (M-1s-1)] determined for both oEDN and mcEDN were similar to that determined previously for hEDN, while omEDN displayed approximately 100-fold less catalytic activity. The relative ribonuclease activities of hEDN/omEDN chimeras pointed to a C-terminal segment as crucial to the enhanced catalytic activity hEDN, and substitution of Arg 132-Ile 133 of hEDN with the Thr-Thr pair at the analogous position in omEDN resulted in an approximately 10-fold reduction in hEDN's catalytic efficiency. However, the reverse substitution, Arg-Ile for Thr-Thr in omEDN, did not enhance the catalytic efficiency of this relatively inactive protein. These results indicate that the Arg and/or Ile residues adjacent to the C-terminus are necessary (but not sufficient) for enhanced ribonuclease activity among the primate EDNs, and will permit prediction of the relative ribonuclease activities based on differences in primary structure.

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.

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.