IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils.

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines, such as IL-5, IL-3, and GM-CSF, are typically present in atopic diseases, including allergic asthma. As a result of the functional redundancy of these three cytokines on eosinophils and the loss of IL-5R on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these three cytokines signal through a common ß-chain receptor but yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce the production of proteins, such as semaphorin-7A, without affecting mRNA levels suggests a unique influence of IL-3 on translation. The purpose of this study was to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared with IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF, or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein S6 (RPS6) and the upstream kinase 90-kDa ribosomal S6 kinase (p90S6K). Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3-enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared with circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations identify IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions.

Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/CCl2, regulate airway inflammation and remodeling in allergic asthma.

Although alveolar macrophages (AMs) from patients with asthma are known to be functionally different from those of healthy individuals, the mechanism by which this transformation occurs has not been fully elucidated in asthma. The goal of this study was to define the mechanisms that control AM phenotypic and functional transformation in response to acute allergic airway inflammation. The phenotype and functional characteristics of AMs obtained from human subjects with asthma after subsegmental bronchoprovocation with allergen was studied. Using macrophage-depleted mice, the role and trafficking of AM populations was determined using an acute allergic lung inflammation model. We observed that depletion of AMs in a mouse allergic asthma model attenuates Th2-type allergic lung inflammation and its consequent airway remodeling. In both human and mouse, endobronchial challenge with allergen induced a marked increase in monocyte chemotactic proteins (MCPs) in bronchoalveolar fluid, concomitant with the rapid appearance of a monocyte-derived population of AMs. Furthermore, airway allergen challenge of allergic subjects with mild asthma skewed the pattern of AM gene expression toward high levels of the receptor for MCP1 (CCR2/MCP1R) and expression of M2 phenotypic proteins, whereas most proinflammatory genes were highly suppressed. CCL2/MCP-1 gene expression was prominent in bronchial epithelial cells in a mouse allergic asthma model, and in vitro studies indicate that bronchial epithelial cells produced abundant MCP-1 in response to house dust mite allergen. Thus, our study indicates that bronchial allergen challenge induces the recruitment of blood monocytes along a chemotactic gradient generated by allergen-exposed bronchial epithelial cells.

Autotaxin production of lysophosphatidic acid mediates allergic asthmatic inflammation.

RATIONALE: Bioactive lipid mediators, derived from membrane lipid precursors, are released into the airway and airspace where they bind high-affinity cognate receptors and may mediate asthma pathogenesis. Lysophosphatidic acid (LPA), a bioactive lipid mediator generated by the enzymatic activity of extracellular autotaxin (ATX), binds LPA receptors, resulting in an array of biological actions on cell proliferation, migration, survival, differentiation, and motility, and therefore could mediate asthma pathogenesis. OBJECTIVES: To define a role for the ATX-LPA pathway in human asthma pathogenesis and a murine model of allergic lung inflammation. METHODS: We investigated the profiles of LPA molecular species and the level of ATX exoenzyme in bronchoalveolar lavage fluids of human patients with asthma subjected to subsegmental bronchoprovocation with allergen. We interrogated the role of the ATX-LPA pathway in allergic lung inflammation using a murine allergic asthma model in ATX-LPA pathway-specific genetically modified mice. MEASUREMENTS AND MAIN RESULTS: Subsegmental bronchoprovocation with allergen in patients with mild asthma resulted in a remarkable increase in bronchoalveolar lavage fluid levels of LPA enriched in polyunsaturated 22:5 and 22:6 fatty acids in association with increased concentrations of ATX protein. Using a triple-allergen mouse asthma model, we showed that ATX-overexpressing transgenic mice had a more severe asthmatic phenotype, whereas blocking ATX activity and knockdown of the LPA2 receptor in mice produced a marked attenuation of Th2 cytokines and allergic lung inflammation. CONCLUSIONS: The ATX-LPA pathway plays a critical role in the pathogenesis of asthma. These preclinical data indicate that targeting the ATX-LPA pathway could be an effective antiasthma treatment strategy.

Safety of and cellular response to segmental bronchoprovocation in allergic asthma.

RATIONALE: Despite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration. METHODS: We performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject's response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms. RESULTS: Among subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×10(4) per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens. CONCLUSIONS: In mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.

Potent synergistic effect of IL-3 and TNF on matrix metalloproteinase 9 generation by human eosinophils.

TNF (designated as TNF-α under previous nomenclature) is the preeminent activator of MMP-9 generation from a variety of cells including eosinophils. We have previously established that TNF strongly synergizes with IFN-γ and IL-4 for eosinophil synthesis of Th1- and Th2-type chemokines respectively. Thus, we sought to determine if TNF-induced synthesis of MMP-9 would be enhanced by the presence of Th1, Th2, or the eosinophil-associated common beta chain (ßc) cytokines. Human blood eosinophils were cultured with TNF alone or in combination with either IFN-γ, IL-4, IL-3, IL-5, or GM-CSF. Concentrations and activities of MMP-9 in eosinophil culture supernates were measured by ELISA and gelatin zymography, mRNA transcription and stabilization by quantitative real-time PCR, and signaling events by immunoblotting and intracellular flow cytometric analysis. Individually, TNF, GM-CSF, or IL-3, but not IL-4 or IFN-γ, induced relatively small (<0.2 ng/ml) but statistically significant quantities of MMP-9. Remarkable synergistic synthesis of MMP-9 (ng/ml levels) occurred in response to TNF plus IL-3, GM-CSF or IL-5, in the order of IL-3>GM-CSF>IL-5. Zymography revealed that eosinophils release MMP-9 in its pro-form. Eosinophil stimulation with the combination of IL-3 plus TNF led to increased steady-state levels of MMP-9 mRNA, prolonged mRNA stabilization, and enhanced activation of ERK1/2 phosphorylation. Inhibition of NF-κB, MEK kinase, or p38 MAP kinase, but not JNK signaling pathways, diminished IL-3/TNF-induced MMP-9 mRNA and protein production. Thus, the synergistic regulation of eosinophil MMP-9 by IL-3 plus TNF likely involves cooperative interaction of multiple transcription factors downstream from ERK, p38, and NF-κB activation as well as post-transcriptional regulation of MMP-9 mRNA stabilization. Our data indicate that within microenvironments rich in ßc-family cytokines and TNF, eosinophils are an important source of proMMP-9 and highlight a previously unrecognized role for synergistic interaction between TNF and ßc-family cytokines, particularly IL-3, for proMMP-9 synthesis.

Potential contribution of IL-7 to allergen-induced eosinophilic airway inflammation in asthma.

The primary function of IL-7 is to promote maturation and survival of T cells. Through microarray expression analysis, we previously observed that human blood eosinophils express mRNA for IL-7R alpha (CD127) and its common gamma chain (CD132). The purpose of this study was to determine whether eosinophils have functional IL-7 receptors and to assess the potential contribution of IL-7 to eosinophilic airway inflammation by evaluating its presence in bronchoalveolar lavage (BAL) fluid of subjects with atopic asthma before and after segmental bronchoprovocation with allergen. Immunoblot analysis revealed that CD127 is present in highly purified human blood eosinophils. Furthermore, eosinophils responded to IL-7 with phosphorylation of STAT5, up-regulation of the activation marker CD69, and prolonged survival. Neutralization of GM-CSF but not IL-5 significantly blunted these functional responses, suggesting that IL-7 mediates its effects by promoting eosinophil release of autologous GM-CSF. Notably, the suppressive effect of anti-GM-CSF on STAT5 phosphorylation occurred within 10 min of eosinophil exposure to IL-7. Thus, IL-7 likely activates eosinophil release of preformed rather than newly synthesized GM-CSF. The biological relevance of IL-7 to eosinophilia in vivo was implicated in a study of airway allergen challenge in patients with allergic asthma. IL-7 concentrations in BAL fluid increased significantly 48 h after segmental allergen challenge and were highly correlated with BAL eosinophils (r = 0.7, p < 0.001). In conclusion, the airway response to allergen is associated with the generation of IL-7, which may contribute to airway inflammation by promoting enhanced eosinophil activation and survival. Activation of eosinophils is a novel function for IL-7.

Up-regulation and activation of eosinophil integrins in blood and airway after segmental lung antigen challenge.

We hypothesized that there are clinically relevant differences in eosinophil integrin expression and activation in patients with asthma. To evaluate this, surface densities and activation states of integrins on eosinophils in blood and bronchoalveolar lavage (BAL) of 19 asthmatic subjects were studied before and 48 h after segmental Ag challenge. At 48 h, there was increased expression of alpha(D) and the N29 epitope of activated beta(1) integrins on blood eosinophils and of alpha(M), beta(2), and the mAb24 epitope of activated beta(2) integrins on airway eosinophils. Changes correlated with the late-phase fall in forced expiratory volume in 1 s (FEV(1)) after whole-lung inhalation of the Ag that was subsequently used in segmental challenge and were greater in subjects defined as dual responders. Increased surface densities of alpha(M) and beta(2) and activation of beta(2) on airway eosinophils correlated with the concentration of IL-5 in BAL fluid. Activation of beta(1) and beta(2) on airway eosinophils correlated with eosinophil percentage in BAL. Thus, eosinophils respond to an allergic stimulus by activation of integrins in a sequence that likely promotes eosinophilic inflammation of the airway. Before challenge, beta(1) and beta(2) integrins of circulating eosinophils are in low-activation conformations and alpha(D)beta(2) surface expression is low. After Ag challenge, circulating eosinophils adopt a phenotype with activated beta(1) integrins and up-regulated alpha(D)beta(2), changes that are predicted to facilitate eosinophil arrest on VCAM-1 in bronchial vessels. Finally, eosinophils present in IL-5-rich airway fluid have a hyperadhesive phenotype associated with increased surface expression of alpha(M)beta(2) and activation of beta(2) integrins.

Generation of Th1 and Th2 chemokines by human eosinophils: evidence for a critical role of TNF-alpha.

Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their microenvironment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-alpha plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-gamma-inducible protein-10), were expressed after stimulation with TNF-alpha plus IFN-gamma. Addition of TNF-alpha appeared to be essential for IFN-gamma-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Th2-type chemokines. Inhibition of NF-kappaB completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-kappaB, STAT6, and STAT1 was induced in eosinophils by TNF-alpha, IL-4, and IFN-gamma, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-alpha plus IL-4 or TNF-alpha plus IFN-gamma. Thus, independently activated signaling cascades appear to lead to activation of NF-kappaB, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-alpha is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils.

Circadian changes in granulocyte-macrophage colony-stimulating factor message in circulating eosinophils.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates eosinophil recruitment, activation, and survival, is expressed by activated eosinophils. Although eosinophil recruitment and enhanced survival have been associated with nocturnal asthma (NA), the contribution of GM-CSF to NA is unknown. OBJECTIVE: To determine whether circulating eosinophil GM-CSF expression correlates with the symptoms of NA. METHODS: The GM-CSF messenger RNA (mRNA) expression at 4 PM and 4 AM was determined by reverse-transcriptase polymerase chain reaction with Southern blot analysis in subjects with and without NA and in controls. RESULTS: A total of 142 asthma subjects were screened for nocturnal asthma with 1-week home peak expiratory flow rate (PEFR) monitoring. Eleven subjects had NA (>20% diurnal variation in PEFR on 4 of 7 days), and 6 met the criteria for non-NA (<10% diurnal variation in PEFR on 7 of 7 days); 8 controls were studied. In subjects with NA, GM-CSF mRNA expression in circulating eosinophils increased 3-fold at 4 AM compared with 4 PM. Diurnal changes in GM-CSF mRNA expression were not detected in the non-NA and control groups. CONCLUSIONS: Day-night variation in eosinophil GM-CSF expression is associated with circadian variation in airway function in asthma, a key manifestation of asthma severity.

Enhanced generation of helper T type 1 and 2 chemokines in allergen-induced asthma.

Allergen-induced asthma is characterized by airway eosinophilia and recruitment of helper T (Th) Type 2 lymphocytes. We hypothesized that lymphocyte-associated chemokines contribute to allergen-induced airway inflammation. Sixteen subjects with asthma were phenotyped according to their response to inhaled antigen as single- or dual-phase responders, and then underwent bronchoscopy and segmental allergen bronchoprovocation. Bronchoalveolar lavage fluids were obtained before and 48 hours after segmental challenge with allergen to determine the cellular response and patterns of Th1 and Th2 chemokines and cytokines. Airway cells, cytokines, and lymphocyte-associated chemokines increased after segmental challenge. Th2 chemokines (thymus and activation-regulated chemokine, macrophage-derived chemokine) correlated with airway eosinophils and concentrations of interleukin-5 and -13. In contrast, airway lymphocytes correlated with both Th2 and Th1 (monokine-induced by IFN-gamma, IFN-gamma-inducible protein-10) chemokines. Notably, when subjects were analyzed according to the presence of a late-phase response, concentrations of both types of lymphocyte-associated chemokines were significantly greater in subjects with a dual-response phenotype. Our findings suggest that both Th2 and Th1 chemokines may be involved in allergen-induced airway inflammation. However, asthma subjects with a dual-responder phenotype have greater generation of chemokines that may lead to enhanced airway inflammation and obstruction after allergen exposure.

Ets-1 regulates TNF-alpha-induced matrix metalloproteinase-9 and tenascin expression in primary bronchial fibroblasts.

Increased subepithelial deposition of extracellular matrix proteins is a key feature in bronchial asthma. Matrix metalloproteinase-9 (MMP-9) is a proteolytic enzyme that degrades the extracellular matrix. Tenascin is an extracellular matrix glycoprotein that is abundant in thickened asthmatic subbasement membrane. The expression of MMP-9 and tenascin reflects disease activity in asthma and airway remodeling. The molecular mechanisms regulating the expression of these proteins remain unknown. Both MMP-9 and tenascin promoters contain an Ets binding site, suggesting control by Ets-1. Thus, we hypothesized that Ets-1 expression is increased in asthma and that it contributed to enhanced MMP-9 and tenascin expression. To test this hypothesis, we determined the expression of Ets-1 in bronchial biopsies obtained from asthmatic subjects and determined the expression of Ets-1, MMP-9, and tenascin by bronchial fibroblasts activated ex vivo. We observed that nuclear extracts from TNF-alpha-activated fibroblasts showed increased Ets-binding activity. In addition, TNF-alpha-activated fibroblasts had increased expression of Ets-1 mRNA and protein, which preceded an increase in MMP-9 and tenascin mRNA. Furthermore, treatment of fibroblasts with Ets-1 antisense oligonucleotides down-regulated TNF-alpha-induced Ets-1, MMP-9, and, to a lesser extent, tenascin protein expression or activity. Taken together, these data demonstrate that TNF-alpha increases MMP-9 and tenascin expression in bronchial fibroblasts via the transcription factor Ets-1, and suggest a role for Ets-1 in airway remodeling in asthma.

Expression of interleukin-5- and granulocyte macrophage-colony-stimulating factor-responsive genes in blood and airway eosinophils.

Because interleukin (IL)-5 family cytokines are critical regulators of eosinophil development, recruitment, and activation, this study was initiated to identify proteins induced by these cytokines in eosinophils. Using oligonucleotide microarrays, numerous transcripts were identified as responsive to both IL-5 and granulocyte macrophage-colony-stimulating factor (GM-CSF), but no transcripts were markedly affected by one cytokine and not the other. Expression of several gene products were seen to be increased following in vitro stimulation of human blood eosinophils, including the IL-3 receptor alpha subunit, lymphotoxin beta, Pim-1, and cyclin D3. Given that eosinophils recovered from the bronchoalveolar lavage fluid of allergic patients after antigen challenge are exposed to IL-5 or GM-CSF in the airway prior to isolation, the hypothesis was tested that selected IL-5- and GM-CSF-responsive genes are upregulated in airway eosinophils relative to the expression in blood cells. Airway eosinophils displayed greater cell surface expression of the IL-3 receptor alpha subunit, CD44, CD25, and CD66e, suggesting that these proteins may be markers of eosinophil activation by IL-5 family cytokines in airway eosinophils. Other genes that were induced by both IL-5 and GM-CSF showed protein expression at similar or decreased levels in airway eosinophils relative to their circulating counterparts (i.e., lymphotoxin beta and CD24). These studies have identified several transcriptional targets of IL-5 and GM-CSF in human eosinophils and suggest that a number of protein products are critical to the responsiveness of airway eosinophils.

Increased thrombin activity after allergen challenge: a potential link to airway remodeling?

In addition to its central role in hemostasis, thrombin may play a role in inflammation and remodeling. To investigate the contribution of thrombin to allergic airway inflammation in asthma, we used an enzymatic assay to determine thrombin activity in bronchoalveolar lavage fluid obtained from 19 subjects with atopic asthma before (Day 0) and 48 hours after (Day 2) segmental bronchoprovocation with antigen. Thrombin activity increased from 0 (0, 2.9) on Day 1 to 41.1 (0.3, 75.6) U x 10(-3)/ml on Day 2 (p = 0.002) and correlated with total protein levels in lavage fluid on Day 2 (r = 0.885, p < 0.001). After antigen challenge, thrombin activity also showed significant correlations with interleukin-5 (r = 0.66, p = 0.002), transforming growth factor beta1 (r = 0.70, p < 0.001), fibronectin (r = 0.85, p < 0.001) and tissue factor (r = 0.55, p = 0.03) levels in lavage fluid. Furthermore, Day 2, but not Day 0 lavage fluid, induced proliferation of human airway fibroblasts. This mitogenic effect was significantly reduced with hirudin, a specific thrombin inhibitor. Taken together, our findings suggest that allergen-driven airway inflammation in asthma is associated with enhanced potential for fibroblast proliferation that is related, at least in part, to increased thrombin activity. We propose that enhanced thrombin activity provides a potential link between allergic inflammation and initiation of airway remodeling.

Chemokine receptor expression on human eosinophils from peripheral blood and bronchoalveolar lavage fluid after segmental antigen challenge.

BACKGROUND: The recruitment of circulating eosinophils to the lung is a characteristic feature of allergic airway inflammation. Chemokine receptors likely play a role in this complex process. However, reports of chemokine receptor expression on human eosinophils are conflicting. OBJECTIVE: The aim of this study was to determine whether the chemokine receptor profile of human eosinophils change when these cells are recruited to the airway after an antigen challenge and development of an allergic inflammatory response. METHODS: Blood and bronchoalveolar lavage (BAL) cells were obtained from 13 allergic subjects 48 hours after segmental bronchoprovocation with antigen. The CC chemokine receptor (CCR) 1 to 7, 9, and CXC chemokine receptor (CXCR) 1 to 4 were determined by flow cytometric analysis of whole blood and unseparated BAL cells. RESULTS: Compared with their circulating counterparts, airway eosinophils had decreased CCR3 and increased CCR4, CCR9, and CXCR3 expression on their cell surface. Furthermore, expression of CCR3, CCR4, and CXCR3 was significantly correlated with the percentage of eosinophils in BAL fluid at 48 hours. Eosinophils also expressed CXCR4, but this receptor did not change after antigen-induced recruitment to the airway. In contrast, the expression of CCR1, CCR2, CCR5, CCR6, CCR7, CXCR1, and CXCR2 remained undetectable on either blood or BAL eosinophils. CONCLUSIONS: Our data suggest that recruitment of eosinophils to the airway is associated with a modulation of their chemokine receptor profiles. These changes in chemokine receptors could be involved in determining eosinophil function and antigen-induced airway inflammation.

Circadian variation of sputum inflammatory cells in mild asthma.

BACKGROUND: Asthma, like many conditions, demonstrates a circadian rhythm with a worsening of lung function in the early morning hours compared with in the late afternoon. OBJECTIVE: Because eosinophilic airway inflammation is a proposed mechanism for worsening asthma, we characterized circadian variation in airway eosinophils and determined its relationship to variability in airway function. METHODS: Pulmonary function testing, sputum induction, and phlebotomy were performed at 7 am and 4 pm in 11 allergic subjects with mild asthma. Sputum was analyzed for cell viability, differential, and eosinophil-derived neurotoxin levels. IL-5 levels in serum were measured by means of ELISA. RESULTS: Subjects had a significant decrease in FEV(1) (median [interquartile range] = 80% [70%-86%] vs 85% [82%-94%], P =.009) and a greater beta-agonist reversibility (median [interquartile range] = 13% [7%-32%] vs 8% [5%-14%], P =.024) in the early morning compared with in the late afternoon. Sputum analysis showed an increase in early morning total sputum leukocytes (median [interquartile range] = 4.3 x 10(6) [2.3 x 10(6) to 6.1 x 10(6)] vs 2.6 x 10(6) [1.7 x 10(6) to 3.6 x 10(6)], P =.044) and eosinophils (median [interquartile range] = 7.0 x 10(4) [2.7 x 10(4) to 18.7 x 10(4)] vs 3.6 x 10(4) [1.0 x 10(4) to 8.2 x 10(4)], P =.024). Furthermore, sputum eosinophils correlated with beta-agonist reversibility (R (s) = 0.665, P =.019). Finally, levels of IL-5 in serum and eosinophil-derived neurotoxin in sputum were significantly increased at 7 am. CONCLUSION: These data suggest that circadian variability in pulmonary function in asthma could be related to changes in airway eosinophil recruitment and activation.

Pathogenesis of asthma.

There is now strong evidence that airway inflammation is a predominant underlying problem in patients with asthma, and it has been suggested that ongoing inflammation may lead to airway injury and remodeling. There is also recent evidence that longstanding asthma could be associated with loss of elastic recoil, which can enhance airway obstruction and worsen asthma control [82,83]. Therefore, the use of anti-inflammatory therapy has been advocated in all guidelines, including the National Asthma Education and Prevention Program (NAEPP) Expert Panel Report [84] and its recent update [85] that recommended inhaled steroids as a first mode of therapy for patients with mild, moderate, or severe, persistent asthma. There is preliminary evidence that early institution of anti-inflammatory therapy might lead to disease modification and limit the progression of subepithelial fibrosis and airway remodeling. The pathogenesis of asthma clearly involves many cells and mediators, although the contribution of each individual factor is probably different from patient to patient depending on the setting and stimulus. Although currently available therapies are highly effective in controlling asthma symptoms and limiting exacerbations in the majority of patients, there is still a subset of patients that proceed to develop severe asthma with decreased lung function, lack of responsiveness to therapy, or frequent exacerbations. It is hoped that rapid progress in the area of asthma genetics and pharmacogenetics will yield a more precise and patient-specific understanding of asthma pathogenesis and allow practitioners to prescribe therapies that are designed for a particular patient or exacerbation. That will undoubtedly help to improve the care of asthma, limit its morbidity, and reduce the side effect of medications.

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge.

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: II. IL-5 down-modulates its receptor via a proteinase-mediated process.

In the accompanying study, we demonstrated that following Ag challenge, membrane (m)IL-5Ralpha expression is attenuated on bronchoalveolar lavage eosinophils, soluble (s)IL-5Ralpha is detectable in BAL fluid in the absence of increased steady state levels of sIL-5Ralpha mRNA, and BAL eosinophils become refractory to IL-5 for ex vivo degranulation. We hypothesized that IL-5 regulates its receptor through proteolytic release of mIL-5Ralpha, which in turn contributes to the presence of sIL-5Ralpha. Purified human peripheral blood eosinophils were incubated with IL-5 under various conditions and in the presence of different pharmacological agents. A dose-dependent decrease in mIL-5Ralpha was accompanied by an increase in sIL-5Ralpha in the supernatant. IL-5 had no ligand-specific effect on mIL-5Ralpha or sIL-5Ralpha mRNA levels. The matrix metalloproteinase-specific inhibitors BB-94 and GM6001 and tissue inhibitor of metalloproteinase-3 partially inhibited IL-5-mediated loss of mIL-5Ralpha, suggesting that sIL-5Ralpha may be produced by proteolytic cleavage of mIL-5Ralpha. IL-5 transiently reduced surface expression of beta-chain, but had no effect on the expression of GM-CSFRalpha. Pretreatment of eosinophils with a dose of IL-5 that down-modulated mIL-5Ralpha rendered these cells unable to degranulate in response to further IL-5 stimulation, but they were fully responsive to GM-CSF. These findings suggest that IL-5-activated eosinophils may lose mIL-5Ralpha and release sIL-5Ralpha in vivo, which may limit IL-5-dependent inflammatory events in diseases such as asthma.