Interleukin (IL)-5 and IL-13 are thought to play key roles in the pathogenesis of asthma. Although both cytokines use eotaxin to regulate eosinophilia, IL-13 is thought to operate a separate pathway to IL-5 to induce airways hyperreactivity (AHR) in the allergic lung. However, identification of the key pathway(s) used by IL-5 and IL-13 in the disease process is confounded by the failure of anti–IL-5 or anti–IL-13 treatments to completely inhibit the accumulation of eosinophils in lung tissue. By using mice deficient in both IL-5 and eotaxin (IL-5/eotaxin−/−) we have abolished tissue eosinophilia and the induction of AHR in the allergic lung. Notably, in mice deficient in IL-5/eotaxin the ability of CD4+ T helper cell (Th)2 lymphocytes to produce IL-13, a critical regulator of airways smooth muscle constriction and obstruction, was significantly impaired. Moreover, the transfer of eosinophils to IL-5/eotaxin−/− mice overcame the intrinsic defect in T cell IL-13 production. Thus, factors produced by eosinophils may either directly or indirectly modulate the production of IL-13 during Th2 cell development. Our data show that IL-5 and eotaxin intrinsically modulate IL-13 production from Th2 cells and that these signaling systems are not necessarily independent effector pathways and may also be integrated to regulate aspects of allergic disease.

The concept that the immune system can recognise tumour cells and either eliminate them (tumour immune surveillance) or select for immunologically resistant variants (immunoediting) is gaining general acceptance by immunologists. In terms of an adaptive immune response to cancer, however, much of the research has focused on the response of cytotoxic CD81 T lymphocytes to tumour-specific antigens and the production of Th1 cytokines by CD4 1 and CD8 1 T cells. In contrast, Th2-mediated immunity has traditionally been viewed as favouring tumour growth, both by promoting angiogenesis and by inhibiting cell-mediated immunity and subsequent tumour cell killing. While there is evidence that components of type 2 inflammation, such as B cells and interleukin-10, do promote tumour growth, there are also many studies demonstrating the anti-tumour activity of CD4 1 Th2 cells, particularly in collaboration with tumour-infiltrating granulocytes, such as eosinophils. In this review, we examine all the components of type 2 immunity and their effects on tumour growth. Collectively, from this analysis, we conclude that there is a great potential for the development of Th2-mediated immunotherapies that harness the cytotoxic activity of eosinophils.

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11−/− and ΔdblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11−/− BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/CCL11−/− and ΔdblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance.

Eosinophil degranulation is thought to play a pivotal role in the pathogenesis of allergic disorders. Although mouse models of allergic disorders have been used extensively to identify the contribution of eosinophils to disease, ultrastructural evidence of active granule disassembly has not been reported. In this investigation, we characterized the degree of eosinophil activation in the bone marrow, blood, lung tissue, and airways lumen [bronchoalveolar lavage fluid (BALF)] of ovalbumin-sensitized and aero-challenged wild-type and interleukin-5 transgenic mice. Degranulation was most prominent in and primarily compartmentalized to the airways lumen. Eosinophils released granule proteins by the process of piecemeal degranulation (PMD). Accordingly, recruitment and activation of eosinophils in the lung correlated with the detection of cell-free eosinophil peroxidase in BALF and with the induction of airways hyper-reactivity. As in previous studies with human eosinophils, degranulation of isolated mouse cells did not occur until after adherence to extracellular matrix. However, higher concentrations of exogenous stimuli appear to be required to trigger adherence and degranulation (piecemeal) of mouse eosinophils when compared with values reported for studies of human eosinophils. Thus, mouse eosinophils undergo PMD during allergic inflammation, and in turn, this process may contribute to pathogenesis. However, the degranulation process in the allergic lung of mice is primarily compartmentalized to the airway lumen. Understanding the mechanism of eosinophil degranulation in the airway lumen may provide important insights into how this process occurs in human respiratory diseases.

Summary Eosinophils play a central role in the pathophysiology of allergic disease. The mechanisms that regulate eosinophil migration are complex; however, chemokines and cytokines produced in both the early and late phases of the asthmatic response appear to cooperate in eosinophil recruitment. In particular, there exists a unique synergy between eotaxin and IL-5. The role of chemokine/cytokine cooperativity has been investigated in the extracellular matrix, adhesion molecule/integrin interactions, receptor polarization and aggregation and the convergence and divergence of intracellular signalling pathways. Understanding the mechanisms whereby eosinophils migrate will allow the development of specific therapeutic strategies aimed at attenuating specific components of the allergic response.

Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin-and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin-and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCEThis study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity. R espiratory syncytial virus (RSV) is a serious lower respiratory tract infection in infants, the elderly, and the immunocompromised (1-4), resulting in Ͼ130,000 hospitalizations in the United States each year (5, 6). Children who experience acute RSV infection of the lower respiratory tract have an increased likelihood of developing childhood asthma (7). To date, there is no safe and effective RSV vaccine available. This is in part linked to the negative outcome of a 1960s clinical trial using a formalin-inactivated alum-precipitated RSV (FI-RSV) vaccine (8). Children immunized with the FI-RSV vaccine experienced more severe disease following subsequent natural exposure to RSV, with one study showing 69% of immunized children developing pneumonia compared to only 9% of children in the unimmunized control group (9). In a second study, 80% of FI-RSV-immunized infants were hospitalized and two died, compared with only 5% hospitalization and no death in the control group (10). FI-RSV vaccineenhanced illness was clinically characterized as severe primary RSV infection, with bronchiolitis, hypoxemia, and pneumonia. However, in contrast to the neutrophilic inflammation observed during primary RSV infection, the more severe a...

GTP binding proteins, heterotrimeric molecules composed of alpha-, beta-, and gamma-subunits, are known to serve as transducers of information from seven-transmembrane receptors. Activation of G-proteins has been generally considered to involve subunit dissociation, with G(alpha) separating from G(betagamma). However, we have found a receptor activation of G(i) in proliferating cells that differs from these models and involves the subcellular translocation of the alpha-subunit from the cell periphery to the nucleus where G(i alpha) binds to chromatin for the duration of mitosis. This report describes the mechanism of G(i) activation in Swiss 3T3 cells in response to serum, thrombin, and epidermal growth factor, and describes a role for G(i2) in the cell cycle. Agonists were found to be unable to induce the physical dissociation of G(i2) subunits. The alpha- and beta-subunits of G(i2) could be coimmunoprecipitated with a G(i alpha) antibody from both the membrane and nuclear fractions of long-term activated cultures, showing that G(i alpha 2) and G(i beta) are induced to comigrate to the nucleus in response to growth factor receptor activation. G(i2) appears to be activated in part by a postreceptor signal that can be mimicked by protein kinase C activation; this signal may be responsible for the convergence of the signaling mechanisms of these distinct seven-transmembrane and tyrosine kinase receptors. We suggest that translocation of G(i alpha) to the nucleus induced by either thrombin or EGF may occur without subunit dissociation. Functional studies of the role of G(i) showed that pertussis toxin does not block DNA synthesis in Swiss 3T3 fibroblasts induced by serum or thrombin, but that cell proliferation is retarded to each. These results provide direct evidence for a novel mechanism of GTP binding protein activation and for an essential role of G(i) in the induction of cell division by a variety of growth factor receptors. G(i) can carry out this role in control of cellular proliferation through its translocation to the nucleus of mitotic cells.

An important feature of chemokines is their ability to bind to the glycosaminoglycan (GAG) side chains of proteoglycans, predominately heparin and heparan sulfate. To date, all chemokines tested bind to immobilized heparin in vitro, as well as cell surface heparan sulfate in vitro and in vivo. These interactions play an important role in modulating the action of chemokines by facilitating the formation of stable chemokine gradients within the vascular endothelium and directing leukocyte migration, by protecting chemokines from proteolysis, by inducing chemokine oligomerization, and by facilitating transcytosis. Despite the importance of eotaxin in eosinophil differentiation and recruitment being well established, little is known about the interaction between eotaxin and GAGs and the functional consequences of such an interaction. Here we report that eotaxin binds selectively to immobilized heparin with high affinity (K d ‫؍‬ 1.23 ؋ 10 ؊8 M), but not to heparan sulfate or a range of other GAGs. The interaction of eotaxin with heparin does not promote eotaxin oligomerization but protects eotaxin from proteolysis directly by plasmin and indirectly by cathepsin G and elastase. In vivo, co-administration of eotaxin and heparin is able to significantly enhance eotaxin-mediated eosinophil recruitment in a mouse air-pouch model. Furthermore, when heparin is coadministered with eotaxin at a concentration that does not normally result in eosinophil infiltration, eosinophil recruitment occurs. In contrast, heparin does not enhance eotaxin-mediated eosinophil chemotaxis in vitro, suggesting protease protection or haptotactic gradient formation as the mechanism by which heparin enhances eotaxin action in vivo. These results suggest a role for mast cell-derived heparin in the recruitment of eosinophils, reinforcing Th2 polarization of inflammatory responses.Chemokines are a superfamily of proteins that control the migration of leukocytes to sites of inflammation during an immune response. A feature of chemokines is their ability to bind to the glycosaminoglycan (GAG) 3 side chains of proteoglycans, predominately heparin and heparan sulfate. To date, all chemokines tested bind to immobilized heparin in vitro, as well as cell surface heparan sulfate in vitro and in vivo (1). In vitro, chemokine-heparin or heparan sulfate binding may be competitively inhibited by the GAGs chondroitin sulfate and dermatan sulfate (1, 2), suggesting that some chemokines may also bind these GAGs. Different chemokines bind heparin or heparan sulfate with varying affinity (1-3), and four different models, or docking modes, have been proposed for the interaction of chemokines with heparin and heparan sulfate (4). Heparin and heparan sulfate play an important role in modulating the function of chemokines (5) and have been proposed to control chemokine action in three ways. First, it is well established that heparan sulfate immobilizes chemokines on the luminal surface of endothelial cells (6, 7), which is essential for the formation of stable chemokine...