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Summary Rhinitis and asthma are linked closely epidemiologically, as has been known for quite some time and emphasized in recent surveys (ISAAC study). Patients with seasonal allergic rhinitis without asthma symptoms can, as a group, be shown to have increased bronchial hyperresponsiveness. During the course of seasonal allergen exposure, patients with allergic rhinitis can be shown to have increased numbers of inflammatory cells (eosinophils and mast cells) in the lower airways, even without symptoms of lower airway disease. Long-standing observations from our laboratory have indicated fluctuations of eosinophil/basophil (Eo-B) progenitors in the peripheral blood in patients with seasonal allergic rhinitis, paralleling changes that are recognized in mature eosinophils and basophils in circulation. The finding of Eo-B progenitor decreases during the height of seasonal allergen exposure led us to hypothesize the concept of ‘in situ haemopoiesis’ to explain a process of systemic activation of haemopoietic mechanisms, in which communication among bone marrow peripheral blood and airway tissue compartments contributed to the allergic inflammatory process at a systemic level. Indeed, allergen challenge to the lower airways in atopic asthmatics elicits an immediate rise in Eo-B progenitors, especially in subjects with late phase responses. These original observations have been buttressed by a series of investigations demonstrating conclusively that exacerbations of asthma are attended by increases in peripheral blood Eo-B progenitors; that allergen challenge to dogs or mice with allergic airways inflammation elicits bone marrow progenitor responses, with trafficking of bone marrow cells to the airway either in pure upper or lower airway disease; and that allergen challenge to atopic asthmatics up-regulates the high affinity receptor for IL-5 on bone marrow progenitors (IL-5Rα), beginning a process of eosinophil differentiation that is probably driven by the release of a serum haemopoietic factor – as we have found in the canine model – which acts on the bone marrow and initiates a series of systemic events. [ABSTRACT FROM AUTHOR]

This article focuses on basophil mast cell and eosinophil growth and differentiation factors in human allergic disease. The synergistic action of cytokines and the involvement of tissue micro-environments, including factors from fibroblasts, endothelial cells and epithelial cells, appear to be important determinants of both the numbers of effector cells, as well as their phenotype and heterogeneity in various tissues such as the nasal mucosa, lung, gastrointestinal tract and skin. It is tempting to speculate that the genetic underpinnings of the allergic diathesis involve basic defects in epithelial, endothelial or other micro-environmental controls, for classical and lineage-specific haemopoietic, and/or pro-inflammatory cytokine production.

Haemopoietic myeloid progenitors contribute to the ongoing recruitment of pro-inflammatory cells, such as eosinophils and basophils (Eo/B), to target tissue sites in allergic diseases. It is apparent that the development of allergic inflammation is critically dependent on the ability of the bone marrow to support the proliferation, differentiation and mobilization of haemopoietic progenitors. The haemopoietic inductive microenvironment in the bone marrow is crucial for providing signals necessary for maintenance of progenitor populations at varying stages of lineage commitment and permitting these cells to circulate in the bloodstream. Progenitors demonstrate responsiveness to specific cytokines, which varies with stage of differentiation. Pro-inflammatory signals, Th2 cytokines in particular, generated following allergen challenge, can impact on haemopoietic progenitor differentiation and mobilization, leading to accelerated Eo/B production. Allergen inhalation by allergic asthmatics induces a time-dependent change in cytokine levels within the bone marrow compartment, influencing differentiation of Eo/B progenitors, as evidenced by the relationship between increased bone marrow IL-5 levels and Eo/B production. It is proposed that inhaled allergen induces trafficking of IL-5-producing T lymphocytes to the bone marrow, further promoting eosinophilopoiesis through IL-5R signalling. In this manner, Th2 lymphocyte trafficking from the airway may regulate events occurring in the bone marrow. Negative regulators of Eo/B differentiation, including Th1 cytokines, may prove to be important for restoring homeostasis. Eo/B progenitors are also altered in cord blood of infants at risk of atopy and asthma, offering a potential biomarker for, and raising the possibility that Eo/B progenitors are directly involved in the development of allergic disease. For example, changes in the expression of haemopoietic cytokine receptors on cord blood progenitor cells are associated with maternal allergic sensitization, atopic risk and its development, suggesting that haemopoietic processes underlying the allergic phenotype may begin to evolve in the perinatal period. [ABSTRACT FROM AUTHOR]

Background Haemopoietic progenitor cells ( HPC) migrate to sites of allergic inflammation where, upon stimulation with epithelial cytokines, they produce Th2 cytokines and differentiate into mature eosinophils and basophils. They also express Toll-like receptors ( TLR) involved in antimicrobial responses. Objective The objective of this study was to compare TLR expression on peripheral blood HPC and TLR-induced responses, in particular changes in epithelial cytokine receptors, in healthy and asthmatic subjects at baseline and following allergen challenge. Methods Ten healthy and 11 allergic asthmatic subjects were studied. HPC-enriched cell populations were stimulated with TLR-2, TLR-4 or TLR-9 ligands. TLR expression by circulating HPC and interleukin ( IL)-25 ( IL-17 RB), IL-33 ( ST2) and thymic stromal lymphopoietin receptor ( TSLPR) expression after TLR ligation were examined by flow cytometry at baseline and, in asthmatics, following allergen challenge. The effects of dexamethasone (Dex) on TLR-induced responses were also assessed. Results Asthmatics had significantly lower circulating HPC expressing TLR-2 and TLR-9 with a similar trend for TLR-4. TLR-4 stimulation of HPC yielded higher numbers of TSLPR+ cells in asthmatics compared with healthy subjects. A similar trend was seen for TLR-9 ligation, an effect further augmented by allergen inhalation. Allergen challenge also enhanced TLR-induced ST2 expression on HPC. Treatment with Dex in vitro increased TLR-4-induced TSLPR expression but had no effect on other epithelial cytokine receptors. Conclusions and Clinical Relevance These data demonstrate an interaction between allergen and TLR ligand exposure in asthmatics. Allergen inhalation augments the TLR-induced inflammatory response by HPC, possibly leading to increased 'in situ haemopoiesis' through up-regulation of TSLPR. These findings show that HPC may be a part of the pro-inflammatory cascade in pathogen-induced asthma exacerbation through their increased responsiveness to TLR stimulation. [ABSTRACT FROM AUTHOR]

To evaluate the effect of a single nasal allergen challenge on peripheral blood eosinophil/basophil (Eo/B) progenitor cells and induced sputum eosinophil counts in subjects with allergic rhinitis.Sixteen adults entered a sequential nasal control and allergen challenge study, outside the pollen season. Blind assessment of peripheral blood Eo/B progenitor colony forming units (CFU), induced sputum and nasal lavage cell counts was made before and 24 h after both challenges. Subjects recorded their rhinitis symptoms and nasal peak inspiratory flow, hourly at home, following both challenges.When comparing the values 24 h after the control vs. the allergen challenge, there were no significant differences in Eo/B progenitor CFU (control (mean, SD): 3.6 (1.0)/106 cells; allergen: 4.4 (1.1)/106 cells) or sputum eosinophils (control (median, inter-quartile range): 1.0 (0.3–1.7)%; allergen: 0.7 (0.0–1.3)%) despite a significant increase in the percentage (median (inter-quartile range) of eosinophils in nasal lavage (control: 0.6 (0.1–0.9)%; allergen; 1.9 (0.9–8.1)% and significant worsening of nasal peak inspiratory flow and rhinitis symptoms.Despite a significant increase in nasal symptoms and lavage eosinophil counts, a single nasal allergen challenge was not sufficient to elicit a measurable haemopoietic response in circulation, or an increase in sputum eosinophil counts. [ABSTRACT FROM AUTHOR]

Increasing levels of proinflammatory cells, including eosinophils and basophils, are seen at the site of allergen challenge in allergic disease of the airways. Mechanisms for the recruitment of these cell types could involve either specific upregulation of adhesion molecule and chemoattraction, or the initiation of proliferation and differentiation of inflammatory cell progenitors derived from the bone marrow. In this study, we demonstrate, in two systems of eosinophilic–basophilic lineage-committed granulocytes of relative immaturity, that eosinophilic differentiation in vivo implies the induction of a distinct adhesion phenotype, characterized by the upregulation of β 7 integrin and downregulation of β 1 and α 5 integrins. Moreover, the eosinophilic differentiation induced an upregulation of complement receptor type 1 and type 3, and the expression was further enhanced upon a short-course in vitro activation with ionomycin. These data indicate a sequential alteration of disparate members of the integrin family during eosinophilic–basophilic differentiation, which may attribute to specific adhesion requirements at distinct stages of cell maturation. [ABSTRACT FROM AUTHOR]

Summary Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)-4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL-4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule-like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34+ cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL-3 and rhIL-5. Cytokine mRNA profiles were analysed by the reverse transcription–polymerase chain reaction (RT–PCR) to determine transcription of IL-4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser-scanning microscopy (CLSM). We found that mRNA sequences encoding IL-4 and RANTES were expressed in freshly prepared, non-differentiated CD34+ cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2R-positive colony cells, as assessed using in situ RT–PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to β2-microglobulin) in rhIL-3- and rhIL-5-treated colony cells (comprising > 90% eosinophil-like cells) up to day 28. Immunoreactivity for IL-4 and RANTES co-localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation. [ABSTRACT FROM AUTHOR]

Background: The sequence of adhesion-molecule expression during eosinophil differentiation remains unclear. Methods: We analyzed the surface expression of α4, β1, and β7 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 β7 integrin expression coincident with a marked upregulation of CD35 and attenuation of CD33 and β1 integrin expression. In addition, adhesion of induced HL-60 clone 15 cells to fibronectin was attenuated by a β7 integrin antibody. Conclusions: Our data show that protein synthesis-dependent upregulation of the functional β7 integrin occurs under conditions when α4 and β1 integrins are fully expressed, indicating a sequential appearance of specific adhesion molecules on differentiating eosinophil progenitors. [ABSTRACT FROM AUTHOR]

Circulating eosinophils, basophils and eosinophil/basophil (Eo/B) progenitors were examined in 12 patients at the time of an exacerbation of asthma accompanied by sputum eosinophilia and after resolution of the exacerbation with inhaled corticosteroid treatment. Differential counts were performed and peripheral blood non-adherent mononuclear cells were cultured for 14 days in methyl-cellulose to determine the number of Eo/B and granulocyte-macrophage (GM) colonies without knowledge of the clinical conditions or findings. With resolution of the asthma exacerbation on beclomethasone therapy, there were significant falls in circulating eosinophils, basophils and Eo/B colonies whereas GM colonies were unchanged. To elucidate whether the observed changes could be due to systemic absorption or local action of inhaled corticosteroid. seven subjects with allergic rhinitis and no current evidence of lower airway inflammation (no symptoms of asthma and normal methacholine airway responsiveness) received 14 days' treatment with the same dose of inhaled beclomethasone or of placebo in a double-blind randomized cross-over study. No significant changes in airway function or in circulating cell counts were observed. The results suggest reduced production of eosinophils and basophils after the resolution of an exacerbation of asthma. This effect may be due to reduced levels of airway-derived eosinophil basophil growth and differentiation factors. [ABSTRACT FROM AUTHOR]