Glucagon-like peptide-1 receptor expression on human eosinophils and its regulation of eosinophil activation.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP-1 also has immune modulatory roles. OBJECTIVES: To measure the expression of the GLP-1 receptor (GLP-1R) on eosinophils and neutrophils in normal and asthmatic subjects and evaluate effects of a GLP-1 analog on eosinophil function. METHODS: Peripheral blood samples were taken from 10 normal and 10 allergic asthmatic subjects. GLP-1R expression was measured on eosinophils and neutrophils. Subsequently, the asthmatic subjects underwent allergen and diluent inhalation challenges, and GLP-1R expression was measured. Purified eosinophils, collected from mild asthmatic subjects, were stimulated with lipopolysaccharide (LPS) and a GLP-1 analog to evaluate eosinophil cell activation markers CD11b and CD69 and cytokine (IL-4, IL-5, IL-8 and IL-13) production. RESULTS: Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. Eosinophil, but not neutrophil, expression of GLP-1R is significantly higher in normal controls compared to allergic asthmatics. The expression of GLP-1R did not change on either eosinophils or neutrophils following allergen challenge. A GLP-1 analog significantly decreased the expression of eosinophil-surface activation markers following LPS stimulation and decreased eosinophil production of IL-4, IL-8 and IL-13, but not IL-5. CONCLUSION AND CLINICAL RELEVANCE: Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. A GLP-1 analog attenuates LPS-stimulated eosinophil activation. GLP-1 agonists may have additional adjunctive indications in treating persons with concomitant type 2 diabetes mellitus and asthma.

Role of local eosinophilopoietic processes in the development of airway eosinophilia in prednisone-dependent severe asthma.

BACKGROUND: In severe asthmatics with persistent airway eosinophilia, blockade of interleukin-5 has significant steroid-sparing effects and attenuates blood and sputum eosinophilia. The contribution of local maturational processes of progenitors within the airways relative to the recruitment of mature cells from the peripheral circulation to the development of airway eosinophilia is not known. We hypothesize that local eosinophilopoiesis may be the predominant process that drives persistent airway eosinophilia and corticosteroid requirement in severe asthmatics. OBJECTIVES: In a cross-sectional study, the number and growth potential of eosinophil-lineage-committed progenitors (EoP) were assayed in 21 severe eosinophilic asthmatics, 19 mild asthmatics, eight COPD patients and eight normal subjects. The effect of anti-IL-5 treatment on mature eosinophils and EoP numbers was made in severe eosinophilic asthmatics who participated in a randomized clinical trial of mepolizumab (substudy of a larger GSK sponsored global phase III trial, MEA115575) where subjects received mepolizumab (100 mg, n = 9) or placebo (n = 8), as six monthly subcutaneous injections. RESULTS: Mature eosinophil and EoP numbers were significantly greater in the sputum of severe asthmatics compared with all other subject groups. In colony-forming assays, EoP from blood of severe asthmatics demonstrated a greater response to IL-5 than mild asthmatics. Treatment of severe asthmatics with mepolizumab significantly attenuated blood eosinophils and increased EoP numbers consistent with blockade of systemic eosinophilopoiesis. There was however no significant treatment effect on mature eosinophils, sputum EoP numbers or the prednisone maintenance dose. CONCLUSIONS AND CLINICAL RELEVANCE: Patients with severe eosinophilic asthma have an exaggerated eosinophilopoeitic process in their airways. Treatment with 100 mg subcutaneous mepolizumab significantly attenuated systemic differentiation of eosinophils, but did not suppress local airway eosinophil differentiation to mature cells. Targeting IL-5-driven eosinophil differentiation locally within the lung maybe of relevance for optimal control of airway eosinophilia and asthma.

Safety and efficacy of an oral CCR3 antagonist in patients with asthma and eosinophilic bronchitis: a randomized, placebo-controlled clinical trial.

BACKGROUND: Several chemokines, notably eotaxin, mediate the recruitment of eosinophils into tissues via the CCR3 receptor. OBJECTIVE: In this study, we investigated the role of CCR3 agonists in asthma by observing the effect of a small molecule antagonist of the CCR3 receptor (GW766994) on sputum eosinophil counts in patients with eosinophilic asthma. METHODS: Clinical and physiological outcomes, the chemotactic activity of sputum supernatant for eosinophils and the presence of eosinophil progenitors in sputum and blood samples were also studied. RESULTS: In a double-blind parallel group study, 60 patients with asthma were randomized to 300 mg of GW766994 twice daily or matching placebo for 10 days followed by prednisone 30 mg for 5 days. Of these patients, 53 had a sputum eosinophil count > 4.9% at baseline. Despite plasma concentrations of drug consistent with > 90% receptor occupancy during the dosing period, the CCR3 antagonist did not significantly reduce eosinophils or eosinophil progenitor cells (CD34(+) 45(+) IL-5Rα(+)) in sputum or in blood. The ex vivo chemotactic effect of sputum supernatants on eosinophils was attenuated by GW766944 compared to placebo. There was no improvement in FEV1 ; however, there was a modest but statistically significant improvement in PC20 methacholine (0.66 doubling dose) and ACQ scores, (0.43). Whilst the improvement in PC20 is statistically significant, it is not of clinical significance. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, this study calls into question the role of CCR3 in airway eosinophilia in asthma and suggests that other cellular mechanisms mediated by the CCR3 receptor may contribute to airway hyperresponsiveness.

Interleukin-4 and interleukin-13 prime migrational responses of haemopoietic progenitor cells to stromal cell-derived factor-1α.

BACKGROUND: Lung-homing of progenitor cells is associated with inflammatory and remodelling changes in asthma. Factors that modulate the increased traffic of progenitor cells to the site of inflammation in asthma remain to be defined. Interleukin (IL)-4 and IL-13 are Th2 cytokines that are key regulators of asthma pathology. OBJECTIVE: We investigated the role of IL-4 and IL-13 in modulating the trans-migrational responses of haemopoietic progenitor cells (HPC). METHODS: HPC were enriched from cord blood (CB) and peripheral blood (PB) samples. Migration of HPC was assessed using transwell migration assays, and responding cells were enumerated by flow cytometry. RESULTS: IL-4 and IL-13 primed migration of CB- and PB-derived HPC (CD34(+) 45(+) cells) to stromal cell-derived factor-1α (SDF-1α), in vitro. However, these cytokines had no effect on migrational responses of eosinophil-lineage committed progenitors (CD34(+) 45(+) IL-5Rα(+) cells) or mature eosinophils to SDF-1α. For HPC, priming effects of IL-4 (0.1 ng/mL) and IL-13 (0.1 ng/mL) were detectable within 1 h and optimal at 18-h post-incubation, and IL-4 was the more effective priming agent. Pre-incubation with IL-4 or IL-13 had no effect on the intensity of cell surface expression of SDF-1α receptor, CXCR4. Disruption of cell membrane cholesterol content by pre-incubation with polyene antibiotics inhibited IL-4 priming of SDF-1α stimulated migration of HPC indicating that increased incorporation of CXCR4 into membrane lipid rafts mediated the cytokine primed migrational response of HPC. This was confirmed by confocal fluorescent microscopy. CONCLUSIONS AND CLINICAL RELEVANCE: IL-4 and IL-13 prime the migrational response of HPC to SDF-1α by enhancing the incorporation of CXCR4 into lipid rafts. The priming effect of these cytokines is specific to primitive HPC. These data suggest that increased local production of IL-4 and IL-13 within the lungs may promote increased SDF-1α mediated homing of HPC to the airways in asthma.

Modulating progenitor accumulation attenuates lung angiogenesis in a mouse model of asthma.

Asthmatic responses are associated with the lung homing of bone marrow (BM)-derived progenitors implicated as effectors of disease pathology. Studies have shown that increases in lung extracted vascular endothelial progenitor cells (VEPCs) correlate with airway angiogenesis and declining lung function. We investigated the effect of modulating lung homing of VEPCs on tissue remodelling and airway hyperresponsiveness (AHR). BALB/c mice were sensitised to ovalbumin, subjected to a chronic exposure protocol and given early concurrent or delayed treatment with a modulator of progenitor traffic, AMD3100 (CXC chemokine receptor 4 antagonist; inhibits chemotactic activity of stromal-derived factor-1α on VEPCs). After ovalbumin challenge, early haemopoietic stem cells (HSCs) and VEPCs were enumerated along with indices of airway inflammation, lung morphometry and AHR. Following ovalbumin challenge, there was a decrease in BM and an associated increase in the lung tissue-extracted HSCs and VEPCs, together with increases in airway eosinophilia, microvessel density and AHR. These outcomes were significantly inhibited by early concurrent treatment with AMD3100. Where lung disease was established, delayed treatment with AMD3100 significantly attenuated HSC numbers and lung angiogenesis but only partially reversed sustained AHR compared with untreated ovalbumin-exposed mice. Progenitor lung homing is associated with the development of asthma pathology, and early modulation of this accumulation can prevent airway remodelling and lung dysfunction.

TPI ASM8 reduces eosinophil progenitors in sputum after allergen challenge.

BACKGROUND: TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides (AON), one targeting the common beta chain (ßc) of the IL-3/IL-5/GM-CSF receptors and the other targeting the chemokine receptor CCR3. Inhalation of TPI ASM8 significantly improves lung function and sputum eosinophilia after allergen inhalation challenge in asthmatics. OBJECTIVE: This study assessed whether TPI ASM8 reduces airway levels of haemopoietic progenitor cells. METHODS: This open-label study was conducted in 14 stable, allergic mild asthmatic subjects with early- and late-phase allergen-induced bronchoconstriction. Subjects underwent allergen challenges after 4-day treatment with placebo, 4 mg b.i.d. and 8 mg o.d. of TPI ASM8. Sputum was induced before, 7 and 24 h after allergen challenges for progenitor measurements. Treatments were separated by 2-3 weeks. RESULTS: TPI ASM8 reduced allergen-induced sputum eosinophils, and the early and late asthmatic responses (P<0.05). TPI ASM8 also reduced the number of CD34(+) CCR3(+) cells (P=0.004) and CD34(+) IL-5Rα(+) cells (P=0.016), and the proportion of CD34(+) cells expressing IL-5Rα (P=0.036). CONCLUSIONS AND CLINICAL RELEVANCE: TPI ASM8 was safe and well tolerated. The results of this study demonstrate blocking of CCR3 and ßc expression by TPI ASM8 significantly inhibits the accumulation of eosinophils and eosinophil progenitors in the airways after allergen challenge. Inhibition of airway progenitor cell accumulation presents a novel therapeutic target.

Human airway smooth muscle promotes eosinophil differentiation.

INTRODUCTION: Human airway smooth muscle (HASM) cells in culture synthesize cytokines and chemokines that may orchestrate the tissue homing and in situ differentiation of haemopoietic progenitor cells from the peripheral circulation. OBJECTIVE: To study the effect of a supernatant from cultured HASM cells on the differentiative and transmigrational responses of haemopoietic progenitor cells. METHODS: HASM cells were grown to confluence and stimulated with a cytomix of TNF-alpha, IL-1beta and IFN-gamma. Peripheral blood-derived progenitors from atopic asthmatics (n=12) and non-atopic controls (n=11) were grown in a methylcellulose culture with a supernatant from stimulated HASM cells to assess clonogenic potential. The ability of HASM cells to stimulate directional migration and adhesion to fibronectin of blood progenitors was also investigated. RESULTS: HASM cells stimulated significant growth of eosinophil/basophil colony forming units (Eo/B CFUs) from blood progenitor cells from both groups of subjects. This activity was significantly attenuated in the presence of anti-IL-5 and anti-granulocyte macrophage-colony forming factor blocking antibodies and by pre-treatment with SB202190 [p38 mitogen-activated protein kinase (MAPK) inhibitor]. An src kinase (srcK) inhibitor (Pyrazolopyrimidine 1) was less effective at attenuating IL-5- and HASM-stimulated Eo/B CFU growth from both groups of subjects. Examination of the phosphorylation of these kinases in CD34(+) cells following co-incubation with the major constituents of HASM showed activation of p38 MAPK but not that of the srcK pathway. The HASM supernatant had no significant effect on the migrational and adhesive responses of haemopoietic progenitor cells in vitro. CONCLUSION: We have shown that HASM cell-derived cytokines promote eosinophil differentiation that is dependent on p38 MAPK but not on the srcK pathway. This study shows that a major structural cell of the lungs, airway smooth muscle, has the capability to direct eosinophil differentiation and maturation from progenitor cells, which in turn may perpetuate an eosinophilic inflammation and consequently tissue remodelling in patients with chronic asthma.

Allergen-induced increases in IL-5 receptor alpha-subunit expression on bone marrow-derived CD34+ cells from asthmatic subjects. A novel marker of progenitor cell commitment towards eosinophilic differentiation.

We have proposed previously that hemopoietic myeloid progenitors contribute to the ongoing recruitment of proinflammatory cells, namely eosinophils, to sites of allergen challenge in allergic diseases such as asthma. In this study, we investigated the involvement of bone marrow-derived progenitors in the development of allergen-induced pulmonary inflammation in mild asthmatic subjects. By flow cytometry, we enumerated the level of expression of CD34, a hemopoietic progenitor cell marker, on bone marrow aspirates taken before and 24 h after allergen challenge. In addition, the coexpression of the alpha-subunits of IL-3 receptor (IL-3R) and IL-5 receptor (IL-5R) on CD34+ cells was investigated. After allergen-challenge, although no significant change in total BM CD34+ cell numbers was observed, a significant increase in the proportion of CD34+ cells expressing IL-5R alpha, but not IL-3R alpha, was detected in the 24-h post-allergen, compared with the pre-allergen bone marrow. This was associated with a significant blood and sputum eosinophilia and increased methacholine airway responsiveness, 24 h post-allergen. Using simultaneous in situ hybridization and immunocytochemistry, we colocalized the expression of messenger RNA for membrane-bound IL-5R alpha to CD34+ cells. In summary, our data suggest that increased expression of IL-5R alpha on CD34+ cells favors eosinophilopoiesis and may thus contribute to the subsequent development of blood and tissue eosinophilia, a hallmark of allergic inflammation.

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.

Regulation of IL-5 receptor on eosinophil progenitors in allergic inflammation: role of retinoic acid.

We and others have shown that IL-5 plays a central role in eosinophil and basophil differentiation, exerting its effects through the IL-5 receptor (IL-5R). Little is currently known concerning regulation of IL-5Ralpha gene transcription in the context of commitment of hemopoietic progenitor cells to the eosinophil and basophil lineages; recent studies have indicated that IL-5 itself can regulate IL-5Ralpha expression on mature eosinophils. We now provide evidence to indicate that IL-5 can upregulate IL-5Ralpha on bone marrow CD34+ progenitors in vitro, as we have demonstrated in vivo in atopic asthmatics. Given that all-trans retinoic acid (ATRA) is known to modulate some granulopoiesis, causing neutrophilic differentiation, we examined the effects of ATRA on eosinophil/basophil differentiation and IL-5Ralpha expression. In cultures of normal human bone marrow, ATRA selectively suppressed eosinophil/basophil differentiation. Similarly, ATRA inhibited eosinophil/basophil differentiation of cord blood CD34+ cells, while neutrophil differentiation proceeded without impediment. Most importantly, these effects of ATRA on CD34+ cells were associated with selective, dose-dependent inhibition of membrane-bound IL-5Ralpha, upregulation of soluble IL-5Ralpha transcription, but no change in GM-CSF receptor expression. These findings indicate that retinoids can differentially regulate membrane and soluble isoforms of IL-5Ralpha, and that these effects have functional consequences in vitro on eosinophil and basophil differentiation. ATRA may be of therapeutic benefit in allergic inflammatory disorders in which eosinophil differentiation and membrane-bound IL-5R are upregulated.

Selective upregulation of a functional beta7 integrin on differentiating eosinophils.

BACKGROUND: The sequence of adhesion-molecule expression during eosinophil differentiation remains unclear. METHODS: We analyzed the surface expression of alpha4, beta1, and beta7 integrins and compared it to established myeloid developmental markers, using the eosinophilic cell line HL-60 clone 15, as well as cord and peripheral blood differentiation assays. RESULTS: Cells induced to eosinophil differentiation by treatment with butyric acid, IL-5, and GM-CSF showed a significant upregulation of beta7 integrin expression coincident with a marked upregulation of CD35 and attenuation of CD33 and beta1 integrin expression. In addition, adhesion of induced HL-60 clone 15 cells to fibronectin was attenuated by a beta7 integrin antibody. CONCLUSIONS: Our data show that protein synthesis-dependent upregulation of the functional beta7 integrin occurs under conditions when beta4 and beta1 integrins are fully expressed, indicating a sequential appearance of specific adhesion molecules on differentiating eosinophil progenitors.

Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects.

We have attempted to determine whether interleukin-5 (IL-5), a cytokine that selectively affects eosinophil (as opposed to neutrophil) differentiation and activation, also modulates eosinophil migrational responses. Using a modified Boyden chemotaxis assay, IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gave a weak locomotory response for eosinophils from normal nonatopic subjects (optimal at 10(-11), 10(-8), and 10(-9) mol/L, respectively), but not for eosinophils from subjects with an eosinophilia associated with asthma and/or allergic rhinitis. In contrast, IL-5 and IL-3 had no effect on neutrophils, while GM-CSF was chemotactic for neutrophils over a limited concentration range, optimal at 10(-8) mol/L. When eosinophils from normal subjects were incubated with IL-5 (10(-9) mol/L), the locomotory response to platelet-activating factor (PAF; 10(-8) mol/L, P less than .05), leukotriene B4 (LTB4; 10(-6) mol/L, P less than .01), and N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-8) mol/L, P less than .01) was significantly enhanced. The percentage enhancement of eosinophil locomotion by IL-5 was greater for eosinophils from normal as compared with subjects with an eosinophilia associated with asthma (P less than .05 for PAF and LTB4; P less than .01 for FMLP). Preincubation of eosinophils from normal subjects with IL-5 (10(-9) mol/L) attenuated the subsequent locomotory response to IL-5 (10(-12) and 10(-11) mol/L, P less than .05). Therefore, the observed refractoriness of eosinophils from eosinophilic subjects to both directional migratory and priming effects of IL-5 in vitro, may reflect a deactivation process resulting from prior exposure in vivo. The selective priming of eosinophil but not neutrophil locomotion by IL-5 suggests that this cytokine may play a significant role in the preferential accumulation of eosinophils at sites of allergic inflammation.

Identification on receptors for leukotriene B4 expressed on guinea-pig peritoneal eosinophils.

We have recently reported that guinea-pig eosinophil chemotactic factor of anaphylaxis (ECF-A), an activity present in diffusates from antigen-challenged sensitized lung, is largely accounted for by leukotriene B4 (LTB4) and to a lesser extent 8(S)15(S)-dihydroxy 5,9,11,13 (Z,E,Z,E) eicosatetraenoic acid. We characterized cell surface receptors for LTB4 on guinea-pig eosinophils in order to demonstrate an association between receptor occupancy and eosinophiliotactic activity of guinea-pig ECF-A. Equilibrium binding studies showed that peritoneal eosinophils bound [3H]LTB4 in a cell concentration and time-dependent fashion. The binding was saturable and specific for LTB4 as other eosinophil chemoattractants, i.e. platelet-activating factor (PAF) and 8(S)15(S)-diHETE, were unable to displace significant amounts of [3H]LTB4. In addition the binding was readily reversed by the LTB4 receptor antagonist LY 255283 (Ki 4.30 nM). Scatchard plot analysis revealed two discrete populations of binding sites, high affinity (Kd1 = 0.30 nM; Bmax = 900 sites/cell) and low-affinity sites (Kd2 = 140 nM; Bmax = 60,000 sites/cell). The major migratory component of LTB4-stimulated eosinophil locomotion was chemotaxis, optimal at 1 x 10(-7) M (P < 0.01) with EC50 value of 3 x 10(-9) M. A comparison of the profile of arachidonic acid metabolism by RP-HPLC analysis showed that following stimulation with calcium ionophore (A23187) guinea-pig eosinophils preferentially synthesized LTB4 (10 ng/10(6) cells) while in contrast human eosinophils synthesized LTC4 (10 ng/10(6) cells). Therefore our data show that guinea-pig eosinophils express both high- and low-affinity receptors for LTB4 and that the chemotactic response to this mediator may be mediated by ligation of the high-affinity binding site. Furthermore guinea-pig peritoneal eosinophils can synthesize LTB4, a mediator which constitutes > 60% of guinea-pig ECF-A.

The role of the bone marrow in allergy and asthma.

The above studies have begun to address the fundamental question of the mechanisms of bone marrow involvement and response to allergen challenge in allergic asthmatics. Further studies in this area should complement our investigations in human asthma--which suggest that a particular bias toward differentiation of Eo-Baso progenitors characterizes the atopic state--as well as our findings in the dog model of allergen-induced airway hyperresponsiveness, which indicate that the bone marrow responds to inhalation of allergen or corticosteroids. Taken in the context of previous indications that IgE and bronchial responsiveness may both be transferrable through bone marrow transplantation (109), these findings indicate a physiologic role for the bone marrow in allergic inflammation. Likewise, these concepts provide a basis for making certain predictions regarding management and novel therapeutic interventions in atopy and asthma.

Interleukin-8 is a chemo-attractant for eosinophils purified from subjects with a blood eosinophilia but not from normal healthy subjects.

Interleukin-8 (IL-8), a pro-inflammatory cytokine with potent neutrophil chemotactic activity, was studied for its effect on eosinophil migration responses, in vitro. Normal density eosinophils were isolated from healthy, non-atopic subjects (<0.35 x 10(9) eosinophils/l) and individuals with various diseases associated with a blood eosinophilia (range 0.56 x 10(9)-12.2 x 10(9) eosinophils/l). IL-8 produced a dose-dependent migrational response for eosinophils from subjects with an eosinophilia, optimal at 10(-8) M (P < 0.01) and the major component of the migrational response was chemokinesis. On a molar basis, IL-8 (EC50 approximately 10(10) M) was 100-fold more potent than platelet activating factor (PAF), although a comparison of the migrational responses showed that at optimal concentrations IL-8 (10(-8) M) produced only 30% maximal responses stimulated by PAF (10(-6) M). In contrast, IL-8 tested over a wide concentration range had a negligible effect on eosinophils from normal subjects. A direct correlation between the total blood eosinophil counts for all subjects and the absolute magnitude of the migrational response to IL-8 (r = 0.727, P < 0.01 at 10(-8) M), PAF (r = 0.551, P < 0.03 at 10(-6) M) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) (r = 0.689, P < 0.02 at 10(-8) M), suggested that heightened eosinophil migrational responses to inflammatory mediators may arise as a consequence of in vivo priming mechanism(s) associated with the development of an eosinophilia. In this regard, eosinophils derived from human cord blood mononuclear cells cultured in the presence of eosinophilopoietic cytokines IL-3 and IL-5, produced migrational responses to IL-8 and PAF, that were comparable with that of eosinophils from eosinophilic subjects. Furthermore, incubation of eosinophils from normal donors with IL-5 (optimal concentration 10(-9) M), significantly enhanced the subsequent migrational responses to both IL-8 (10(-8) M, P < 0.01) and PAF (10(-8) M, P < 0.05). Therefore, the increased responsiveness of eosinophils from eosinophilic subjects may reflect in vivo priming by IL-5 and this phenomenon may contribute partly to the mechanism(s) by which eosinophils preferentially accumulate at sites of allergic inflammation.

Reduced expression of hemopoietic cytokine receptors on cord blood progenitor cells in neonates at risk for atopy.

BACKGROUND: Recent findings point to an association between allergic asthma in adults and increased responsiveness of myeloid progenitor cells to certain hemopoietic growth factors. However, it is not clear at what age these changes in progenitor cells first become manifest, although increasing evidence suggests that the allergic phenotype may begin to emerge in very early life. OBJECTIVE: We sought to compare expression of hemopoietic cytokine receptors on CD34(+) progenitor cells in cord blood from normal infants ("low risk" for subsequent atopy) and infants with at least one atopic first degree relative ("at risk" for subsequent atopy). METHODS: Cord blood was obtained from 21 neonates. Nonadherent mononuclear cells were stained with mAbs directed against CD45, CD34, and the alpha-chains of the GM-CSF, IL-3, and IL-5 receptors and analyzed by flow cytometry. RESULTS: No differences in absolute CD34(+) numbers were observed between the 2 groups. However, expression of GM-CSF receptor on CD34(+) cells was reduced in the "at-risk" compared with the "low- risk" group (P =.021), although no significant differences were noted between the 2 groups with respect to IL-3 and IL-5 receptor expression. CONCLUSION: The functional sequelae of reduced GM-CSF receptor expression on CD34(+) cells remain to be determined. Nonetheless, these findings show an association between genetic risk for atopy and changes in the expression of hemopoietic cytokine receptors on cord blood progenitor cells and support the notion that the allergic phenotype may begin to evolve in the perinatal period.

Hemopoietic mechanisms in allergic airway inflammation.

The bone marrow actively participates in the production of IgER-positive inflammatory cells (eosinophils, basophils and mast cells), which are typically recruited to tissues in atopic individuals. Understanding the signalling between the tissue and the bone marrow at the molecular level may well open up new avenues of therapy for allergic inflammation.

Regulation of IL-5 and IL-5 receptor expression in the bone marrow of allergic asthmatics.

BACKGROUND: Following consistent demonstrations of the clinical relevance of fluctuations in eosinophil-basophil (Eo-B) progenitors in the blood of patients with a variety of allergic airway disorders, we have turned our attention recently to hemopoietic events occurring in the bone marrow of allergic asthmatic subjects, utilizing a model of airway allergen challenge. METHODS: Flow-cytometric analyses of CD34/45+ progenitors for coexpression of surface alpha-receptor subunits for IL-3, IL-5 and GM-CSF, as well as in situ hybridization and in situ PCR methodologies to detect mRNA for IL-5 and GM-CSF in developing Eo-B in colony and liquid culture assays were employed before and after in vivo allergen challenge. RESULTS: An early, specific upregulation of IL-5R alpha expression on CD34/45 progenitors was observed after allergen challenge, concomitant with the development of the late-phase asthmatic response. Protein and mRNA for both GM-CSF and IL-5 were expressed in a time-dependent manner ex vivo, in developing (beta 7-integrin-positive), colony-derived Eo-B after allergen challenge in vivo. Both retinoic acid and corticosteroids were able to downregulate IL-3- and IL-5-induced expression of IL-5R on cord-blood-derived as well as HL-60 cloned Eo-B progenitors. CONCLUSION: These studies indicate the critical involvement of IL-5 and IL-5R in the induction of Eo-B differentiation and eosinophilic airway inflammation in allergic asthmatics, and point to these events as potential targets for long-term therapy of atopic disease.

Identification of guinea pig eosinophil chemotactic factor of anaphylaxis as leukotriene B4 and 8(S),15(S)-dihydroxy-5,9,11,13(Z,E,Z,E)-eicosatetraenoic acid.

We have purified and characterized the guinea pig eosinophil chemotactic factor of anaphylaxis (ECF-A), an activity previously described in diffusates from sensitized lung challenged with specific Ag that appeared to selectively attract eosinophils from mixed leukocyte populations. Time course studies showed that the release of ECF-A from challenged presensitized guinea pig lung fragments closely paralleled the release of immunoreactive leukotriene B4 (iLTB4) and histamine. However, the majority of ECF-A (greater than 80%) and iLTB4 (greater than 79%) was extractable with the lipid fraction from the methanol wash of Sep-Pak-extracted diffusate, whereas histamine remained in the aqueous phase. A comparable neutrophil chemotactic activity was also found in the methanol extracts of the anaphylactic diffusates. By using a combination of HPLC and specific RIA, greater than 60% of ECF-A was attributable to LTB4. A second eosinophil chemotactic activity was also identified and coeluted (on both reverse phase and straight phase HPLC) with the synthetic standard 8(S),15(S)-dihydroxy-5,9,11,13(Z,E,Z,E)eicosatetraenoic acid (8(S),15(S)-diHETE). This was confirmed as 8(S),15(S)-diHETE by gas chromatography-mass spectrometry. Platelet-activating factor and histamine had negligible activity for guinea pig eosinophils, compared with synthetic LTB4 (p less than 0.05, 10(-9) and 10(-8) M; p less than 0.01, 10(-7) to 5 x 10(-6) M). In addition, synthetic 8(S),15(S)-diHETE had 3 times less activity than LTB4 at optimal chemotactic concentrations (10(-6) and 10(-7) M, respectively). Thus, guinea pig ECF-A appears to be largely attributable to lipoxygenase products of arachidonic acid, namely LTB4 and 8(S),15(S)-diHETE. Because guinea pig ECF-A was equally active on neutrophils (greater than 96% purity), it can no longer be considered a selective eosinophil chemoattractant.