The effects of allergen and anti-allergic drugs on murine hemopoietic cells: moving targets, unusual mechanisms, and changing paradigms

We used a variety of techniques to evaluate the effects of airway allergen exposure in mice on the responses of hemopoietic cells to cytokines and drugs in vitro and in vivo. Initial studies have shown that allergen exposure of sensitized mice leads to release of circulating mediators, that induce rapid upregulation of bone-marrow responses to IL-5 and GM-CSF. This may be related to glucocorticoids, because exogenous dexamethasone has similar effects on cultured murine bone-marrow, and because stress-induced glucocorticoids, in na?ve or sensitized mice, have effects indistinguishable from those of allergen challenge in sensitized animals. Upregulation of eosinophil production is associated with an increased expression of alpha4 integrins, which may contribute to retention of these cells in the bone-marrow. Glucocorticoids regulate the adhesiveness, maturation and survival of eosinophils in murine bone-marrow culture, partly by counteracting the actions of Prostaglandin E2 and possibly other prostanoids. Allergen exposure of sensitized mice leads to accumulation of hemopoietic progenitors in the lungs, which differ from those in bone-marrow in growth properties and sensitivity to glucocorticoids. Lung transplantation has been used to demonstrate that the lung acts as a source of endocrine factors that promote hemopoietic cell accumulation, independently of damage caused by local allergic inflammation.     

Upregulation by glucocorticoids of responses to eosinopoietic cytokines in bone-marrow from normal and allergic mice

Since the production of eosinopoietic cytokines (GM-CSF, IL-3, IL-5) is inhibited by glucocorticoids, while responsiveness to these cytokines is enhanced in bone-marrow of allergic mice, we studied the ability of glucocorticoids to modulate murine bone-marrow eosinopoiesis. Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized and challenged mice or (c) dexamethasone (1-5 mg kg(-1)) injected mice. Cultures were established with GM-CSF (2 ng ml(-1)) or IL-5 (1 ng ml(-1)), respectively, alone or associated with dexamethasone, hydrocortisone or corticosterone. Total myeloid colony numbers, frequency and size of eosinophil colonies, and numbers of eosinophil-peroxidase-positive cells were determined at day 7. In BALB/c mice, dexamethasone (10(-7) M) increased GM-CSF-stimulated myeloid colony formation (P = 0.01), as well as the frequency (P=0.01) and size (P<0.01) of eosinophil colonies. Dexamethasone (10(-7) M) alone had no effect. Dexamethasone (10(-7)-10(-10) M) increased (P<0.002) eosinophil precursor responses to IL-5. Potentiation by dexamethasone was still detectable: (a) on low density, immature, nonadherent BALB/c bone-marrow cells, (b) on bone-marrow from other strains, and (c) on cells from allergic mice. Hydrocortisone and corticosterone had similar effects. Dexamethasone administered in vivo, 24 h before bone-marrow harvest, increased subsequent progenitor responses to GM-CSF (P = 0.001) and precursor responses to IL-5 (P<0.001). These effects were blocked by RU 486 (20 mg kg(-1), orally, 2 h before dexamethasone, or added in vitro at 10 microM, P<0.001). Glucocorticoids, acting in vivo or in vitro, through glucocorticoid receptors, enhance bone-marrow eosinopoiesis in na?ve and allergic mice.     

Novel lineage- and stage-selective effects of retinoic acid on mouse granulopoiesis: Blockade by dexamethasone or inducible NO synthase inactivation

Despite the close relationship of eosinophils and neutrophils, these granulocyte lineages respond to distinct cytokines and play unique roles in immune responses. They nevertheless respond to shared physiological/pharmacological regulators, including glucocorticoids and retinoids, and to ubiquitous mediators, including NO. Others showed that, in humans, all-trans retinoic acid (ATRA) suppresses eosinophil differentiation, but promotes neutrophil differentiation. Mechanisms of dual co-regulation of physiological granulopoiesis were here examined in murine bone-marrow, a model system suitable for exploration of immunopharmacological mechanisms, given the availability of experimental resources, including mutant/knockout mouse strains. We examined the effects of ATRA on mouse eosinophil and neutrophil production, using wild-type (BALB/c, C57BL/6) and mutant (iNOS-, CD95L-, or CD95-KO) bone-marrow cultures, further assessing the modification of ATRA activity by dexamethasone and iNOS blockade. ATRA (10^− 6–10^− 8 M) significantly decreased eosinophil production relative to IL-5 controls. This effect was iNOS-independent, but CD95L- and caspase-dependent, and prevented by dexamethasone (10^− 7 M in vitro; 1–20 mg·kg^− 1 in vivo). In myeloid colony formation assays, ATRA markedly suppressed GM-CSF-responsive progenitors, through an iNOS-dependent, CD95-independent, dexamethasone-sensitive mechanism. By contrast, ATRA potently enhanced GM-CSF-dependent neutropoiesis in liquid culture from BALB/c or C57BL/6 bone-marrow. This novel stimulatory effect was resistant to dexamethasone and abolished in iNOS-KO bone-marrow. ATRA injections also induced lineage- and stage-selective effects on granulopoiesis in vivo. ATRA therefore co-regulates eosinophil and neutrophil production in murine bone-marrow through multiple lineage- and stage-selective mechanisms.     

Inhibitory effect of olopatadine on antigen-induced eosinophil infiltration and the LFA-1 and Mac-1 expression in eosinophils

The inhibitory effect of olopatadine, a new antiallergic drug, on antigen-induced eosinophil infiltration and its mechanisms were examined using the local sensitized rat allergic rhinitis model and isolated IL-5-stimulated rat peritoneal eosinophils. Olopatadine dose-dependently inhibited antigen-induced eosinophil infiltration in the nasal mucosa. Olopatadine dose-dependently repressed the IL-5-induced expressions of CD11a/CD18 (LFA-1) and CD11b/CD18 (Mac-1) on rat peritoneal eosinophils. However, olopatadine had no effect on IL-5-induced CD49d/CD29 (VLA-4) expression. These results suggest that olopatadine may inhibit antigen-induced eosinophil infiltration through repression of LFA-1 and Mac-1 expression.     

Allergenic sensitization prevents upregulation of haemopoiesis by cyclo-oxygenase inhibitors in mice

1. We evaluated whether immunization affects bone-marrow responses to indomethacin, because allergenic sensitization and challenge upregulate responses to haemopoietic cytokines (including IL-5-driven eosinopoiesis) in murine bone-marrow, while indomethacin upregulates haemopoiesis and protects bone-marrow from radiation damage. 2. Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized mice, with or without intranasal challenge. Cultures were established with GM-CSF (2 ng ml(-1)) or IL-5 (1 ng ml(-1)), respectively, alone or associated with indomethacin (10(-7) - 10(-11) M) or aspirin (10(-7) - 10(-8) M). Total myeloid colony numbers and numbers of eosinophil-peroxidase-positive cells were determined at day 7. 3. In na?ve BALB/c mice, indomethacin (10(-7) - 10(-9) M) increased GM-CSF-stimulated myeloid colony formation (P=0.003 and P=0.009, respectively). In contrast, it had no effect on bone-marrow of ovalbumin-sensitized and challenged mice. Indomethacin (10(-7) - 10(-9) M) also increased eosinophil precursor responses to IL-5 in bone-marrow of na?ve (P<0.001 and P=0.002 respectively), but not sensitized-challenged mice. Aspirin (10(-7) M) had similar effects, equally abolished by sensitization. Enhancement of haemopoiesis by indomethacin required adherent cells from na?ve bone-marrow. Nonadherent cells responded to IL-5 but not to indomethacin. Indomethacin was effective on bone-marrow from sham-sensitized, ovalbumin-challenged, but not from sensitized, saline-challenged mice. Plasma transfer from immune mice abolished eosinophil precursor responses to indomethacin in bone-marrow of na?ve recipients. This was not prevented by previous removal of antibody from immune plasma. 4. COX inhibitors enhance haemopoiesis in na?ve but not allergic mice. Responsiveness to indomethacin can be abolished either by active sensitization or by immune plasma transfer. Specific antibody is not involved.     

Eosinophilopoiesis at the cross-roads of research on development, immunity and drug discovery

Eosinophils are a minority subpopulation of leukocytes whose roles in host defense against infection remain controversial, but which have been implicated in the pathogenesis of both acute allergic inflammation and the chronic bronchopulmonary remodelling in asthma. Eosinophilia, a hallmark of both helminth infections and atopic diseases, is maintained through upregulation of eosinophilopoiesis by means of increased production and effectiveness of Interleukin-5 (IL-5), a major Th2 cytokine. These mechanisms are further modulated by a wide variety of agents, including glucocorticoids, nonsteroidal anti-inflammatory drugs and mediators of inflammation. We review recent progress made by different groups in the study of eosinophilopoiesis that led to the identification of the heterogeneous targets for developmental regulation by IL-5 and other agents, and to the ongoing characterization of the molecular mechanisms that ensure their commitment to the eosinophil lineage. We argue that the study of eosinophilopoiesis provides insight into basic developmental processes, and especially into how modulators influence the constitutive rate of eosinophil production by controlling the rates of apoptosis and terminal differentiation. The mechanisms underlying the apparently paradoxical effects of dexamethasone, a drug widely employed to control inflammation, as well as the role of specific molecular targets (including inducible NO synthase and CD95/Fas) in developmental regulation, are discussed in detail. We further argue that eosinophilopoiesis offers unique insights of how immune and endocrine effector loops interact to control both the steady-state responses to IL-5 and the susceptibility to modulation of these responses by drugs and cytokines. We also review the existing evidence on the recruitment of circulating stem cells and progenitors into inflammatory sites, and on a critical role for IL-5 in the accumulation of eosinophil lineage-committed progenitors in lungs of allergic mice. Finally, we review recent progress in the study of the regulatory T cell populations present in bone-marrow, and discuss alternative mechanisms through which cellular immunity may influence eosinophilopoiesis.     

Modification of eosinophil function by suplatast tosilate (IPD), a novel anti-allergic drug

Suplatast tosilate (IPD), a new dimethylsulfonium agent, is used therapeutically in allergic diseases. Suplatast has been reported to attenuate airway hyperresponsiveness in guinea pigs, human IgE synthesis, and murine peritoneal eosinophilia. However, the effect of suplatast on human eosinophils is not known. In this study, we examined the effects of suplatast in human eosinophils on platelet activating factor (PAF, 1 microM)-induced chemotaxis by the blind well chamber technique, eosinophil adhesion to TNF-alpha (10 ng/ml) or IL-4 (10 ng/ml)-stimulated human umbilical vein endothelial cells (HUVECs), and expression of very late antigen-4 (VLA-4) on eosinophils and vascular cell adhesion molecule-1 (VCAM-1) on HUVECs by flow cytometry. Suplatast suppressed IL-4-induced eosinophil adhesion to HUVECs in a dose-dependent manner. Eosinophils from the normal subjects did not express VLA-4. However, there was a significant increase (P < 0.01) in the basal expression of VLA-4 in allergic patients. PAF or IL-4 did not enhance VLA-4 expression on eosinophils, and there was no significant effect of suplatast on VLA-4 expression in allergic patients. Suplatast did not affect TNF-alpha-induced VCAM-1 expression. Interestingly, suplatast significantly suppressed IL-4 induced VCAM-1 expression on HUVECs and PAF-induced eosinophil chemotaxis. These data suggest that suplatast may modify eosinophil participation in airway inflammation by attenuating inflammatory mediators-induced chemotaxis and adhesion to endothelial cells, and thus might be useful in the treatment of bronchial asthma.     

Hemopoietic progenitor cells and hemopoietic factors: potential targets for treatment of allergic inflammatory diseases

Eosinophilic infiltration is a cardinal feature of allergic inflammation; based upon its biological actions, the eosinophil has assumed the role as the principal inflammatory cell in asthma. In assessing the mechanisms by which eosinophils are recruited to sites of inflammation, a sizeable body of evidence exists supporting the proposal that expansion of hemopoietic compartments in the bone marrow stimulates an increased turnover and traffic of mature eosinophils to the site of allergic inflammation. In addition, recent findings point to the possible egress and traffic of primitive progenitor cells to the site of inflammation where in-situ differentiation may provide a continued supply of pro-inflammatory cells. In the present article, we will review the evidence for these findings, and discuss the rationale for targeting hemopoiesis and migrational pathways of hemopoietic cells in the treatment of allergic disease. In this context, we will discuss the effect of corticosteroid treatment on hemopoietic mechanisms; the effects of therapies that inhibit the actions of cysteinyl leukotrienes (CysLTs); the effects of in vivo blockade of the eosinophil-active cytokine, interleukin (IL)-5; and, the effects of antihistamines on hemopoiesis. In addition, we will address the potential role that small molecular weight chemokine receptor antagonists may play in modulating progenitor cell trafficking to tissue sites of inflammation.     

Pharmacological regulation of human eosinophil apoptosis

Eosinophilic inflammation of the airways is a key characteristic of asthma. The balance between eosinophil recruitment into the lung and their removal from the lungs determines the number of eosinophils in the airways. Apoptosis or programmed cell death is of importance in the removal of eosinophils from the lungs. In asthma, eosinophil apoptosis is delayed. Glucocorticoids enhance eosinophil apoptosis, whereas beta(2)-agonists may delay apoptosis in eosinophils. Detailed knowledge on the mechanisms that regulate this process gives an opportunity to develop specific asthma therapies targeting the eosinophil. This review aims to focus on the signalling leading to or preventing apoptosis in human eosinophils as well as reviews the current evidence on the regulation of eosinophil apoptosis and/or survival in allergic diseases.     

Eosinophil Apoptosis as a Therapeutic Target in Allergic Asthma

Asthma is a chronic inflammatory disease of the airways manifesting in many different phenotypes. Allergic asthma, comprising approximately half of patients with asthma, is characterized by the accumulation of eosinophils into the lungs. Eosinophils release factors that damage the surrounding cells and participate in the maintenance and exacerbation of inflammation. In the absence of any inflammatory survival‐prolonging factors, eosinophils die by apoptosis in few days but in inflamed airways, eosinophil survival is thought to be prolonged due to the surrounding pro‐inflammatory factors such as IL‐5, IL‐3 and GM‐CSF. Resolution of eosinophilic inflammation is an important goal in the treatment of allergic asthma. Apoptosis is a physiological and non‐inflammatory way to eliminate these harmful cells, and development of drugs targeting eosinophil apoptosis is one possible strategy for the therapy of allergic asthma. Importance of this strategy is supported by the fact that promotion of eosinophil apoptosis is a property of many anti‐asthmatic agents such as glucocorticoids, the current main anti‐inflammatory therapy of asthma, theophylline and leukotriene modifiers. β₂ agonists have been shown to modulate eosinophil longevity by increasing survival. Also, anti‐IL‐5 antibody mesolizumab has shown efficacy in reducing asthma exacerbations in patients with severe eosinophilic asthma. Many potential future anti‐asthmatic agents, such as Siglec‐8 activating antibody and novel humanized anti‐IL‐5 antibody MEDI‐563, have the property of inducing eosinophil apoptosis. This MiniReview aims to present eosinophil apoptosis as a therapeutic target in the treatment of allergic asthma. We summarize the effects and mechanisms of current and potential future anti‐asthmatic drugs on eosinophil apoptosis and additionally, discuss the potential factors that promote eosinophil longevity in the lungs.     

Role of the Mac-1 and VLA-4 integrins, and concomitant Th2-cytokine production, in nitric oxide modulated eosinophil migration from bone marrow to lungs in allergic mice

Although numerous studies demonstrate the participation of nitric oxide (NO) in various inflammatory diseases, the precise function of NO in allergic asthma remains unclear. We investigated whether iNOS inhibition could interfere with the kinetics of VLA-4 and Mac-1 expression and adhesion properties of bone marrow and peripheral blood eosinophils of sensitized mice after antigen exposure. Treatment of allergic mice with 1400 W (iNOS inhibitor) increased the adhesion of bone marrow eosinophils to ICAM-1, but not blood eosinophils, at 24h and 48 h after OVA-challenge. Conversely, adhesion of blood eosinophils from 1400 W-treated mice to VCAM-1 diminished at 24h and was almost completely blocked at 48 h. 1400 W did not induce any change in the adhesion of bone marrow eosinophils to VCAM-1, at 24h, but cells collected 48 h after challenge showed significantly lower adherence. Flow cytometry demonstrated that 1400 W resulted in a significantly increased Mac-1 expression on bone marrow eosinophils at 24h, as compared to control mice. However, at 24h, 1400 W significantly decreased Mac-1 and VLA-4 expressions on blood eosinophils. At 48 h, the expressions of both Mac-1 and VLA-4 returned to previous levels. Results show a temporal effect of iNOS upon Mac-1 expression and function, the chief adhesion molecule involved in the eosinophil efflux from the bone marrow at 24h. In contrast, Mac-1 and VLA-4 were involved in eosinophil mobilization from blood to lungs at 48 h after antigen challenge. Data suggest an important role of the Mac-1 and VLA-4 in the iNOS-modulated migration of eosinophils to the lungs of allergic mice.     

Suppressive effect of tranilast on interleukin-5 prolonged eosinophils survival via apoptosis.

Tranilast has long been used clinically to treat allergic diseases such as bronchial asthma. To further clarify the antiinflammatory machanism, we examined the ability of tranilast to counteract the prolongation of eosinophil survival induced by interleukin (IL)-5. Tranilast reduced the IL-5 prolonged survival of eosinophils at the concentration range of 30 microg/ml to 100 microg/ml. The DNA extracted from eosinophils cultured with tranilast showed signs of fragmentation that was comparable with apoptosis. Electron-microscopic analysis of activated eosinophils cultured with 100 microg/ml of tranilast also revealed morphologic features of apoptosis. These data suggest that tranilast may act in vivo on activated eosinophils to reduce inflammation in allergic diseases.     

大鼠过敏性气道炎症中VCAM—1表达、嗜酸细胞浸润和药理调节

目的:探讨过敏性气道炎症大鼠肺内VCAM-1表达和嗜酸细胞浸润,以及速激肽NK-1受体拮抗剂SR140333和地塞米松的可能调节方式.方法:致敏大鼠以1％卵白蛋白气雾吸入攻击后24小时,测定VCAM-1在不同组别大鼠肺内的表达和支气管周围嗜酸细胞浸润.抗原攻击前3天,每天2次腹腔注射SR140333(0.01,0.10mg/kg)或地塞米松(0.50mg/kg).结果:与未致敏大鼠相比VCAM-1在致敏大鼠肺内表达增加(P<0.05),预先用地塞米松处理可抑制其增加(P<0.05),SR140333无此作用(P>0.05).此外,抗原攻击致敏大鼠促使支气管周围嗜酸细胞浸润,但不能进一步增加VCAM-1的表达(P>0.05).SR140333抑制嗜酸细胞浸润(P<0.05),但不影响VCAM-1表达(P>0.05);地塞米松则抑制这两种反应(P<0.05).结论:VCAM-1表达在抗原致敏后增加,地塞米松和SR140333抑制大鼠过敏性气道炎症的机制可能不同.     

Interleukin-5, eosinophilic diseases and therapeutic intervention

Interleukin 5 (IL-5) is the key cytokine in an eosinophil's life span: it supports eosinophilopoiesis and eosinophil differentiation, contributes to eosinophil migration, tissue localisation and function, and prevents eosinophil apoptosis. Given the likely role of eosinophils in chronic inflammatory diseases, a lot of research over the past decade was aimed at antagonising IL-5 function. It appears from recent studies that, although this can easily be achieved in vitro, blocking IL-5 function in vivo is much more difficult than originally anticipated. Here, we review the current status of IL-5 and IL-5 receptor research, with emphasis on strategies to interfere with IL-5 function.     

Cysteinyl leukotrienes mediate the enhancing effects of indomethacin and aspirin on eosinophil production in murine bone marrow cultures

BACKGROUND: Prostaglandin E(2) (PGE(2)) suppresses, while indomethacin and aspirin enhance, eosinophil production in murine liquid bone-marrow cultures. Because cysteinyl leukotrienes (cys-LTs) enhance human eosinophil colony formation, we investigated whether the effects of indomethacin and aspirin on murine bone-marrow were due to blockade of PGE(2) production alone, or involved further promotion of cys-LTs production/signalling. EXPERIMENTAL APPROACH: BALB/c liquid bone-marrow cultures were established with IL-5, alone or associated with indomethacin, aspirin, or cys-LTs. The effects of preventing cys-LT production or signalling were assessed. KEY RESULTS: Indomethacin and aspirin counteracted the suppression of eosinophil production by exogenous PGE(2). LTD(4), LTC(4) and LTE(4) enhanced IL-5-dependent eosinophil production and further counteracted the effect of exogenous PGE(2). The 5-lipoxygenase activating protein (FLAP) inhibitor, MK886, a leukotriene synthesis inhibitor, zileuton, the CysLT(1) receptor antagonists, MK571 and montelukast, or inactivation of the LTC(4) synthase gene, abolished effects of indomethacin and aspirin. MK886 and zileuton were ineffective but MK571 and montelukast were effective, against LTD(4). Indomethacin, aspirin and LTD(4) failed to enhance eosinophil production in bone-marrow from CysLT1 receptor-deficient mice. Indomethacin, aspirin and LTD(4) no longer counteracted the effects of exogenous PGE(2) in the presence of MK571 and montelukast. MK886, MK571 and montelukast had no effect by themselves, or in association with PGE(2). CONCLUSIONS AND IMPLICATIONS: Dependence on the FLAP/5-lipoxygenase/LTC(4) synthase pathway and receptor signalling shows that cyclo-oxygenase inhibitors act here through endogenous cys-LTs. While PGE(2) does not act by suppressing cys-LT production, cys-LTs override PGE(2) signalling. Eosinophil production is therefore coordinately regulated by both pathways.     

Role of eosinophils in allergic inflammation

Eosinophils are one of the cells that play a critical role in the pathogenesis of allergic diseases. The increase in the number of eosinophils in such diseases is regulated by interleukin-5 (IL-5). The author have prepared recombinant rat IL-5 using a baculovirus expression system and examined its biological activities in rat eosinophils. It was demonstrated that recombinant rat IL-5 prolongs the survival of mature eosinophils and differentiates immature eosinophils into mature eosinophils, suggesting that rat IL-5 is a factor for eosinophilia in rats. Recombinant rat eosinophil-associated ribonuclease (Ear)-1 and Ear-2 were also prepared. Eosinophil granule proteins are thought to cause tissue damage due to their cytotoxic activity, but using recombinant rat Ear-1 and Ear-2, it was found that rat Ear-1 and Ear-2 have strong RNase A activity and bactericidal activity, suggesting that these proteins play critical roles in host defense. Finally, the important role of acetylation of histones was clarified in the differentiation of HL-60 clone 15 cells into eosinophils using the histone deacetylase inhibitors sodium n-butyrate, apicidin, and trichostatin A. These findings would be useful for further investigations of the role of eosinophils in allergic inflammation.     

Control of eosinophil toxicity in the lung

The inappropriate accumulation of eosinophils and the subsequent release of their potent pro-inflammatory mediator arsenal are thought to be important contributors to the pathogenesis of asthma and other allergic diseases. It is also becoming apparent that eosinophils may play a role in the orchestration of immune responses in the asthmatic lung. There is therefore much interest in the development of strategies to limit or prevent eosinophil-induced toxicity. The mechanisms by which eosinophils accumulate in the peribronchial tissues of the lung are complex and include enhanced differentiation and release from the bone marrow, selective adhesion and transendothelial migration, directed movement in response to specific chemotactic mediators and finally prolonged survival as a consequence of delayed apoptosis. Thus it can be appreciated that there are many points at which the toxicity of eosinophils can be limited or even prevented. Important areas for potential advances in glucocorticoid (GC) development include efforts to dissociate their anti-inflammatory effects from unwanted side effects. Other areas include the development of humanized monoclonal antibodies against IL-4, IL-13 and IL-5 together with the inhibition of adhesion pathways and/or chemokines responsible for eosinophil accumulation in the asthmatic lung. Several avenues of research are currently underway in an attempt to define mechanisms by which pro-inflammatory cells such as eosinophils can be safely removed from the asthmatic lung through apoptosis induction and their subsequent ingestion by phagocytes. This review will discuss both the potential and shortcomings of these diverse approaches to limit eosinophil toxicity in the asthmatic lung.     

α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95

Background and Purpose

α-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored.

Experimental Approach

α-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD₄; and dexamethasone.

Key Results

α-GalCer (10⁻⁶–10⁻⁸M) suppressed IL-5-stimulated eosinopoiesis by inducing apoptosis. α-GalCer pretreatment in vivo (100 μg·kg⁻¹, i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD₄ and NSAIDs.

Conclusions and Implications

α-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD₄, NSAIDs and aminoguanidine, and modified by dexamethasone.     

Interactions between eotaxin and interleukin-5 in the chemotaxis of primed and non-primed human eosinophils

This study was designed to understand the relationship between interleukin-5 and eotaxin in modulating the chemotaxis of eosinophils obtained from healthy subjects and subjects with allergic rhinitis. Chemotaxis of eosinophils from patients with allergic rhinitis toward interleukin-5 (0.25 ng/ml) was 78% higher than that of healthy subjects. Incubation of eosinophils with eotaxin (100 ng/ml) did not change the interleukin-5-induced chemotaxis of eosinophils from healthy subjects, but it reversed the enhanced chemotaxis seen in eosinophils from allergic patients. Chemotaxis of eosinophils from patients with allergic rhinitis toward eotaxin (100 ng/ml) was 65% higher than that of eosinophils from healthy subjects. Incubation of eosinophils with interleukin-5 (100 ng/ml) significantly increased the eotaxin-induced chemotaxis in both subject groups, but such increases were markedly higher for cells from patients with allergic rhinitis. Our finding that eotaxin inhibits the enhanced eosinophil chemotaxis toward interleukin-5 in primed cells suggests that this chemokine may downregulate eosinophil accumulation in the nasal mucosa of allergic patients.     

Effects of saiboku-to on the survival of human eosinophils.

In recent years, bronchial asthma has come to be regarded as a chronic inflammatory disease of the respiratory tract, with mast cells, lymphocytes and eosinophils playing important roles in its pathogenesis. Proteins contained in eosinophil granules, especially major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase (EPO), can cause tissue injury. When stimulated, eosinophils release mediators such as leukotriene C4 (LTC4) and platelet activating factors (PAF). Thus, they are recognized as effector cells that are actively involved in the development of allergic inflammation. In this study, eosinophils from healthy volunteers were used to investigate the effects of Saiboku-to on eosinophils whose survival had been prolonged through stimulation with eosinophil-activating cytokines such as interleukin (IL)-3, IL-5 and granulocyte macrophage colony stimulating factors (GM-CSF). As a result, the cytokine-enhanced survival of eosinophils was significantly shortened by the addition of Saiboku-to. These findings suggest that Saiboku-to has the potential to inhibit allergic responses by directly affecting eosinophils which are related to allergic inflammation.