Lorna J. Wood

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.

Interaction between haemopoietic regulation and airway inflammation

Asthma is characterized by reversible airway narrowing, by airway hyperresponsiveness, and by airway inflammation. Inhaled allergens are the most important of the stimuli known to cause asthma. Methods for studying inhaled allergen in the laboratory have been well standardized and extensively used for the investigation of the pathophysiology and the pharmacological modulation of allergen‐induced airway responses. Allergen inhalation by a sensitized subject results in an early asthmatic response, and, in the majority of subjects, a late asthmatic response and airway hyperresponsiveness. The late response and airway hyperresponsiveness are associated with increases in airway eosinophils and metachromatic cells. Allergen‐induced airway inflammation in dogs (predominantly neutrophilic) is associated with increased granulocyte‐macrophage progenitors in bone marrow, which is dependent on the effects of a circulating serum factor stimulating the bone marrow. The newly formed cells traffic to the airways. These increases in granulocyte‐macrophage progenitors are blocked by inhaled corticosteroids. In human subjects, allergen‐induced eosinophilic inflammation is associated with increases in Eo/B progenitors, mediated through up‐regulation if the IL‐5 receptor on progenitors and increases responsiveness to IL‐5. Inhaled corticosteroids also attenuate all allergen‐induced physiological responses and airway inflammation, an effect possibly mediated, in part, through inhibition of eosinophil and basophil maturation or release from the bone marrow.

Bone marrow progenitors in allergic airways diseases: studies in canine and human models.

In a canine model of Ascaris suum-inducible bronchial hyperresponsiveness, we previously demonstrated that bone marrow-derived myeloid progenitors rise within 24 h of allergen inhalation; this effect is abolished by pretreatment with inhaled budesonide. We now report that this allergen-induced bone marrow response is observable in human asthmatics, and involves increases in both neutrophil-macrophage and eosinophil-basophil progenitors, within 6 h of allergen inhalation, as measured either by hematopoietic colony assays or by flow cytometric analyses of CD34+, IL-3R alpha+, and/or IL-5-responsive cell populations. In dogs, but not in humans, a transferrable serum hematopoietic activity accounts for the marrow response to inhaled allergen. These findings suggest that allergen-induced increases in bone marrow progenitor formation depend either on a serum hematopoietic factor(s) released after allergen challenge, or upon constitutive marrow upregulation of specific progenitors in allergic airway disease. Further studies to characterize the serum hematopoietic factor(s) and to determine the nature of any atopy-related progenitor profile are in progress.

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 α–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 α 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 (β 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.

INTERLEUKIN-5 AND ALLERGIC INFLAMMATION

Since the identification of human eosinophil differentiation factor as interleukin (IL)-5 in the mid-1980s [1], it has become increasingly clear that, in contrast with other cytokines thought to be involved in allergic inflammation, such as granulocyte macrophage-colony stimulating factor (GM-CSF) or IL-3, the biological activity of IL-5 is very specifically focused on the development [2], differentiation [3], recruitment [4], activation [5] and survival [6] of a single cell type, the eosinophil. This has stimulated great interest, as increased eosinophil production and recruitment into the upper or lower airways appears to be essential for the clinical manifestations of allergen inhalation to these sites. The specificity of IL-5 has also raised the possibility that blocking its activity may be a useful therapy for allergic diseases. Allergen inhalation challenge has been a very useful clinical model to study the pathophysiology of allergen inhalation into the upper or lower airways. Allergen inhalation into the lower airways by allergic subjects results in the development of acute bronchoconstriction mainly mediated by cysteinyl leucotriene and histamine released from mast cells [7] (the early response), and in more than half of subjects, the development of late bronchoconstrictor responses beginning 2–4 h after inhalation, which can persist for more that 24 h [8]. These bronchoconstrictor responses are again mainly mediated by cysteinyl leucotriene and histamine [7], likely released from inflammatory cells newly recruited into the airways. The cells recruited during the late response are eosinophils [9] and metachromatic cells [10]. The increased number of eosinophils can persist for up to 1 week after a single allergen inhalation, and this is temporally associated with the persistence of allergen-induced airway hyperresponsiveness. Allergen inhalation increases the production of IL-5 in the airways as measured in bronchoalveolar lavage (BAL) cells [11], and induced sputum [12]. Interestingly eosinophils are one of the cells responsible for this increase [11]. Also, as reported in this issue of the Journal by Hallde ́n et al. [13], allergen inhalation increases the number of peripheral blood eosinophils and lymphocytes containing intracellular IL-5. The relatively high number of lymphocytes positive for IL-5 suggested to the authors that not only CD4 þ TH2 cells contained IL-5, but also other lymphocytes including CD8 and CD4CD8 cells. Eosinophils are produced in the bone marrow from eosinophil/basophil colony forming units (Eo/B-CFU). These progenitors, in common with other marrow progenitors express the cell surface marker CD34. An important aspect of allergic inflammatory responses is the induction of increases in inflammatory cell progenitors, which contribute to disease through the continued production of inflammatory effector cells. Higher numbers of both circulating Eo/B-CFUs and CD34 þ haemopoietic progenitor cells are demonstrable in the blood of atopic subjects compared with normals [14]. In addition, the numbers of Eo/B-CFU in the circulation of asthmatic subjects at the time of an acute exacerbation is significantly higher than those measured after resolution of the exacerbation [15]. In vivo studies in atopic subjects have shown that there are fluctuating numbers ofcirculating Eo/B-CFU during seasonal exposure to allergen [16] and significantly higher numbers 24 h following allergen inhalation [17]. Increases in bone marrow Eo/B-CFU have also been demonstrated in allergen asthmatic subjects after allergen inhalation [18]. In this study, subjects who developed early and late (dual) asthmatic responses and who had significantly greater numbers of eosinophils in the airways, had significantly greater numbers of bone marrow Eo/B-CFU when the cells were incubated with a suboptimal concentration of IL-5, when compared with subjects with isolated early asthmatic responses, with fewer airway eosinophils. This indicates that after allergen challenge, the bone marrow of the dual responders is more responsive to IL-5, which may reflect either a specific induction of a population of more committed eosinophil/basophil progenitors, or an upregulation of the IL-5 receptor on the surface of these cells. This issue was resolved by Sehmi et al. [19], who confirmed an increase in the proportion of CD34 þ cells expressing the alpha subunit of the IL-5 receptor, following allergen, in dual but not isolated early responders. These results may indicate that the responsiveness of the bone marrow to IL-5 after allergen is a determinant of the magnitude of the eosinophilic responses to inhaled allergen, and of the degree of the subsequent physiological abnormalities. Other studies have supported an important role for IL-5 in the development of allergen-induced airway inflammation. In studies of transgenic mice which overexpress IL-5, there is marked circulating and tissue eosinophilia [20], while in animal models, antibodies to IL-5 can block allergeninduced local and systemic eosinophilia and airway hyperresponsiveness for periods lasting up to 6 months. In murine [21], guinea-pig [22] and primate models [23], treatment Clinical and Experimental Allergy, 1999, Volume 29, pages 573–575

Bone marrow contribution to eosinophilic inflammation

Allergen-induced bone marrow responses are observable in human allergic asthmatics, involving specific increases in eosinophil-basophil progenitors (Eo/B-CFU), measured either by hemopoietic assays or by flow cytometric analyses of CD34-positive, IL-3R alpha-positive, and/or IL-5-responsive cell populations. The results are consistent with the upregulation of an IL-5-sensitive population of progenitors in allergen-induced late phase asthmatic responses. Studies in vitro on the phenotype of developing eosinophils and basophils suggest that the early acquisition of IL-5R alpha, as well as the capacity to produce cytokines such as GM-CSF and IL-5, are features of the differentiation process. These observations are consistent with findings in animal models, indicating that allergen-induced increases in bone marrow progenitor formation depend on hemopoietic factor(s) released post-allergen. The possibility that there is constitutive marrow upregulation of eosinophilopoiesis in allergic airways disease is also an area for future investigation.