Helene F. Rosenberg

Eosinophils: Biological Properties and Role in Health and Disease

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the growing properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil‐mediated mucosal diseases are conceptualized.

Eosinophils: changing perspectives in health and disease

Eosinophils have been traditionally perceived as terminally differentiated cytotoxic effector cells. Recent studies have profoundly altered this simplistic view of eosinophils and their function. New insights into the molecular pathways that control the development, trafficking and degranulation of eosinophils have improved our understanding of the immunomodulatory functions of these cells and their roles in promoting homeostasis. Likewise, recent developments have generated a more sophisticated view of how eosinophils contribute to the pathogenesis of different diseases, including asthma and primary hypereosinophilic syndromes, and have also provided us with a more complete appreciation of the activities of these cells during parasitic infection.

Adaptive evolution of a duplicated pancreatic ribonuclease gene in a leaf-eating monkey

Although the complete genome sequences of over 50 representative species have revealed the many duplicated genes in all three domains of life, the roles of gene duplication in organismal adaptation and biodiversity are poorly understood. In addition, the evolutionary forces behind the functional divergence of duplicated genes are often unknown, leading to disagreement on the relative importance of positive Darwinian selection versus relaxation of functional constraints in this process. The methodology of earlier studies relied largely on DNA sequence analysis but lacked functional assays of duplicated genes, frequently generating contentious results. Here we use both computational and experimental approaches to address these questions in a study of the pancreatic ribonuclease gene (RNASE1) and its duplicate gene (RNASE1B) in a leaf-eating colobine monkey, douc langur. We show that RNASE1B has evolved rapidly under positive selection for enhanced ribonucleolytic activity in an altered microenvironment, a response to increased demands for the enzyme for digesting bacterial RNA. At the same time, the ability to degrade double-stranded RNA, a non-digestive activity characteristic of primate RNASE1, has been lost in RNASE1B, indicating functional specialization and relaxation of purifying selection. Our findings demonstrate the contribution of gene duplication to organismal adaptation and show the power of combining sequence analysis and functional assays in delineating the molecular basis of adaptive evolution.

Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2–MyD88 signal pathway in dendritic cells and enhances Th2 immune responses

Eosinophil-derived neurotoxin (EDN) is an eosinophil granule–derived secretory protein with ribonuclease and antiviral activity. We have previously shown that EDN can induce the migration and maturation of dendritic cells (DCs). Here, we report that EDN can activate myeloid DCs by triggering the Toll-like receptor (TLR)2–myeloid differentiation factor 88 signaling pathway, thus establishing EDN as an endogenous ligand of TLR2. EDN activates TLR2 independently of TLR1 or TLR6. When mice were immunized with ovalbumin (OVA) together with EDN or with EDN-treated OVA-loaded DCs, EDN enhanced OVA-specific T helper (Th)2-biased immune responses as indicated by predominant production of OVA-specific interleukin (IL)-5, IL-6, IL-10, and IL-13, as well as higher levels of immunoglobulin (Ig)G1 than IgG2a. Based on its ability to serve as a chemoattractant and activator of DCs, as well as the capacity to enhance antigen-specific immune responses, we consider EDN to have the properties of an endogenous alarmin that alerts the adaptive immune system for preferential enhancement of antigen-specific Th2 immune responses.

Eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens

Eosinophils remain among the most enigmatic of cells, as our appreciation of their detrimental activities—e.g., asthma and allergic disease—far outweighs our understanding of their beneficial effects. Among the major secretory effector proteins of eosinophils are the ribonucleases eosinophil‐derived neurotoxin (EDN) and eosinophil cationic protein (ECP) in primates and their orthologs, the eosinophil‐associated ribonucleases (EARs) in rodents. The rapid diversification observed among these ribonucleases suggested that the ultimate target(s) might be similarly efficient at generating sequence diversity while maintaining an unalterable susceptibility to ribonucleolytic cleavage. This has prompted us to consider a role for these proteins and by extension, for eosinophils, in host defense against single‐stranded RNA virus pathogens. We detail our studies of the antiviral activity of eosinophils and eosinophil ribonucleases against respiratory syncytial virus (RSV) in vitro and the related, natural rodent pathogen, pneumonia virus of mice (PVM), in vivo, and consider the possibility that antiviral host defense and the dysregulated responses leading to asthma represent opposing sides of an eosinophil‐mediated double‐edged sword.

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.

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 α-chain, and transcripts encoding mouse eosinophil peroxidase, CCR3, the IL-3/IL-5/GM-CSF receptor common β-chain, and the transcription factor GATA-1. BmEos are functionally competent: they undergo chemotaxis toward mouse eotaxin-1 and produce characteristic cytokines, including IFN-γ, IL-4, MIP-1α, 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.

The Chemokine Macrophage-Inflammatory Protein-1α and Its Receptor CCR1 Control Pulmonary Inflammation and Antiviral Host Defense in Paramyxovirus Infection

In this work, we explore the responses of specific gene-deleted mice to infection with the paramyxovirus pneumonia virus of mice (PVM). We have shown previously that infection of wild type mice with PVM results in pulmonary neutrophilia and eosinophilia accompanied by local production of macrophage-inflammatory protein-1α (MIP-1α). Here we examine the role of MIP-1α in the pathogenesis of this disease using mice deficient in MIP-1α or its receptor, CCR1. The inflammatory response to PVM in MIP-1α-deficient mice was minimal, with ∼10–60 neutrophils/ml and no eosinophils detected in bronchoalveolar lavage fluid. Higher levels of infectious virus were recovered from lung tissue excised from MIP-1α-deficient than from fully competent mice, suggesting that the inflammatory response limits the rate of virus replication in vivo. PVM infection of CCR1-deficient mice was also associated with attenuated inflammation, with enhanced recovery of infectious virus, and with accelerated mortality. These results suggest that the MIP-1α/CCR1-mediated acute inflammatory response protects mice by delaying the lethal sequelae of infection.

Animal Pneumoviruses: Molecular Genetics and Pathogenesis

SUMMARY Pneumoviruses are single-stranded, negative-sense, nonsegmented RNA viruses of the family Paramyxoviridae, subfamily Pneumovirinae, and include pathogens that infect humans (respiratory syncytial virus and human metapneumovirus), domestic mammals (bovine, ovine, and caprine respiratory syncytial viruses), rodents (pneumonia virus of mice), and birds (avian metapneumovirus). Among the topics considered in this review are recent studies focused on the roles of the individual virus-encoded components in promoting virus replication as well as in altering and evading innate antiviral host defenses. Advances in the molecular technology of pneumoviruses and the emergence of recombinant pneumoviruses that are leading to improved virus-based vaccine formulations are also discussed. Since pneumovirus infection in natural hosts is associated with a profound inflammatory response that persists despite adequate antiviral therapy, we also review the recent experimental treatment strategies that have focused on combined antiviral, anti-inflammatory, and immunomodulatory approaches.

Interleukin-5 (IL-5) augments the progression of liver fibrosis by regulating IL-13 activity.

ABSTRACT Eosinophils are frequently found in increased numbers in a variety of chronic fibrotic diseases; however, their role in the development of hepatic fibrosis has not been dissected in vivo. Here, we used interleukin-5 (IL-5) knockout (KO) mice to determine whether eosinophils contribute to the progressive liver fibrosis that develops in response to chronic Schistosoma mansoni infection. Although infection intensities were similar in C57BL/6 and IL-5 KO mice, the average size of granulomas was significantly smaller in both acutely and chronically infected IL-5 KO mice. Their granulomas were also completely devoid of eosinophils. In addition, the knockout mice displayed over a 40% reduction in hepatic fibrosis by week 16 postinfection. The reduced fibrosis was associated with increased production of the antifibrotic cytokine gamma interferon. Moreover, although IL-13 production did not decrease consistently in the absence of IL-5, IL-13-triggered responses were substantially reduced in the granulomatous tissues. This was confirmed by analyzing the expression of several genes associated with alternative macrophage activation, including arginase 1, Fizz-1, and YM-1. Importantly, all of these IL-13-regulated genes have been linked with the mechanisms of wound healing and fibrosis. In addition to IL-5 polarizing the antigen-specific CD4+ Th2 cell response, we found that granuloma eosinophils were themselves a significant source of IL-13. Thus, by producing profibrotic mediators and polarizing the Th2 response, these findings illustrate both direct and indirect roles for eosinophils and IL-5 in the pathogenesis of schistosomiasis-induced liver fibrosis. Thus, inhibiting the activity of IL-5 or eosinophils may prove effective for a variety of chronic fibrotic diseases.