Priming of the Respiratory Tract with Immunobiotic Lactobacillus plantarum Limits Infection of Alveolar Macrophages with Recombinant Pneumonia Virus of Mice (rK2-PVM).

UNLABELLED: Pneumonia virus of mice (PVM) is a natural rodent pathogen that replicates in bronchial epithelial cells and reproduces many clinical and pathological features of the more severe forms of disease associated with human respiratory syncytial virus. In order to track virus-target cell interactions during acute infection in vivo, we developed rK2-PVM, bacterial artificial chromosome-based recombinant PVM strain J3666 that incorporates the fluorescent tag monomeric Katushka 2 (mKATE2). The rK2-PVM pathogen promotes lethal infection in BALB/c mice and elicits characteristic cytokine production and leukocyte recruitment to the lung parenchyma. Using recombinant virus, we demonstrate for the first time PVM infection of both dendritic cells (DCs; CD11c(+) major histocompatibility complex class II(+)) and alveolar macrophages (AMs; CD11c(+) sialic acid-binding immunoglobulin-like lectin F(+)) in vivo and likewise detect mKATE2(+) DCs in mediastinal lymph nodes from infected mice. AMs support both active virus replication and production of infectious virions. Furthermore, we report that priming of the respiratory tract with immunobiotic Lactobacillus plantarum, a regimen that results in protection against the lethal inflammatory sequelae of acute respiratory virus infection, resulted in differential recruitment of neutrophils, DCs, and lymphocytes to the lungs in response to rK2-PVM and a reduction from ∼ 40% to <10% mKATE2(+) AMs in association with a 2-log drop in the release of infectious virions. In contrast, AMs from L. plantarum-primed mice challenged with virus ex vivo exhibited no differential susceptibility to rK2-PVM. Although the mechanisms underlying Lactobacillus-mediated viral suppression remain to be fully elucidated, this study provides insight into the cellular basis of this response. IMPORTANCE: Pneumonia virus of mice (PVM) is a natural mouse pathogen that serves as a model for severe human respiratory syncytial virus disease. We have developed a fully functional recombinant PVM strain with a fluorescent reporter protein (rK2-PVM) that permits us to track infection of target cells in vivo. With rK2-PVM, we demonstrate infection of leukocytes in the lung, notably, dendritic cells and alveolar macrophages. Alveolar macrophages undergo productive infection and release infectious virions. We have shown previously that administration of immunobiotic Lactobacillus directly to the respiratory mucosa protects mice from the lethal sequelae of PVM infection in association with profound suppression of the virus-induced inflammatory response. We show here that Lactobacillus administration also limits infection of leukocytes in vivo and results in diminished release of infectious virions from alveolar macrophages. This is the first study to provide insight into the cellular basis of the antiviral impact of immunobiotic L. plantarum.

Eosinophil-associated ribonuclease 11 is a macrophage chemoattractant.

RNase A is the prototype of an extensive family of divergent proteins whose members share a unique disulfide-bonded tertiary structure, conserved catalytic motifs, and the ability to hydrolyze polymeric RNA. Several members of this family maintain independent roles as ribonucleases and modulators of innate immunity. Here we characterize mouse eosinophil-associated RNase (Ear) 11, a divergent member of the eosinophil ribonuclease cluster, and the only known RNase A ribonuclease expressed specifically in response to Th2 cytokine stimulation. Mouse Ear 11 is differentially expressed in somatic tissues at baseline (brain ≪ liver < lung < spleen); systemic stimulation with IL-33 results in 10-5000-fold increased expression in lung and spleen, respectively. Ear 11 is also expressed in response to protective priming of the respiratory mucosa with Lactobacillus plantarum; transcripts are detected both locally in lung as well as systemically in bone marrow and spleen. Mouse Ear 11 is enzymatically active, although substantially less so than mEar 1 and mEar 2; the relative catalytic efficiency (kcat/Km) of mEar 11 is diminished ∼1000-1500-fold. However, in contrast to RNase 2/EDN and mEar 2, which have been characterized as selective chemoattractants for CD11c(+) dendritic cells, mEar 11 has prominent chemoattractant activity for F4/80(+)CD11c(-) tissue macrophages. Chemoattractant activity is not dependent on full enzymatic activity, and requires no interaction with the pattern recognition receptor, Toll-like receptor 2 (TLR2). Taken together, this work characterizes a divergent RNase A ribonuclease with a unique expression pattern and function, and highlights the versatility of this family in promoting innate immunity.

Activated mouse eosinophils protect against lethal respiratory virus infection.

Eosinophils are recruited to the airways as a prominent feature of the asthmatic inflammatory response where they are broadly perceived as promoting pathophysiology. Respiratory virus infections exacerbate established asthma; however, the role of eosinophils and the nature of their interactions with respiratory viruses remain uncertain. To explore these questions, we established acute infection with the rodent pneumovirus, pneumonia virus of mice (PVM), in 3 distinct mouse models of Th2 cytokine-driven asthmatic inflammation. We found that eosinophils recruited to the airways of otherwise naïve mice in response to Aspergillus fumigatus, but not ovalbumin sensitization and challenge, are activated by and degranulate specifically in response to PVM infection. Furthermore, we demonstrate that activated eosinophils from both Aspergillus antigen and cytokine-driven asthma models are profoundly antiviral and promote survival in response to an otherwise lethal PVM infection. Thus, although activated eosinophils within a Th2-polarized inflammatory response may have pathophysiologic features, they are also efficient and effective mediators of antiviral host defense.

B cells are not essential for Lactobacillus-mediated protection against lethal pneumovirus infection.

We have shown previously that priming of respiratory mucosa with live Lactobacillus species promotes robust and prolonged survival from an otherwise lethal infection with pneumonia virus of mice, a property known as heterologous immunity. Lactobacillus priming results in a moderate reduction in virus recovery and a dramatic reduction in virus-induced proinflammatory cytokine production; the precise mechanisms underlying these findings remain to be elucidated. Because B cells have been shown to promote heterologous immunity against respiratory virus pathogens under similar conditions, in this study we explore the role of B cells in Lactobacillus-mediated protection against acute pneumovirus infection. We found that Lactobacillus-primed mice feature elevated levels of airway Igs IgG, IgA, and IgM and lung tissues with dense, B cell (B220(+))-enriched peribronchial and perivascular infiltrates with germinal centers consistent with descriptions of BALT. No B cells were detected in lung tissue of Lactobacillus-primed B cell deficient µMT mice or Jh mice, and Lactobacillus-primed µMT mice had no characteristic infiltrates or airway Igs. Nonetheless, we observed diminished virus recovery and profound suppression of virus-induced proinflammatory cytokines CCL2, IFN-γ, and CXCL10 in both wild-type and Lactobacillus-primed µMT mice. Furthermore, Lactobacillus plantarum-primed, B cell-deficient µMT and Jh mice were fully protected from an otherwise lethal pneumonia virus of mice infection, as were their respective wild-types. We conclude that B cells are dispensable for Lactobacillus-mediated heterologous immunity and were not crucial for promoting survival in response to an otherwise lethal pneumovirus infection.

Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice.

Eosinophil activities are often linked with allergic diseases such as asthma and the pathologies accompanying helminth infection. These activities have been hypothesized to be mediated, in part, by the release of cationic proteins stored in the secondary granules of these granulocytes. The majority of the proteins stored in these secondary granules (by mass) are major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX). Unpredictably, a knockout approach targeting the genes encoding these proteins demonstrated that, unlike in mice containing a single deficiency of only MBP-1 or EPX, the absence of both granule proteins resulted in the near complete loss of peripheral blood eosinophils with no apparent impact on any other hematopoietic lineage. Moreover, the absence of MBP-1 and EPX promoted a concomitant loss of eosinophil lineage-committed progenitors in the marrow, identifying a specific blockade in eosinophilopoiesis as the causative event. Significantly, this blockade of eosinophilopoiesis is also observed in ex vivo cultures of marrow progenitors and is not rescued in vivo by adoptive bone marrow engraftment, suggesting a cell-autonomous defect in marrow progenitors. These observations implicate a role for granule protein gene expression as a regulator of eosinophilopoiesis and provide another strain of mice congenitally deficient of eosinophils.

Chemotaxis of bone marrow derived eosinophils in vivo: a novel method to explore receptor-dependent trafficking in the mouse.

Here, we describe a novel method via which ex vivo cultured mouse bone marrow derived eosinophils (bmEos) can be adoptively transferred into recipient mice in order to study receptor-dependent recruitment to lung tissue in vivo. Intratracheal instillation of recombinant human eotaxin-2 (hCCL24) prior to introduction of bmEos via tail vein injection resulted in an approximately fourfold increase in Siglec F-positive/CD11c-negative eosinophils in the lungs of eosinophil-deficient ΔdblGATA recipient mice compared with controls. As anticipated, bmEos generated from CCR3-gene-deleted mice did not migrate to the lung in response to hCCL24 in this model, indicating specific receptor dependence. BmEos generated from GFP-positive BALB/c mice responded similarly to hCCL24 in vitro and were detected in lung tissue of BALB/c WT as well as BALB/c ΔdblGATA eosinophil-deficient recipient mice, at approximately fourfold (at 5 h post-injection) and approximately threefold (at 24 h postinjection) over baseline, respectively. Comparable results were obtained with GFP-positive C57BL/6 bmEos responding to intratracheal hCCL24 in C57BL/6 ΔdblGATA recipient mice. The use of ex vivo cultured bmEos via one or more of these methods offers the possibility of manipulating bmEos prior to transfer into a WT or gene-deleted recipient host. Thus, this chemotaxis model represents a novel and robust tool for pharmacological studies in vivo.

Lactobacillus-mediated priming of the respiratory mucosa protects against lethal pneumovirus infection.

The inflammatory response to respiratory virus infection can be complex and refractory to standard therapy. Lactobacilli, when targeted to the respiratory epithelium, are highly effective at suppressing virus-induced inflammation and protecting against lethal disease. Specifically, wild-type mice primed via intranasal inoculation with live or heat-inactivated Lactobacillus plantarum or Lactobacillus reuteri were completely protected against lethal infection with the virulent rodent pathogen, pneumonia virus of mice; significant protection (60% survival) persisted for at least 13 wk. Protection was not unique to Lactobacillus species, and it was also observed in response to priming with nonpathogenic Gram-positive Listeria innocua. Priming with live lactobacilli resulted in diminished granulocyte recruitment, diminished expression of multiple proinflammatory cytokines (CXCL10, CXCL1, CCL2, and TNF), and reduced virus recovery, although we have demonstrated clearly that absolute virus titer does not predict clinical outcome. Lactobacillus priming also resulted in prolonged survival and protection against the lethal sequelae of pneumonia virus of mice infection in MyD88 gene-deleted (MyD88(-/-)) mice, suggesting that the protective mechanisms may be TLR-independent. Most intriguing, virus recovery and cytokine expression patterns in Lactobacillus-primed MyD88(-/-) mice were indistinguishable from those observed in control-primed MyD88(-/-) counterparts. In summary, we have identified and characterized an effective Lactobacillus-mediated innate immune shield, which may ultimately serve as critical and long-term protection against infection in the absence of specific antiviral vaccines.

Genetic diversity of human RNase 8.

BACKGROUND: Ribonuclease 8 is a member of the RNase A family of secretory ribonucleases; orthologs of this gene have been found only in primate genomes. RNase 8 is a divergent paralog of RNase 7, which is lysine-enriched, highly conserved, has prominent antimicrobial activity, and is expressed in both normal and diseased skin; in contrast, the physiologic function of RNase 8 remains uncertain. Here, we examine the genetic diversity of human RNase 8, a subject of significant interest given the existence of functional pseudogenes (coding sequences that are otherwise intact but with mutations in elements crucial for ribonucleolytic activity) in non-human primate genomes. RESULTS: RNase 8 expression was detected in adult human lung, spleen and testis tissue by quantitative reverse-transcription PCR. Only two single-nucleotide polymorphisms and four unique alleles were identified within the RNase 8 coding sequence; nucleotide sequence diversity (π = 0.00122 ± 0.00009 per site) was unremarkable for a human nuclear gene. We isolated transcripts encoding RNase 8 via rapid amplification of cDNA ends (RACE) and RT-PCR which included a distal potential translational start site followed by sequence encoding an additional 30 amino acids that are conserved in the genomes of several higher primates. The distal translational start site is functional and promotes RNase 8 synthesis in transfected COS-7 cells. CONCLUSIONS: These results suggest that RNase 8 may diverge considerably from typical RNase A family ribonucleases and may likewise exhibit unique function. This finding prompts a reconsideration of what we have previously termed functional pseudogenes, as RNase 8 may be responding to constraints that promote significant functional divergence from the canonical structure and enzymatic activity characteristic of the RNase A family.

Mouse and human eosinophils degranulate in response to platelet-activating factor (PAF) and lysoPAF via a PAF-receptor-independent mechanism: evidence for a novel receptor.

Platelet-activating factor (PAF [1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine]) is a phospholipid mediator released from activated macrophages, mast cells, and basophils that promotes pathophysiologic inflammation. Eosinophil responses to PAF are complex and incompletely elucidated. We show in this article that PAF and its 2-deacetylated metabolite (lysoPAF) promote degranulation (release of eosinophil peroxidase) via a mechanism that is independent of the characterized PAFR. Specifically, we demonstrate that receptor antagonists CV-3988 and WEB-2086 and pertussis toxin have no impact on PAF- or lysoPAF-mediated degranulation. Furthermore, cultured mouse eosinophils from PAFR(-/-) bone marrow progenitors degranulate in response to PAF and lysoPAF in a manner indistinguishable from their wild-type counterparts. In addition to PAF and lysoPAF, human eosinophils degranulate in response to lysophosphatidylcholine, but not phosphatidylcholine, lysophosphatidylethanolamine, or phosphatidylethanolamine, demonstrating selective responses to phospholipids with a choline head-group and minimal substitution at the sn-2 hydroxyl. Human eosinophils release preformed cytokines in response to PAF, but not lysoPAF, also via a PAFR-independent mechanism. Mouse eosinophils do not release cytokines in response to PAF or lysoPAF, but they are capable of doing so in response to IL-6. Overall, our work provides the first direct evidence for a role for PAF in activating and inducing degranulation of mouse eosinophils, a crucial feature for the interpretation of mouse models of PAF-mediated asthma and anaphylaxis. Likewise, we document and define PAF and lysoPAF-mediated activities that are not dependent on signaling via PAFR, suggesting the existence of other unexplored molecular signaling pathways mediating responses from PAF, lysoPAF, and closely related phospholipid mediators.

Pneumoviruses infect eosinophils and elicit MyD88-dependent release of chemoattractant cytokines and interleukin-6.

Eosinophils are recruited to the lung in response to infection with pneumovirus pathogens and have been associated with both the pathophysiologic sequelae of infection and, more recently, with accelerated virus clearance. Here, we demonstrate that the pneumovirus pathogens, respiratory syncytial virus (RSV) and pneumonia virus of mice (PVM), can infect human and mouse eosinophils, respectively, and that virus infection of eosinophils elicits the release of disease-related proinflammatory mediators from eosinophils. RSV replication in human eosinophils results in the release of infectious virions and in the release of the proinflammatory mediator, interleukin-6 (IL-6). PVM replication in cultured bone marrow eosinophils (bmEos) likewise results in release of infectious virions and the proinflammatory mediators IL-6, IP-10, CCL2, and CCL3. In contrast to the findings reported in lung tissue of RSV-challenged mice, PVM replication is accelerated in MyD88 gene-deleted bmEos, whereas release of cytokines is diminished. Interestingly, exogenous IL-6 suppresses virus replication in MyD88 gene-deleted bmEos, suggesting a role for a MyD88-dependent cytokine-mediated feedback circuit in modulating this response. Taken together, our findings suggest that eosinophils are targets of virus infection and may have varied and complex contributions to the pathogenesis and resolution of pneumovirus disease.

Isolation of human eosinophils: microbead method has no impact on IL-5 sustained viability.

BACKGROUND/PURPOSE: In a recent issue of Experimental Dermatology (18, 2009, 654), Schefzyk et al. concluded that multi-antibody eosinophil isolation (Miltenyi) should be abandoned, as differential purity was minimal, and eosinophils underwent accelerated apoptosis when compared with those isolated with traditional anti-CD16 microbeads. Our intent was to investigate the universality of these findings. METHODS: We isolated eosinophils from normal donor granulocyte packs using two methods, and evaluated purity, viability and annexin-V/propidium-iodide staining. RESULTS: Purity was substantially greater when multi-antibody isolation was used for eosinophil isolation from granulocyte packs (98% vs 69%). No differential survival was detected when eosinophils were maintained in culture with or without interleukin-5. CONCLUSIONS: Multi-antibody eosinophil isolation represents a substantial advantage over anti-CD-16 microbeads when isolating large numbers of eosinophils from concentrated leucocyte preparations. No differential survival was observed. While appropriate consideration of methods is always crucial, multi-antibody eosinophil isolation should not be abandoned completely.

Functionally competent eosinophils differentiated ex vivo in high purity from normal mouse bone marrow.

We have devised an ex vivo culture system which generates large numbers of eosinophils at high purity (>90%) from unselected mouse bone marrow progenitors. In response to 4 days of culture with recombinant mouse FLT3-L and recombinant mouse stem cell factor followed by recombinant mouse IL-5 alone thereafter, the resulting bone marrow-derived eosinophils (bmEos) express immunoreactive major basic protein, Siglec F, IL-5R alpha-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common beta-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-gamma, IL-4, MIP-1alpha, and IL-6. The rodent pathogen pneumonia virus of mice replicates in bmEos and elevated levels of IL-6 are detected in supernatants of bmEos cultures in response to active infection. Finally, differentiating bmEos are readily transfected with lentiviral vectors, suggesting a means for rapid production of genetically manipulated cells.

Interferon-gamma coordinates CCL3-mediated neutrophil recruitment in vivo.

BACKGROUND: We have shown previously that acute infection with the respiratory pathogen, pneumonia virus of mice (PVM), results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice. RESULTS: In this work, we demonstrate that CCL3-mediated neutrophil recruitment is coordinated by interferon-gamma (IFNgamma). Neutrophil recruitment in response to PVM infection was diminished five-fold in IFNgamma receptor gene-deleted mice, although neutrophils from IFNgammaR -/- mice expressed transcripts for the CCL3 receptor, CCR1 and responded functionally to CCL3 ex vivo. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNgamma. Interestingly, although supplemental IFNgamma restored neutrophil recruitment and resulted in a sustained weight loss among CCL3-overexpressing IFNgamma -/- mice, CCL3-mediated neutrophil recruitment alone did not result in the pulmonary edema or respiratory failure characteristic of severe viral infection, suggesting that CCL3 and IFN-gamma together are sufficient to promote neutrophil recruitment but not pathologic activation. CONCLUSION: Our findings reveal a heretofore unrecognized hierarchical interaction between the IFNgamma and CCL3, which demonstrate that IFNgamma is crucial for CCL3-mediated neutrophil recruitment in vivo.

Eosinophils and their interactions with respiratory virus pathogens.

Eosinophils are implicated in the pathophysiology of respiratory virus infection, most typically in negative roles, such as promoting wheezing and bronchoconstriction in conjunction with virus-induced exacerbations of reactive airways disease and in association with aberrant hypersensitivity responses to viral vaccines. However, experiments carried out in vitro and in vivo suggest positive roles for eosinophils, as they have been shown to reduce virus infectivity in tissue culture and promote clearance of the human pathogen, respiratory syncytial virus in a mouse challenge model. The related natural rodent pathogen, pneumonia virus of mice (PVM), is highly virulent in mice, and is not readily cleared by eosinophils in vivo. Interestingly, PVM replicates in eosinophils and promotes cytokine release. The molecular basis of virus infection in eosinophils and its relationship to disease outcome is currently under study.

RNase 1 genes from the family Sciuridae define a novel rodent ribonuclease cluster.

The RNase A ribonucleases are a complex group of functionally diverse secretory proteins with conserved enzymatic activity. We have identified novel RNase 1 genes from four species of squirrel (order Rodentia, family Sciuridae). Squirrel RNase 1 genes encode typical RNase A ribonucleases, each with eight cysteines, a conserved CKXXNTF signature motif, and a canonical His(12)-Lys(41)-His(119) catalytic triad. Two alleles encode Callosciurus prevostii RNase 1, which include a Ser(18)<-->Pro, analogous to the sequence polymorphisms found among the RNase 1 duplications in the genome of Rattus exulans. Interestingly, although the squirrel RNase 1 genes are closely related to one another (77-95% amino acid sequence identity), the cluster as a whole is distinct and divergent from the clusters including RNase 1 genes from other rodent species. We examined the specific sites at which Sciuridae RNase 1s diverge from Muridae/Cricetidae RNase 1s and determined that the divergent sites are located on the external surface, with complete sparing of the catalytic crevice. The full significance of these findings awaits a more complete understanding of biological role of mammalian RNase 1s.

Efficient replication of pneumonia virus of mice (PVM) in a mouse macrophage cell line.

Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-alpha (TNF-alpha), interferon-beta (IFN-beta), macrophage inflammatory proteins 1alpha and 1beta (MIP-1alpha and MIP-1beta) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems.

The mouse RNase 4 and RNase 5/ang 1 locus utilizes dual promoters for tissue-specific expression.

The ribonuclease A (RNase A) superfamily has been the subject of extensive studies in the areas of protein evolution, structure and biochemistry and are exciting molecules in that they appear to be responding to unique selection pressures, generating proteins capable of multiple and diverse activities. The RNase 4 and RNase 5/ang 1 shared locus breaks a pattern that is otherwise canonical among the members of the RNase A gene superfamily. Conserved among humans, mice and rats, the locus includes two non-coding exons followed by two distinct exons encoding RNase 4 and RNase 5/ang 1. Transcription from this locus is controlled by differential splicing and tissue-specific expression from promoters located 5' to each of the non-coding exons. Promoter 1, 5' to exon I, is universally active, while Promoter 2, 5' to exon II, is active only in hepatic cells in promoter assays in vitro. Transcription from Promoter 2 is dependent on an intact HNF-1 consensus binding site which binds the transcription factor HNF-1alpha. In summary, RNase 4 and RNase 5/ang 1 are unique among the RNase A ribonuclease genes in that they maintain a complex gene locus that is conserved across species with transcription initiated from tissue-specific dual promoters followed by differential exon splicing.

RNase 8, a novel RNase A superfamily ribonuclease expressed uniquely in placenta.

We report the identification and characterization of the gene encoding the eighth and final human ribonuclease (RNase) of the highly diversified RNase A superfamily. The RNase 8 gene is linked to seven other RNase A superfamily genes on chromosome 14. It is expressed prominently in the placenta, but is not detected in any other tissues examined. Phylogenetic analysis suggests that RNase 7 is the closest relative of RNase 8 and that the pair likely resulted from a recent gene duplication event in primates. Further analysis reveals that the RNase 8 gene has incorporated non-silent mutations at an elevated rate (1.3 x 10(-9) substitutions/site/year) and that orthologous RNase 8 genes from 6 of 10 primate species examined have been deactivated by frameshifting deletions or point mutations at crucial structural or catalytic residues. The ribonucleolytic activity of recombinant human RNase 8 is among the lowest of members of this superfamily and it exhibits neither antiviral nor antibacterial activities characteristic of some other RNase A ribonucleases. The rapid evolution, species-limited deactivation and tissue-specific expression of RNase 8 suggest a unique physiological function and reiterates the evolutionary plasticity of the RNase A superfamily.

Human RNase 7: a new cationic ribonuclease of the RNase A superfamily.

Here we report on the expression and function of RNase 7, one of the final RNase A superfamily ribonucleases identified in the human genome sequence. The human RNase 7 gene is expressed in various somatic tissues including the liver, kidney, skeletal muscle and heart. Recombinant RNase 7 is ribonucleolytically active against yeast tRNA, as expected from the presence of eight conserved cysteines and the catalytic histidine-lysine- histidine triad which are signature motifs of this superfamily. The protein is atypically cationic with an isoelectric point (pI) of 10.5. Expression of recombinant RNase 7 in Escherichia coli completely inhibits the growth of the host bacteria, similar to what has been observed for the cationic RNase, eosinophil cationic protein (ECP/RNase 3, pI 11.4). An in vitro assay demonstrates dose-dependent cytotoxicity of RNase 7 against bacteria E.coli, Pseudomonas aeruginosa and Staphylococcus aureus. While RNase 7 and ECP/RNase 3 are both cationic and share this particular aspect of functional similarity, their protein sequence identity is only 40%. Of particular interest, ECP/RNase 3's cationicity is based on an (over)abundance of arginine residues, whereas RNase 7 includes an excess of lysine. This difference, in conjunction with the independent origins and different expression patterns, suggests that RNase 7 and ECP/RNase 3 may have been recruited to target different pathogens in vivo, if their physiological functions are indeed host defenses.

Characterization of a ribonuclease gene and encoded protein from the reptile, Iguana iguana.

In this work we identify an intronless open reading frame encoding an RNase A ribonuclease from genomic DNA from the Iguana iguana IgH2 cell line. The iguana RNase is expressed primarily in pancreas, and represents the majority of the specific enzymatic activity in this tissue. The encoded sequence shares many features with its better-known mammalian counterparts including the crucial His12, Lys40 and His114 catalytic residues and efficient hydrolytic activity against yeast tRNA substrate (k(cat)/K(m)=6 x 10(4) M(-1) s(-1)), albeit at a reduced pH optimum (pH 6.0). Although the catalytic activity of the iguana RNase is not diminished by human placental RI, iguana RNase is not bactericidal nor is it cytotoxic even at micromolar concentrations. Phylogenetic analysis indicates moderate (46%) amino acid sequence similarity to a pancreatic RNase isolated from Chelydra serpentina (snapping turtle) although no specific relationship could be determined between these RNases and the pancreatic ribonucleases characterized among mammalian species. Further analysis of ribonucleases from non-mammalian vertebrate species is needed in order to define relationships and lineages within the larger RNase A gene superfamily.