Depletion of eosinophils by anti-IL-5 monoclonal antibody treatment of mice infected with Trichinella spiralis does not alter parasite burden or immunologic resistance to reinfection.

Mechanisms of parasite killing by eosinophils are widely studied and are often implicated in mediating resistance to parasitic infection, especially in conjunction with specific antibodies. Evidence for the eosinophil as an anti-parasite killer cell in vivo is limited and may not justify the belief that eosinophils engage and/or kill infective helminths. We reexamined this question in a mouse model of trichinosis in which antisera to eosinophils were previously used to show the requirement for eosinophils in resistance to this nematode. The current studies used mAb to IL-5 to suppress eosinophil levels in CF1 mice infected with Trichinella spiralis. In mice given a primary infection and injected with an isotype control mAb or left untreated, the medullary and peripheral blood eosinophil numbers peaked at 3 wk postinfection (PI) and returned to baseline levels by 4 wk PI. Peripheral blood eosinophil numbers in infected mice injected with anti-IL-5 were maintained at levels below those of uninfected normal mice through 4 wk of infection. Histologically, there was a prominent eosinophil accumulation in infected, untreated, or control-mAb-treated mice associated with nurse cell complexes containing infective juveniles in skeletal muscle at 3 and 4 wk PI. This was largely eliminated in mice treated with anti-IL-5 mAb. However, the number of muscle stage juvenile worms recovered 3 and 4 wk PI after acid pepsin digestion was unaffected by eosinophil depletion. Challenge infections, in which mice were infected at day 0 with 125 muscle stage worms and challenged at day 28 PI with 350 muscle stage worms, developed peak eosinophil numbers in bone marrow and peripheral blood 3 wk after primary infection and 2 wk after challenge infection in mice receiving either no treatment or control mAb. In challenged mice receiving anti-IL-5 mAb, medullary and peripheral blood eosinophil numbers remained at or below those of uninfected animals. Although all groups exhibited significant resistance measured as muscle stage worm burdens 56 days PI, eosinophil depletion did not affect resistance of muscle worm recovery. These results suggest that eosinophils are not essential in the control of T. spiralis in either primary or challenge infections of CF1 mice. This in vivo study illustrates the questionable value of in vitro killing assays to assign effector function to any single inflammatory cell type.     

Bronchopulmonary hyperreactivity and lung eosinophil sequestration but not their migration to the alveolar compartment are independent of interleukin-5 in allergic mice

IL-5 is present in the lung and in the circulation following allergenic challenges in humans and in animals, but its role in bronchopulmonary hyperreactivity (BHR) and lung and bronchoalveolar lavage fluid (BALF) eosinophilia remains unclear. Because compartmentalization of IL-5 is recognized, the anti-IL-5 monoclonal antibody TRFK-5 or its isotype control GL113 were delivered selectively intranasally (i.n.) and/or intravenously (i.v.) before the prior i.n. challenge with 10 mug OVA in BALB/c and BP2 "Biozzi" mice immunized according to optimized protocols with read-outs taken 24 h later. IL-5 in the BALF was suppressed by i.n. TRFK-5, whereas its production persisted in the serum. Conversely, i.v. TRFK-5 suppressed IL-5 in the serum but not in the BALF. IL-5 was suppressed in conditioned medium from lung explants from mice treated with i.n. TRFK-5, which did not affect the other Th2 cytokines, IL-4 and IL-13. IL-5 is thus present in the alveolar, pulmonary and circulatory compartments following an i.n. allergenic challenge. When specific anti-IL-5 antibodies were delivered by the same i.n. route, BALF eosinophilia was markedly reduced, whereas BHR and lung eosinophil sequestration persisted totally or mostly, in both strains. The passage of eosinophils from lungs to alveoli depends on IL-5 released into the BALF, but not into circulation, whereas their lung sequestration and BHR are mostly IL-5-independent. IL-5 alone does not account for the complexities of BHR or of eosinophil tissue trapping, and lung-targeted immunobiologicals should be delivered into the appropriate compartment in order to assess the role of specific mediators in experimental airways/lung allergy.     

Eosinophilopoiesis in a murine model of allergic airway eosinophilia: involvement of bone marrow IL-5 and IL-5 receptor alpha

The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVA-sensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2'-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3+ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2'-deoxyuridine positive. After three allergen exposures, both CD3+ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R alpha-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment.     

The effect of IL-5 and eotaxin expression in the lung on eosinophil trafficking and degranulation and the induction of bronchial hyperreactivity

The mechanisms regulating the selective migration and degranulation of eosinophils in the asthmatic lung and the subsequent development of airways hyperreactivity (AHR) have not been fully delineated. In this investigation, we have employed a novel transgene model to facilitate the dissection of the contributions of IL-5 and/or eotaxin to eosinophil function in the absence of complex tissue signals derived from the allergic lung. Gene transfer of IL-5 and/or eotaxin to the lungs of naive mice induced a pronounced and selective airways eosinophilia, but did not result in eosinophil degranulation or AHR. Airways eosinophilia occurred independently of the induction of a blood eosinophilia, but was markedly augmented by the coexpression of both cytokines and/or by the transient mobilization of eosinophils from the bone marrow by the administration of i.v. IL-5. However, for eosinophil degranulation and AHR to occur, the inhalation of Ag was required in association with IL-5 and eotaxin expression. Investigations in IL-5-deficient mice linked eosinophilia, and not solely IL-5 and eotaxin, with the induction of AHR. Furthermore, eosinophil degranulation and AHR were dependent on CD4+ T cells. Importantly, this investigation shows that IL-5 regulates eosinophilia within the lung as well as in the circulation and also amplifies eotaxin-induced chemotaxis in the airway compartment. Moreover, the interplay between these cytokines, CD4+ T cells, and factors generated by Ag inhalation provides fundamental signals for eosinophil degranulation and the induction of AHR.     

CD8 T cells are essential in the development of respiratory syncytial virus-induced lung eosinophilia and airway hyperresponsiveness

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways. The immune cell requirements for these responses to RSV infection are not well defined. To delineate the role of CD8 T cells in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice depleted of CD8 T cells to develop these symptoms of RSV infection. BALB/c mice were depleted of CD8 T cells using anti-CD8 Ab treatment before intranasal administration of infectious RSV. Six days postinfection, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and levels of IFN-gamma, IL-4, and IL-5 in bronchoalveolar lavage fluid were monitored. RSV infection resulted in airway eosinophilia and AHR in control mice, but not in CD8-depleted animals. Further, whereas RSV-infected mice secreted increased amounts of IL-5 into the airways as compared with noninfected controls, no IL-5 was detectable in both bronchoalveolar lavage fluid and culture supernatants from CD8-depleted animals. Treatment of CD8-depleted mice with IL-5 fully restored both lung eosinophilia and AHR. We conclude that CD8 T cells are essential for the influx of eosinophils into the lung and the development of AHR in response to RSV infection.     

IL-5 and eosinophils are essential for the development of airway hyperresponsiveness following acute respiratory syncytial virus infection

Viral respiratory infections can cause bronchial hyperresponsiveness and exacerbate asthma. In mice, respiratory syncytial virus (RSV) infection, which induces an immune response dominated by IFN-gamma, results in airway hyperresponsiveness (AHR) and eosinophil influx into the airways, both of which are prevented by pretreatment with anti-IL-5 Ab. To delineate the role of IL-5, IL-4, and IFN-gamma in the development of RSV-induced AHR and lung eosinophilia, we tested the ability of mice deficient in each of these cytokines to develop these symptoms of RSV infection. Mice deficient in either IL-5, IL-4, or IFN-gamma were administered infectious RSV intranasally, and 6 days later, airway responsiveness to inhaled methacholine was assessed by barometric body plethysmography, and numbers of lung eosinophils and production of IFN-gamma, IL-4, and IL-5 by mononuclear cells from peribronchial lymph nodes were monitored. RSV infection resulted in airway eosinophilia and AHR in both IL-4- and IFN-gamma-deficient mice, but not in IL-5-deficient mice. Reconstitution of IL-5-deficient mice with IL-5 restored these responses and enhanced the responses in IL-4-deficient mice. Anti-VLA-4 (very late Ag-4) treatment prevented lung eosinophilia and AHR following RSV infection and IL-5 reconstitution. We conclude that in response to RSV, IL-5 is essential for the influx of eosinophils into the lung and that eosinophils in turn are critical for the development of AHR. IFN-gamma and IL-4 are not essential for these responses to RSV infection.     

Analysis of eosinophil turnover in vivo reveals their active recruitment to and prolonged survival in the peritoneal cavity

Eosinophils are potent effector cells associated with allergic inflammation and parasite infections. However, limited information exists about their turnover, migration, and survival in vivo. To address these important questions, we determined murine eosinophil turnover under steady state and inflammatory conditions by flow cytometric analysis of BrdU incorporation and analyzed their migration pattern and survival in different tissues after adoptive transfer into recipient mice. In naive mice approximately 50% of bone marrow eosinophils were labeled with BrdU during a 15-h pulse, whereas only 10% of splenic eosinophils were labeled within this time frame. Unexpectedly, the rate of eosinophil production did not change during acute infection with the helminth parasite Nippostrongylus brasiliensis despite massive eosinophilia in several tissues. Eosinophils present in lung and peritoneum remained largely BrdU negative, indicating that eosinophilia in end organs was mainly caused by increased survival of already existing eosinophils rather than increased production of new eosinophils in the bone marrow. Adoptive transfer experiments revealed that eosinophils preferentially migrated to the peritoneum in a macrophage-independent and pertussis toxin-sensitive manner, where they survived for several days. Peritoneal eosinophils expressed high levels of the inhibitory receptor Siglec-F, released less eosinophil peroxidase compared with eosinophils from the spleen, and could recirculate to other organs. These results demonstrate that the peritoneum serves as reservoir for eosinophils.     

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2'-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Airway allergen exposure stimulates bone marrow eosinophilia partly via IL-9

Background

Interleukin (IL)-9 is a Th2-derived cytokine with pleiotropic biological effects, which recently has been proposed as a candidate gene for asthma and allergy. We aimed to evaluate the therapeutic effect of a neutralizing anti-IL-9 antibody in a mouse model of airway eosinophilic inflammation and compared any such effect with anti-IL-5 treatment.

Methods

OVA-sensitized Balb/c mice were intraperitoneally pretreated with a single dose (100 μg) of an anti-mouse IL-9 monoclonal antibody (clone D9302C12) or its vehicle. A third group was given 50 μg of a monoclonal anti-mouse IL-5 antibody (TRFK-5) or its vehicle. Animals were subsequently exposed to OVA on five days via airways. Newly produced eosinophils were labelled using 5-bromo-2''-deoxyuridine (BrdU). BrdU⁺ eosinophils and CD34⁺ cell numbers were examined by immunocytochemistry. After culture and stimulation with OVA or PMA+IC, intracellular staining of IL-9 in bone marrow cells from OVA-exposed animals was measured by Flow Cytometry. The Mann-Whitney U-test was used to determine significant differences between groups.

Results

Anti-IL-9 significantly reduced bone marrow eosinophilia, primarily by decrease of newly produced (BrdU⁺) and mature eosinophils. Anti-IL-9 treatment also reduced blood neutrophil counts, but did not affect BAL neutrophils. Anti-IL-5 was able to reduce eosinophil numbers in all tissue compartments, as well as BrdU⁺ eosinophils and CD34⁺ progenitor cells, and in all instances to a greater extent than anti-IL-9. Also, FACS analysis showed that IL-9 is over-expressed in bone marrow CD4⁺ cells after allergen exposure.

Conclusions

Our data shows that a single dose of a neutralizing IL-9 antibody is not sufficient to reduce allergen-induced influx of newly produced cells from bone marrow to airways. However, in response to allergen, bone marrow cells over-express IL-9. This data suggest that IL-9 may participate in the regulation of granulocytopoiesis in allergic inflammation.     

Pathogenesis of murine experimental allergic rhinitis: a study of local and systemic consequences of IL-5 deficiency

Recent studies have demonstrated an important role for IL-5-dependent bone marrow eosinophil progenitors in allergic inflammation. However, studies using anti-IL-5 mAbs in human asthmatics have failed to suppress lower airway hyperresponsiveness despite suppression of eosinophilia; therefore, it is critical to examine the role of IL-5 and bone marrow responses in the pathogenesis of allergic airway disease. To do this, we studied the effects of IL-5 deficiency (IL-5(-/-)) on bone marrow function as well as clinical and local events, using an established experimental murine model of allergic rhinitis. Age-matched IL-5(+/+) and IL-5(-/-) BALB/c mice were sensitized to OVA followed by 2 wk of daily OVA intranasal challenge. IL-5(-/-) OVA-sensitized mice had significantly higher nasal mucosal CD4(+) cells and basophilic cell counts as well as nasal symptoms and histamine hyperresponsiveness than the nonsensitized group; however, there was no eosinophilia in either nasal mucosa or bone marrow; significantly lower numbers of eosinophil/basophil CFU and maturing CFU eosinophils in the presence of recombinant mouse IL-5 in vitro; and significantly lower expression of IL-5Ralpha on bone marrow CD34(+)CD45(+) progenitor cells in IL-5(-/-) mice. These findings suggest that IL-5 is required for normal bone marrow eosinophilopoiesis, in response to specific Ag sensitization, during the development of experimental allergic rhinitis. However, the results also suggest that suppression of the IL-5-eosinophil pathway in this model of allergic rhinitis may not completely suppress clinical symptoms or nasal histamine hyperresponsiveness, because of the existence of other cytokine-progenitor pathways that may induce and maintain the presence of other inflammatory cell populations.     

IL-13 is required for eosinophil entry into the lung during respiratory syncytial virus vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.     

Purinergic P2Y12 Receptor Activation in Eosinophils and the Schistosomal Host Response

Identifying new target molecules through which eosinophils secrete their stored proteins may reveal new therapeutic approaches for the control of eosinophilic disorders such as host immune responses to parasites. We have recently reported the expression of the purinergic P2Y12 receptor (P2Y12R) in human eosinophils; however, its functional role in this cell type and its involvement in eosinophilic inflammation remain unknown. Here, we investigated functional roles of P2Y12R in isolated human eosinophils and in a murine model of eosinophilic inflammation induced by Schistosoma mansoni (S. mansoni) infection. We found that adenosine 5’-diphosphate (ADP) induced human eosinophils to secrete eosinophil peroxidase (EPO) in a P2Y12R dependent manner. However, ADP did not interfere with human eosinophil apoptosis or chemotaxis in vitro. In vivo, C57Bl/6 mice were infected with cercariae of the Belo Horizonte strain of S. mansoni. Analyses performed 55 days post infection revealed that P2Y12R blockade reduced the granulomatous hepatic area and the eosinophilic infiltrate, collagen deposition and IL-13/IL-4 production in the liver without affecting the parasite oviposition. As found for humans, murine eosinophils also express the P2Y12R. P2Y12R inhibition increased blood eosinophilia, whereas it decreased the bone marrow eosinophil count. Our results suggest that P2Y12R has an important role in eosinophil EPO secretion and in establishing the inflammatory response in the course of a S. mansoni infection.     

Pathological and haematological responses of cats experimentally infected with Toxocara canis larvae

Responses of eight adult cats to one or two infections with larvae of Toxocara canis were studied up to 39 days post infection (DPI). Clinically, all cats remained normal throughout the study. The major necropsy finding was multifocal, white to grey nodules mainly within the liver, lungs and kidneys; live larvae were found in liver nodules. Histologically, the nodules were eosinophilic granulomas. Granulomas containing a larval section were observed mainly within the liver. All infected cats had variably severe, eosinophilic arteritis and bronchiolitis and medial hypertrophy and hyperplasia of the pulmonary arteries. No inflammatory eye lesions were detected. Circulating eosinophil levels increased in all infected cats; peak values of 15,790 and 10,050 eosinophils microliters-1 were observed at 25 or 32 DPI in cats receiving a single or double infection, respectively. Bone marrow of all infected cats exhibited marked eosinophilic hyperplasia which did not correlate with the level of circulating eosinophilia. Thus, infection of cats by the larvae of T. canis causes disseminated eosinophilic and granulomatous disease with marked pulmonary artery and airway lesions.     

Regulation of eosinophilopoiesis in a murine model of asthma

Eosinophilic inflammation plays a key role in tissue damage that characterizes asthma. Eosinophils are produced in bone marrow and recent observations in both mice and humans suggest that allergen exposure results in increased output of eosinophils from hemopoietic tissue in individuals with asthma. However, specific mechanisms that alter eosinophilopoiesis in this disease are poorly understood. The current study used a well-characterized murine animal model of asthma to evaluate alterations of eosinophil and eosinophil progenitor cells (CFU-eo) in mice during initial sensitization to allergen and to determine whether observed changes in either cell population were regulated by T lymphocytes. Following the first intranasal installation of OVA, we observed sequential temporal elevation of eosinophils in bone marrow, blood, and lung. In immunocompetent BALB/c mice, elevation of bone marrow eosinophils was accompanied by transient depletion of CFU-eo in that tissue. CFU-eo rebounded to elevated numbers before returning to normal baseline values following intranasal OVA exposure. In T cell-deficient BALB/c nude (BALB/c(nu/nu)) mice, CFU-eo were markedly elevated following allergen sensitization, in the absence of bone marrow or peripheral blood eosinophilia. These data suggest that eosinophilia of asthma results from alterations in two distinct hemopoietic regulatory mechanisms. Elevation of eosinophil progenitor cells in the bone marrow is T cell independent and likely results from altered bone marrow stromal cell function. Differentiation of eosinophil progenitor cells and phenotypic eosinophilia is T cell dependent and does not occur in athymic nude mice exposed to intranasal allergen.     

Interleukin-5 modulates interleukin-8 secretion in eosinophilic inflammation.

Serum and BALF (bronchoalveolar lavage fluid) IL-8 levels and serum levels were investigated in Toxocara canis infected guinea-pigs and the role of IL-5 as a modulator of cytokine secretion was studied. Serum levels increased early in infected animals, exceeding control levels 4 h after infection, peaked between days 6 and 18, and continued to exceed control levels after 48 days of infection. Serum and BALF IL-8 levels showed the same profile as blood eosinophilia, increasing 6 days post-infection and peaking between days 18 and 24. Treatment of infected animals with anti-IL-5 Ab suppressed eosinophilia with a parallel increase in blood IL-8 levels, whereas no change was found in levels. To support our in vivo observation we carried out experiments in vitro using guinea-pig LPS-stimulated adherent peritoneal cells which release large amounts of IL-8 into the supernatants. When rIL-5 was added to LPS-stimulated cells, 65% inhibition of IL-8 release into the supernatants was observed. Pre-incubation of cells with anti-IL-5 Ab prevented the inhibition of IL-8 release into the supernatants induced by rIL-5. Our results demonstrate for the first time that TNF-alpha and IL-8 are released concomitant with or after IL-5 in the eosinophilic inflammation induced by T. canis. Moreover, in addition to showing that IL-5 is fundamental for the induction of blood eosinophilia, the present results suggest that this cytokine may play a new biological role by acting as modulator of IL-8 secretion.     

Eosinophil differentiation in response to Fasciola hepatica and its excretory/secretory antigens

Bone marrow cells from mice infected with Fasciola hepatica, from mice injected with F. hepatica excretory/secretory (ES) antigens, and from uninfected or uninjected control animals were cultured in the presence of F. hepatica ES antigens or the eosinophil differentiation cytokine IL-5. Eosinophil maturation in cultures was assessed quantitatively by measuring eosinophil peroxidase (EPO) activity and qualitatively by visual appraisal in stained preparations over a week. It was found that the presence in all cultures (including those from control animals) of either ES antigens at an optimal concentration of 100 μml⁻¹ (established in preliminary trials) or IL-5 at 500 units ml⁻¹ led to enhanced EPO activity. EPO activity in cultures without IL-5 or ES antigens remained static or fell over the culture period. At day 3 in all cultures containing IL-5 or ES antigens, there was maintenance of, or only a slight decline in, the number of eosinophils that were present when cultures were initiated, and more of them were mature than at day 0 as evidenced by their EPO activity. However, there was a marked fall in eosinophil numbers in all cultures in the absence of IL-5 or ES antigens. The results indicate that F. hepatica ES antigens, like IL-5, stimulate eosinophil maturation in bone marrow with a consequent rise in EPO activity in the cells. Whether the antigen(s) acts directly or indirectly on the eosinophils or their precursors has yet to be established. Nevertheless, it seems clear that F. hepatica produces a molecule with a functionally similar effect to that of IL-5.     

Generation of rat eosinophils by recombinant rat interleukin-5 in vitro and in vivo

The addition of recombinant rat interleukin-5 (IL-5), which was purified from the hemolymph of silkworm Bombyx mori larvae infected with IL-5-expressing recombinant virus, to cultures of rat bone marrow cells resulted in an increase in the number of Luxol-fast-blue staining eosinophils in a time- and concentration-dependent manner. After 6 days culture with 100 pM recombinant rat IL-5, more than 90% of the bone marrow cells were eosinophil. The contents of major basic protein (MBP) in the bone marrow cells determined by Western blot analysis using a polyclonal antibody to rat MBP were also increased by recombinant rat IL-5 (100 pM). Furthermore, intravenous injections of recombinant rat IL-5 twice a day for six consecutive days increased the population of eosinophils in peripheral blood cells and in bone marrow cells. These findings indicate that rat IL-5 induces terminal differentiation and proliferation of progenitor cells to mature eosinophils in rats.     

Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with loss of IL-5 responsiveness,but up-regulate IL-3 receptor alpha expression

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5R alpha, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3R alpha was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor alpha was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3R alpha by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5R alpha down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.     

A causative relationship exists between eosinophils and the development of allergic pulmonary pathologies in the mouse

Asthma and mouse models of allergic respiratory inflammation are invariably associated with a pulmonary eosinophilia; however, this association has remained correlative. In this report, a causative relationship between eosinophils and allergen-provoked pathologies was established using eosinophil adoptive transfer. Eosinophils were transferred directly into the lungs of either naive or OVA-treated IL-5(-/-) mice. This strategy resulted in a pulmonary eosinophilia equivalent to that observed in OVA-treated wild-type animals. A concomitant consequence of this eosinophil transfer was an increase in Th2 bronchoalveolar lavage cytokine levels and the restoration of intracellular epithelial mucus in OVA-treated IL-5(-/-) mice equivalent to OVA-treated wild-type levels. Moreover, the transfer also resulted in the development of airway hyperresponsiveness. These pulmonary changes did not occur when eosinophils were transferred into naive IL-5(-/-) mice, eliminating nonspecific consequences of the eosinophil transfer as a possible explanation. Significantly, administration of OVA-treated IL-5(-/-) mice with GK1.5 (anti-CD4) Abs abolished the increases in mucus accumulation and airway hyperresponsiveness following adoptive transfer of eosinophils. Thus, CD4(+) T cell-mediated inflammatory signals as well as signals derived from eosinophils are each necessary, yet alone insufficient, for the development of allergic pulmonary pathology. These data support an expanded view of T cell and eosinophil activities and suggest that eosinophil effector functions impinge directly on lung function.     

The effect of specific immunization or infection with Trichostrongylus colubriformis on production of eosinophil differentiation factors in guinea pigs.

The cultivation of bone marrow was used to quantitate the levels of eosinophil differentiation factors (EDF) produced in conditioned medium (CM) by incubation of mesenteric lymph node cells (MLNC) with mitogens or specific antigens from the intestinal nematode parasite, Trichostrongylus colubriformis. In liquid cultures with 20 units ml⁻¹ recombinant murine interleukin-5 (IL-5), bone marrow cells (BMC) from either normal or infected donors contained <5% eosinophils and differentiated to> 50% eosinophils over 2–3 weeks. Conditioned medium from 3–4 week infected donors produced between 20 and 50% eosinophils when donor MLNC were stimulated with the specific antigen preparation SP3, but macrophages predominated when using CM from MLNC incubated with Concanavalin A (ConA). CM from MLNC of challenged donors incubated with SP3 produced 30–70% eosinophils in BMC assays, with highest levels induced by CM from high responder (HR) donors. Marrow from parasitized or normal donors gave rise to comparable proportions of eosinophils. CM was also produced from LNC of donors given protein or parasite antigens in adjuvant where between 28 and 35% eosinophils were produced in culture. There were no differences between activities attributable to the antigen, but Freund's complete adjuvant induced earlier differentiation of BMC than alum-induced CM. The results confirm that high levels of EDF activity are specifically induced by parasitic infection, and can also be produced by intraperitoneal and subcutaneous inoculation of adjuvanted antigens. Consistent with the greater eosinophilia exhibited by HR guinea pigs to infection with T.colubriformis L3, their MLNC also produced the highest levels of EDF activity.