Background: The sequence of adhesion-molecule expression during eosinophil differentiation remain... more Background: The sequence of adhesion-molecule expression during eosinophil differentiation remains unclear. Methods: We analyzed the surface expression of a 4 , b 1 , and b 7 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 signi®cant upregulation of b 7 integrin expression coincident with a marked upregulation of CD35 and attenuation of CD33 and b 1 integrin expression. In addition, adhesion of induced HL-60 clone 15 cells to ®bronectin was attenuated by a b 7 integrin antibody. Conclusions: Our data show that protein synthesis-dependent upregulation of the functional b 7 integrin occurs under conditions when a 4 and b 1 integrins are fully expressed, indicating a sequential appearance of speci®c adhesion molecules on differentiating eosinophil progenitors.
The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting throug... more The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting through a receptor (cysLT1-R) which can be targeted in rhinitis and asthma. We investigated the effects of cysLT1-R antagonism in experimental allergic rhinitis, focusing on bone marrow eosinophil progenitor responses. BALB ⁄c mice were sensitized, then given daily intranasal ovalbumin for 2 weeks, with montelukast sodium (5 mg ⁄kg or 2AE5 mg ⁄kg) or placebo by gavage. Bone marrow eosinophil ⁄basophil colonies were enumerated, and colony cells were morphologically assessed as indices of eosinophil differentiation and maturation. Montelukast treatment resulted in a significant decrease of eosinophils in the nasal mucosa, and in either bone marrow interleukin (IL)-5-, but not IL-3-, or granulocyte-macrophage colony-stimulating factor-responsive eosinophil ⁄basophil colony-forming units, and IL-5-stimulated eosinophil maturation. These results indicate that cysLT1-R antagonism in vivo limits both IL-5-responsive eosinophilopoiesis, acting at several stages of eosinophil differentiation and maturation. The anti-allergic effects of cysLT1-R antagonists are consistent with the concept that cysLTs and IL-5 act together in the recruitment of eosinophils and eosinophil progenitors from the marrow during upper airway allergic inflammation.
Background: Allergic asthma is an inflammatory airway disease in which Th2 cytokines play an impo... more Background: Allergic asthma is an inflammatory airway disease in which Th2 cytokines play an important role. Epithelial-derived interleukin (IL)-25 has been suggested to be important in the maintenance of Th2-type responses. The effects of IL-25 are mediated by the IL-25 receptor, composed of two subunits, IL-17RA and IL-17RB. Eosinophils are effector cells in allergic asthma. The role of IL-25 in eosinophil function is unknown. This study examined the expression of IL-25 and its receptor on eosinophils in allergic asthmatics compared to atopic nonasthmatics and normal controls. Methods: The expression of IL-25, IL-17RA and IL-17RB on eosinophils, and levels of plasma IL-25 were measured in 14 normal control subjects, 15 atopic nonasthmatics and 14 mild allergic asthmatics. Results: The expression of IL-17RB on eosinophils was significantly higher in allergic asthmatics (43.08%, range 33.96-59.98%) than in atopic nonasthmatics (11.98%, range 6.33-27.11%, p = 0.002) and normal contro...
Adhesion Molecule Expression on Human Cord Blood Progenitors During Early Eosinophilic Commitment... more Adhesion Molecule Expression on Human Cord Blood Progenitors During Early Eosinophilic Commitment: Upregulation of b 7 Integrins. Scand J Immunol 2002;56:161±167
RATIONALE: Increased vascularity is an important element of airway remodeling in patients with br... more RATIONALE: Increased vascularity is an important element of airway remodeling in patients with bronchial asthma. However, the molecular mechanism involved in this process remains unclear. In order to clarify it, in vitro responses of human lung microvascular endothelial cell (HMVEC-L) to several cytokines were examined. METHODS: HMVEC-L was incubated with a combination of TNFalpha and IL-4 (Th2 condition) or a combination of TNF-alpha and IFNgamma (Th1 condition) in the presence or absence of VEGF and FGF2 for 48 hours. Cell proliferation was measured by 3 H-thymidine uptake. Tube formation was determined by light microscopy after cells were seeded on Matrigel for 18 hours. Comprehensive gene expression profiles of HMVEC-L in Th1 or Th2 condition were analyzed by using high-density oligonucleotide probe arrays (GeneChip) and real-time PCR. RESULTS: Both Th1 and Th2 conditions significantly reduced VEGF-or FGF2-mediated HMVEC-L proliferation. However, Th2 condition but not Th1 condition markedly promotes tube formation of HMVEC-L both in the presence and absence of VEGF or FGF2. GeneChip analysis revealed that CXCL1, CXCL2, CXCL5, CXCL6 and CXCL8 which are known to bind CXCR2 and to possess angiogenic activity, were predominantly upregulated in the Th2 condition. In contrast, CXCL9, CXCL10 and CXCL11 which bind to CXCR3 and possess angiostatic activity, were significantly up-regulated only in the Th1 condition. We also found that the induction of these chemokines was not inhibited by dexamethasone treatment at all. CONCLUSIONS: Our results suggest a possible role of CXC chemokines in the pathogenesis of airway remodeling in asthma through their angiogenic/angiostatic activities. Funding: RATIONALE: IL-13 is an important effector molecule in asthma pathology. Contrary to initial characterizations of asthma as a "Th2" disease, both IL-13 and IFN-gamma levels are also elevated in the airways of asthmatics. In PBL, numbers of IL-13 + cells can be increased by antigen/CD3-mediated stimulation or independent of antigen in response to IL-2 or IL-15, whereas increases in IFN-gamma + T cells generally require co-stimulation with IL-12. We tested whether asthmatic lymphocytes differ in their response to antigen -dependent or -independent stimulation for accumulation of IL-13 + and IFN-gamma + T cells. METHODS: PBL obtained from asthmatic and control, non-asthmatic subjects were stimulated for cytokine production with PMA, Calcimycin, and monensin before and after 5-d culture with CD3+CD28 mAb and IL-2, or 6-d culture with IL-2. Expression of IL-13 and IFN-gamma in gated T cells was analyzed by flow cytometry. RESULTS: There were no significant differences in day 0 proportions of IL-13 + and IFN-gamma + T cells between asthmatics and controls. Mean increase in IL-13 + T cells was significantly greater in asthmatics than controls in response to IL-2 (2-fold greater) but not CD3-mediated stimulation. Although IFN-gamma + cells did not increase in response to IL-2 in controls, in asthmatics, IFN-gamma + cells increased significantly in mean. The difference between asthmatics and controls for mean numbers of IFN-gamma + cells after CD3-mediated stimulation was not significant. CONCLUSIONS: IL-2-induced accumulation of IL-13 + and IFNgamma + T cells is greater in asthmatics than control subjects. These results may implicate IL-2/IL-15-mediated allergen-independent processes in the seemingly paradoxical co-elevation of IL-13 and IFN-gamma levels in asthmatics. RATIONALE: Interleukin-5 (IL-5) is a critical hematopoietic growth factor for eosinophil differentiation, activation and survival. Previous studies have demonstrated that IL-5R␣ levels on peripheral blood eosinophils are downregulated following exposure to IL-5. METHODS: Peripheral blood (PB) and bone marrow (BM) samples were collected from 8 allergic asthmatics for collection of eosinophils and non-adherent mononuclear cells. Cells were incubated with IL-5 (1ng/ml) for 10 min and 120 min, stained for IL-5R␣, and analyzed by flow cytometry. Eosinophils were identified by gating on the CD16-population, and CD34 + progenitors were identified using a sequential gating strategy. RESULTS: BM and PB eosinophils expressing IL-5R␣ decreased to 22.30±15.70% and 18.38±26.34%, respectively, after 120 min incubation with IL-5, compared to control 54.96±10.54% and 70.41±19.51%, respectively (p=0.002 and 0.001). There was a trend towards significance in the fall in IL-5R␣ after 120 min between BM and PB eosinophils (p=0.056). BM CD34 + progenitor expression of IL-5R␣ was 7.70±11.00%, lower than BM or PB eosinophils, being 54.96±10.54% and 70.41±19.51%, respectively (p<0.001). There was no effect of IL-5 on BM CD34+ progenitors after 10 min (p=0.227) or 120 min (p=0.251) compared to control. CONCLUSIONS: BM and PB eosinophils downregulate IL-5R␣ after IL-5 incubation, but BM CD34+ progenitors, do not. This suggests that IL-5R␣ is regulated differently in BM CD34+ progenitors. There is very low expression of IL-5R␣ on BM CD34+. RATIONALE: Qualitative protein array analysis indicates that patients with severe asthma (i.e. those with persistent symptoms and frequent medical encounters despite high dose, inhaled corticosteroids [ICS]) have apparently greater amounts of BDNF, CXCL13, and MIP-3␣ in their sputum compared with patients with mild persistent asthma. Whether these increased mediator levels are associated with increased inflammation is not known. METHODS: After informed consent, 173 asthmatics (37 characterized as "severe" on high dose ICS [SA], 136 "nonsevere" [NSA] by NHLBI & SARP criteria) underwent assessment of cells from induced sputum and ELISAs on sputum supernates for BDNF, CXCL13, BMP-4 and MIP-3␣. Statistical analysis was performed with Sigmastat (ANOVA and t-test as appropriate).
Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34 ... more Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34 + progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates BM to release these effector cells in increased numbers. We hypothesize that mast cells (MCs) may play a predominant role in this process.
Background: The use of combination inhaled budesonide and formoterol as maintenance and reliever ... more Background: The use of combination inhaled budesonide and formoterol as maintenance and reliever therapy significantly improves the risk and the time to exacerbations in asthma. Objectives: To explore the mechanisms underlying the effect of the reliever dose on exacerbations by examining the effect of combination therapy on the allergen challenge model when given after allergen exposure. Methods: In a randomized, double-blind crossover study, single doses of budesonide/formoterol (400/12 mg), formoterol (12 mg), budesonide (400 mg), or placebo were administered during the acute bronchoconstriction response (early airway response) immediately after allergen inhalation in 15 patients with mild asthma. Allergen-induced late airway response (LAR), sputum inflammatory markers, airway hyperresponsiveness, and exhaled nitric oxide were measured. Results: All active treatments significantly attenuated the LAR, with budesonide/formoterol significantly better than its monocomponents (maximum FEV 1 fall: placebo, [mean 6 SEM] 21.2% 6 3.1%; budesonide/formoterol, 4.2% 6 1.4%; formoterol, 7.5% 6 1.7%; budesonide, 10.4% 6 1.6%). Allergen-induced change in methacholine PC 20 was significantly attenuated by budesonide/formoterol, but not by its monocomponents. Sputum cell counts and exhaled nitric oxide increased significantly after all allergen challenges, with no significant attenuation by any of the treatments. Therapy with combination and formoterol alone, but not budesonide, significantly reduced the early airway response. Conclusion: A single dose of budesonide/formoterol was superior to its monocomponents in attenuating the allergeninduced LAR and airway hyperresponsiveness. These effects may represent the contribution of the reliever dose to the budesonide/formoterol maintenance and reliever regimen. Clinical implications: The protective effect against allergic airway responses with a single reliever dose of budesonide/ formoterol is predominantly related to greater functional antagonism of airway smooth muscles. (J Allergy Clin Immunol 2007;119:322-7.)
RATIONALE: Priming of eosinophils by cytokines such as GM-CSF leading to augmented adhesion and r... more RATIONALE: Priming of eosinophils by cytokines such as GM-CSF leading to augmented adhesion and response to chemoattractants is essential for optimal eosinophil activation and development of allergic inflammation. The actin reorganization and integrin activation are implicated in eosinophil priming but its mechanism of regulation by GM-CSF is unknown. We investigated the role of recently identified eosinophil phosphoprotein, an actin-bundling L-plastin, in GM-CSF-induced upregulation of aMb2 integrin CD11b/CD18 and priming for chemotaxis. METHODS: Coimmunoprecipitation studies with subsequent MALDI-TOF-TOF or western blot analysis addressed L-plastin phosphorylation and protein-protein interactions. Chemotaxis to suboptimal concentrations of eotaxin (Boyden chamber) and expression of CD11b/CD18 (flow cytometry) was determined to assess eosinophil priming by GM-CSF. RESULTS: Phosphoproteomic analysis revealed constitutive phosphorylation of L-plastin on Ser7 which was further upregulated by GM-CSF. Coimmunoprecipitation studies showed interaction of L-plastin with the a chain of the GM-CSF receptor, PKCb II , vimentin and cophilin. Pharmacological inhibition of PKCb II blocked GM-CSF-inducible phosphorylation of L-plastin. Flow cytometric analysis showed upregulation of constitutively expressed aMb2 integrin which was sensitive to PKCb II inhibitor. Similarly, GM-CSF allowed eosinophils to respond to suboptimal concentrations of eotaxin in chemotaxis assay and this effect was abrogated by PKCb II inhibitor. On the other hand, internalization of L-plastin-derived phosphorylated peptide encompassing Ser7 upregulated aMb2 integrin expression and increased eosinophil chemotaxis to eotaxin independently on GM-CSF stimulation. CONCLUSIONS: Our data establish a novel role of L-plastin in linking GM-CSF signaling to eosnophil priming and suggest that signaling events implicated in integrin function act via induction of PKCb II -mediated L-plastin phosphorylation.
Background: In steady state, hemopoietic progenitors constantly egress from the bone marrow (BM) ... more Background: In steady state, hemopoietic progenitors constantly egress from the bone marrow (BM) into the blood and circulate through the peripheral tissues. In allergic diseases, the BM releases increased numbers of CD34 1 progenitor cells that migrate to the site of allergic inflammation, where they differentiate into tissue-dwelling and classic effector cells of allergy, such as mast cells, eosinophils, and basophils. Objective: To examine whether peripheral blood CD34 1 cells in addition to being progenitors may also directly function as inflammatory effector cells. Methods: Highly purified neonatal or adult blood CD34 1 cells were examined for the expression of thymic stromal lymphopoietin (TSLP) and IL-33 receptors and for their response to these cytokines as well as to supernatants of primary small airway epithelial cells and nasal explants from rhinosinusitis and control subjects. Sputum of patients with asthma was examined before and after allergen inhalation for the presence of IL-5 and IL-13-containing CD34 1 cells. Results: Circulating CD34 1 cells expressed receptors for TSLP and IL-33 and responded to these cytokines by rapidly releasing high levels of proinflammatory T H 2-like cytokines and chemokines. These cells were activated in a TSLP-dependent manner by the supernatant fluids from activated primary human small airway epithelial cells and from nasal explants of patients with chronic rhinosinusitis. Moreover, activated CD34 1 cells containing IL-5 and IL-13 could be detected in the sputum of individuals with allergic asthma, with numbers increasing in response to specific allergen inhalation challenge. Conclusion: Blood CD34 1 cells, in addition to being progenitors, may act as proinflammatory effector cells by themselves and directly contribute to the allergic inflammation. (J Allergy Clin Immunol 2009;123:472-8.)
Mobilization of hemopoietic progenitor cells from the bone marrow (BM) is a feature of inflammato... more Mobilization of hemopoietic progenitor cells from the bone marrow (BM) is a feature of inflammatory asthmatic responses. Understanding the mechanisms regulating progenitor cell mobilization and trafficking to the peripheral circulation might be important for the development of effective asthma therapies. We investigated the role of adhesion molecules in the mobilization of hemopoietic progenitor cells from the BM during an allergen-induced asthmatic response. BM and peripheral blood samples were obtained from dual-responders with mild asthma before and at several time points after allergen challenge. Fluctuations in expression and adhesive properties of beta1- and beta2-integrins on CD34(+)CD45(+) progenitor cells were assessed by using flow cytometry and adhesion to protein-coated wells, respectively. On BM-derived CD34(+)CD45(+) cells, expression of very late antigen (VLA) 4, but not VLA-5 or Mac-1, decreased significantly 24 hours after allergen challenge and had begun to recover by 48 hours after challenge. In peripheral blood allergen challenge induced a significant decrease in VLA-4 levels after 6 hours, which had not recovered by 96 hours after challenge. Similarly, VLA-5 expression decreased, most prominently at 72 to 96 hours after allergen challenge. In contrast, Mac-1 levels did not change. Chemokine-stimulated adhesion of BM-derived CD34(+)CD45(+) cells to fibronectin was significantly attenuated 24 hours after challenge. Furthermore, adhesion to fibronectin and vascular cell adhesion molecule 1 was greatly reduced by anti-VLA-4 or anti-VLA-5 antibodies. Preferential downregulation of beta1-integrin expression on progenitor cells can reduce the tethering forces to BM components, thus facilitating their egress to the peripheral circulation during an allergic inflammatory response.
Background: IL-5 plays a central role in eosinophil and basophil differentiation, exerting its ef... more Background: IL-5 plays a central role in eosinophil and basophil differentiation, exerting its effects through the IL-5 receptor (IL-5Rα). Currently, little is known concerning regulation of IL-5Rα expression in the context of commitment of hemopoietic progenitor cells to the eosinophil and basophil lineages. Objective: Because all-trans retinoic acid (ATRA) is known to modulate some aspects of hemopoietic differentiation, we examined the effects of ATRA on eosinophil-basophil differentiation and IL-5Rα expression. Methods: Progenitor cells were obtained from bone marrow aspirates and cord blood samples. Enriched populations of CD34 + cells were isolated by means of positive immunomagnetic selection with MACS beads. Results: In semisolid methylcellulose cultures of normal human bone marrow, ATRA (10 -6 mol/L) selectively suppressed eosinophil-basophil colony-forming units but had no effect on granulocyte-macrophage colony-forming units. Similarly, ATRA (10 -6 mol/L) inhibited eosinophil-basophil differentiation of cord blood CD34 + cells in liquid culture, whereas neutrophil differentiation proceeded without impediment. Most importantly, these effects of ATRA (10 -8 to 10 -6 mol/L) on CD34 + cells were associated with a dose-dependent inhibition of IL-5Rα but no change in GM-CSF receptor expression, as detected with flow cytometry. Conclusions: These findings indicate that retinoids can differentially regulate expression of IL-5Rα, but not GM-CSF receptor, and that these effects have functional consequences in vitro on eosinophil and basophil differentiation. (J Allergy Clin Immunol 2002;109:307-13.)
The role of in¯ammatory effector cells in the pathogenesis of airway allergy has been the subject... more The role of in¯ammatory effector cells in the pathogenesis of airway allergy has been the subject of much investigation. However, whether systemic factors are involved in the development of local responses in both upper and lower airways has not been fully clari®ed. The present study was performed to investigate aspects of the pathogenesis of isolated allergic rhinitis in a murine model sensitized to ovalbumin (OVA). Both upper-and lower-airway physiological responsiveness and in¯ammatory changes were assessed, as well as bone marrow progenitor responses, by culture and immunohistological methods. Signi®cant nasal symptoms and hyper-responsiveness appeared after intranasal OVA challenge (P<0 . 0001 and P<0 . 01, respectively), accompanied with signi®cant nasal mucosal changes in CD4 + cells (P<0 . 001), interleukin (IL)-4 + cells (P<0 . 01), IL-5 + cells (P<0 . 01), basophilic cells (P<0 . 02) and eosinophils (P<0 . 001), in the complete absence of hyper-responsiveness or in¯ammatory changes in the lower airway. In the bone marrow, there were signi®cant increases in CD34 + cells, as well as in eosinophils and basophilic cells. In the presence in vitro of mouse recombinant IL-5, IL-3 or granulocyte±macrophage colony-stimulating factor (GM-CSF), the level of bone marrow eosinophil/basophil (Eo/Baso) colony-forming cells increased signi®cantly in the OVA-sensitized group. We conclude that, in this murine model of allergic rhinitis, haemopoietic progenitors are upregulated, which is consistent with the involvement of bone marrow in the pathogenesis of nasal mucosal in¯ammation. Both local and systemic events, initiated in response to allergen provocation, may be required for the pathogenesis of allergic rhinitis. Understanding these events and their regulation could provide new therapeutic targets for rhinitis and asthma.
American Journal of Respiratory Cell and Molecular Biology, 1998
Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of at... more Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of atopic individuals, and can be enhanced in atopic asthmatics by allergen-inhalation challenge. It is known that mature basophils and eosinophils synthesize cytokines relevant to allergic inflammation. To investigate the potential role of growth factors in allergic disease we examined the expression of the hemopoietic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5, in differentiating Eo/B colony cells from normal and atopic individuals, and from atopic asthmatics before and after allergen-inhalation challenge. Peripheral blood was collected from two normal and 12 atopic individuals, and also from 25 atopic asthmatics before and 24 h after allergen challenge. Nonadherent mononuclear cells were isolated and grown in semisolid growth medium. Eo/B colonies were selected and cytospins were prepared for immunocytochemical analysis of colony cells. Eo/B colonies, especially carbol chromotrope 2R+ cells, selected at Days 10, 14, and 18 from atopic donors contained messenger RNA for GM-CSF by combined in situ reverse transcription-polymerase chain reaction and cytochemistry, and demonstrated time-dependent expression of GM-CSF by immunocytochemistry (P = 0.007). Atopic individuals demonstrated a higher percentage of cells expressing GM-CSF than did normal subjects under all growth conditions when examined at Day 14 (P = 0. 04). Atopic asthmatics challenged with inhaled allergen who demonstrated a dual airway response, an increase in the number of blood eosinophils (P = 0.0001), and an increase in the number of Eo/B CFU (P = 0.02) also demonstrated a significant increase in the percentage of colony cells expressing immunostainable GM-CSF (P = 0. 0009), but only a variable effect on those expressing IL-5, 24 h after allergen. These results suggest that GM-CSF expression by differentiating Eo/Bs may provide an additional stimulus in vivo to enhance Eo/B progenitor differentiation in atopic and asthmatic individuals, especially after allergen challenge. The concept of microenvironmental differentiation, where blood progenitor cells may aid in their own differentiation, is supported by these ex vivo findings.
American Journal of Respiratory and Critical Care Medicine, 2008
The drug product TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides desig... more The drug product TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides designed to inhibit allergic inflammation by down-regulating human CCR3 and the common beta chain (beta(c)) of IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors. This study examined the effects of inhaled TPI ASM8 on sputum cellular influx, CCR3 and beta(c) mRNA and protein levels, and the airway physiologic response after inhaled allergen. Seventeen subjects with mild atopic asthma were randomized in a crossover study to inhale 1,500 microg TPI ASM8 or placebo by nebulizer, once daily for 4 days. On Day 3, subjects underwent allergen inhalation challenge. Sputum samples were collected before and after allergen. CCR3 and beta(c) protein levels were measured by flow cytometry, mRNA was measured using real-time quantitative polymerase chain reaction, and the FEV1 was measured over 7 hours after challenge. Compared with placebo, TPI ASM8 inhibited sputum eosinophil influx by 46% (P = 0.02) and blunted the increase in total cells (63%) after allergen challenge. TPI ASM8 significantly reduced the early asthmatic response (P = 0.04) with a trend for the late asthmatic response (P = 0.08). The allergen-induced (Day 2 to Day 3) levels of beta(c) mRNA and CCR3 mRNA in sputum-derived cells were inhibited by TPI ASM8 (P = 0.039 and P = 0.054, respectively), with no significant effects on the cell surface protein expression of CCR3 and beta(c) (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05). No serious adverse events were reported. TPI ASM8 attenuates the allergen-induced increase in target gene mRNA and airway responses in subjects with mild asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 00264966).
Peroxisome proliferator-activated receptor (PPAR) agonists have been suggested as novel therapeut... more Peroxisome proliferator-activated receptor (PPAR) agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists has been shown to inhibit airway eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including attenuated generation of chemoattractants (e.g. eotaxin) and decreased eosinophil migrational responses. In addition, studies report that PPAR agonists can inhibit the differentiation of several cell types. To date, no studies have examined the effects of PPAR agonists on interleukin-5 (IL-5) -induced eosinophil differentiation from haemopoietic progenitor cells. Non-adherent mononuclear cells or CD34 + cells isolated from the peripheral blood of allergic subjects were grown for 2 weeks in Methocult â cultures with IL-5 (10 ng/ml) and IL-3 (25 ng/ml) in the presence of 1-1000 nM PPARa agonist (GW9578), PPARb/d agonist (GW501516), PPARc agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony-forming units (Eo/B CFU) was quantified by light microscopy. The signalling mechanism involved was assessed by phosphoflow. Blood-extracted CD34 + cells cultured with IL-5 or IL-5 + IL-3 formed Eo/B CFU, which were significantly inhibited by rosiglitazone (100 nM, P < 0Á01) but not GW9578 or GW501516. In addition, rosglitazone significantly inhibited IL-5-induced phosphorylation of extracellular signal-regulated kinase 1/2. We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, mediated by attenuation of the extracellular signal-regulated kinase 1/2 signalling pathway. These findings indicate that the PPARc agonist can attenuate tissue eosinophilia by interfering with local differentiative responses.
Background: The sequence of adhesion-molecule expression during eosinophil differentiation remain... more Background: The sequence of adhesion-molecule expression during eosinophil differentiation remains unclear. Methods: We analyzed the surface expression of a 4 , b 1 , and b 7 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 signi®cant upregulation of b 7 integrin expression coincident with a marked upregulation of CD35 and attenuation of CD33 and b 1 integrin expression. In addition, adhesion of induced HL-60 clone 15 cells to ®bronectin was attenuated by a b 7 integrin antibody. Conclusions: Our data show that protein synthesis-dependent upregulation of the functional b 7 integrin occurs under conditions when a 4 and b 1 integrins are fully expressed, indicating a sequential appearance of speci®c adhesion molecules on differentiating eosinophil progenitors.
The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting throug... more The cysteinyl leukotrienes (cysLTs) are potent lipid mediators in allergic disease, acting through a receptor (cysLT1-R) which can be targeted in rhinitis and asthma. We investigated the effects of cysLT1-R antagonism in experimental allergic rhinitis, focusing on bone marrow eosinophil progenitor responses. BALB ⁄c mice were sensitized, then given daily intranasal ovalbumin for 2 weeks, with montelukast sodium (5 mg ⁄kg or 2AE5 mg ⁄kg) or placebo by gavage. Bone marrow eosinophil ⁄basophil colonies were enumerated, and colony cells were morphologically assessed as indices of eosinophil differentiation and maturation. Montelukast treatment resulted in a significant decrease of eosinophils in the nasal mucosa, and in either bone marrow interleukin (IL)-5-, but not IL-3-, or granulocyte-macrophage colony-stimulating factor-responsive eosinophil ⁄basophil colony-forming units, and IL-5-stimulated eosinophil maturation. These results indicate that cysLT1-R antagonism in vivo limits both IL-5-responsive eosinophilopoiesis, acting at several stages of eosinophil differentiation and maturation. The anti-allergic effects of cysLT1-R antagonists are consistent with the concept that cysLTs and IL-5 act together in the recruitment of eosinophils and eosinophil progenitors from the marrow during upper airway allergic inflammation.
Background: Allergic asthma is an inflammatory airway disease in which Th2 cytokines play an impo... more Background: Allergic asthma is an inflammatory airway disease in which Th2 cytokines play an important role. Epithelial-derived interleukin (IL)-25 has been suggested to be important in the maintenance of Th2-type responses. The effects of IL-25 are mediated by the IL-25 receptor, composed of two subunits, IL-17RA and IL-17RB. Eosinophils are effector cells in allergic asthma. The role of IL-25 in eosinophil function is unknown. This study examined the expression of IL-25 and its receptor on eosinophils in allergic asthmatics compared to atopic nonasthmatics and normal controls. Methods: The expression of IL-25, IL-17RA and IL-17RB on eosinophils, and levels of plasma IL-25 were measured in 14 normal control subjects, 15 atopic nonasthmatics and 14 mild allergic asthmatics. Results: The expression of IL-17RB on eosinophils was significantly higher in allergic asthmatics (43.08%, range 33.96-59.98%) than in atopic nonasthmatics (11.98%, range 6.33-27.11%, p = 0.002) and normal contro...
Adhesion Molecule Expression on Human Cord Blood Progenitors During Early Eosinophilic Commitment... more Adhesion Molecule Expression on Human Cord Blood Progenitors During Early Eosinophilic Commitment: Upregulation of b 7 Integrins. Scand J Immunol 2002;56:161±167
RATIONALE: Increased vascularity is an important element of airway remodeling in patients with br... more RATIONALE: Increased vascularity is an important element of airway remodeling in patients with bronchial asthma. However, the molecular mechanism involved in this process remains unclear. In order to clarify it, in vitro responses of human lung microvascular endothelial cell (HMVEC-L) to several cytokines were examined. METHODS: HMVEC-L was incubated with a combination of TNFalpha and IL-4 (Th2 condition) or a combination of TNF-alpha and IFNgamma (Th1 condition) in the presence or absence of VEGF and FGF2 for 48 hours. Cell proliferation was measured by 3 H-thymidine uptake. Tube formation was determined by light microscopy after cells were seeded on Matrigel for 18 hours. Comprehensive gene expression profiles of HMVEC-L in Th1 or Th2 condition were analyzed by using high-density oligonucleotide probe arrays (GeneChip) and real-time PCR. RESULTS: Both Th1 and Th2 conditions significantly reduced VEGF-or FGF2-mediated HMVEC-L proliferation. However, Th2 condition but not Th1 condition markedly promotes tube formation of HMVEC-L both in the presence and absence of VEGF or FGF2. GeneChip analysis revealed that CXCL1, CXCL2, CXCL5, CXCL6 and CXCL8 which are known to bind CXCR2 and to possess angiogenic activity, were predominantly upregulated in the Th2 condition. In contrast, CXCL9, CXCL10 and CXCL11 which bind to CXCR3 and possess angiostatic activity, were significantly up-regulated only in the Th1 condition. We also found that the induction of these chemokines was not inhibited by dexamethasone treatment at all. CONCLUSIONS: Our results suggest a possible role of CXC chemokines in the pathogenesis of airway remodeling in asthma through their angiogenic/angiostatic activities. Funding: RATIONALE: IL-13 is an important effector molecule in asthma pathology. Contrary to initial characterizations of asthma as a "Th2" disease, both IL-13 and IFN-gamma levels are also elevated in the airways of asthmatics. In PBL, numbers of IL-13 + cells can be increased by antigen/CD3-mediated stimulation or independent of antigen in response to IL-2 or IL-15, whereas increases in IFN-gamma + T cells generally require co-stimulation with IL-12. We tested whether asthmatic lymphocytes differ in their response to antigen -dependent or -independent stimulation for accumulation of IL-13 + and IFN-gamma + T cells. METHODS: PBL obtained from asthmatic and control, non-asthmatic subjects were stimulated for cytokine production with PMA, Calcimycin, and monensin before and after 5-d culture with CD3+CD28 mAb and IL-2, or 6-d culture with IL-2. Expression of IL-13 and IFN-gamma in gated T cells was analyzed by flow cytometry. RESULTS: There were no significant differences in day 0 proportions of IL-13 + and IFN-gamma + T cells between asthmatics and controls. Mean increase in IL-13 + T cells was significantly greater in asthmatics than controls in response to IL-2 (2-fold greater) but not CD3-mediated stimulation. Although IFN-gamma + cells did not increase in response to IL-2 in controls, in asthmatics, IFN-gamma + cells increased significantly in mean. The difference between asthmatics and controls for mean numbers of IFN-gamma + cells after CD3-mediated stimulation was not significant. CONCLUSIONS: IL-2-induced accumulation of IL-13 + and IFNgamma + T cells is greater in asthmatics than control subjects. These results may implicate IL-2/IL-15-mediated allergen-independent processes in the seemingly paradoxical co-elevation of IL-13 and IFN-gamma levels in asthmatics. RATIONALE: Interleukin-5 (IL-5) is a critical hematopoietic growth factor for eosinophil differentiation, activation and survival. Previous studies have demonstrated that IL-5R␣ levels on peripheral blood eosinophils are downregulated following exposure to IL-5. METHODS: Peripheral blood (PB) and bone marrow (BM) samples were collected from 8 allergic asthmatics for collection of eosinophils and non-adherent mononuclear cells. Cells were incubated with IL-5 (1ng/ml) for 10 min and 120 min, stained for IL-5R␣, and analyzed by flow cytometry. Eosinophils were identified by gating on the CD16-population, and CD34 + progenitors were identified using a sequential gating strategy. RESULTS: BM and PB eosinophils expressing IL-5R␣ decreased to 22.30±15.70% and 18.38±26.34%, respectively, after 120 min incubation with IL-5, compared to control 54.96±10.54% and 70.41±19.51%, respectively (p=0.002 and 0.001). There was a trend towards significance in the fall in IL-5R␣ after 120 min between BM and PB eosinophils (p=0.056). BM CD34 + progenitor expression of IL-5R␣ was 7.70±11.00%, lower than BM or PB eosinophils, being 54.96±10.54% and 70.41±19.51%, respectively (p<0.001). There was no effect of IL-5 on BM CD34+ progenitors after 10 min (p=0.227) or 120 min (p=0.251) compared to control. CONCLUSIONS: BM and PB eosinophils downregulate IL-5R␣ after IL-5 incubation, but BM CD34+ progenitors, do not. This suggests that IL-5R␣ is regulated differently in BM CD34+ progenitors. There is very low expression of IL-5R␣ on BM CD34+. RATIONALE: Qualitative protein array analysis indicates that patients with severe asthma (i.e. those with persistent symptoms and frequent medical encounters despite high dose, inhaled corticosteroids [ICS]) have apparently greater amounts of BDNF, CXCL13, and MIP-3␣ in their sputum compared with patients with mild persistent asthma. Whether these increased mediator levels are associated with increased inflammation is not known. METHODS: After informed consent, 173 asthmatics (37 characterized as "severe" on high dose ICS [SA], 136 "nonsevere" [NSA] by NHLBI & SARP criteria) underwent assessment of cells from induced sputum and ELISAs on sputum supernates for BDNF, CXCL13, BMP-4 and MIP-3␣. Statistical analysis was performed with Sigmastat (ANOVA and t-test as appropriate).
Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34 ... more Background: Shortly after allergen exposure, the number of bone marrow (BM) and circulating CD34 + progenitors increases. We aim to analyze the possible mechanism whereby the allergic reaction stimulates BM to release these effector cells in increased numbers. We hypothesize that mast cells (MCs) may play a predominant role in this process.
Background: The use of combination inhaled budesonide and formoterol as maintenance and reliever ... more Background: The use of combination inhaled budesonide and formoterol as maintenance and reliever therapy significantly improves the risk and the time to exacerbations in asthma. Objectives: To explore the mechanisms underlying the effect of the reliever dose on exacerbations by examining the effect of combination therapy on the allergen challenge model when given after allergen exposure. Methods: In a randomized, double-blind crossover study, single doses of budesonide/formoterol (400/12 mg), formoterol (12 mg), budesonide (400 mg), or placebo were administered during the acute bronchoconstriction response (early airway response) immediately after allergen inhalation in 15 patients with mild asthma. Allergen-induced late airway response (LAR), sputum inflammatory markers, airway hyperresponsiveness, and exhaled nitric oxide were measured. Results: All active treatments significantly attenuated the LAR, with budesonide/formoterol significantly better than its monocomponents (maximum FEV 1 fall: placebo, [mean 6 SEM] 21.2% 6 3.1%; budesonide/formoterol, 4.2% 6 1.4%; formoterol, 7.5% 6 1.7%; budesonide, 10.4% 6 1.6%). Allergen-induced change in methacholine PC 20 was significantly attenuated by budesonide/formoterol, but not by its monocomponents. Sputum cell counts and exhaled nitric oxide increased significantly after all allergen challenges, with no significant attenuation by any of the treatments. Therapy with combination and formoterol alone, but not budesonide, significantly reduced the early airway response. Conclusion: A single dose of budesonide/formoterol was superior to its monocomponents in attenuating the allergeninduced LAR and airway hyperresponsiveness. These effects may represent the contribution of the reliever dose to the budesonide/formoterol maintenance and reliever regimen. Clinical implications: The protective effect against allergic airway responses with a single reliever dose of budesonide/ formoterol is predominantly related to greater functional antagonism of airway smooth muscles. (J Allergy Clin Immunol 2007;119:322-7.)
RATIONALE: Priming of eosinophils by cytokines such as GM-CSF leading to augmented adhesion and r... more RATIONALE: Priming of eosinophils by cytokines such as GM-CSF leading to augmented adhesion and response to chemoattractants is essential for optimal eosinophil activation and development of allergic inflammation. The actin reorganization and integrin activation are implicated in eosinophil priming but its mechanism of regulation by GM-CSF is unknown. We investigated the role of recently identified eosinophil phosphoprotein, an actin-bundling L-plastin, in GM-CSF-induced upregulation of aMb2 integrin CD11b/CD18 and priming for chemotaxis. METHODS: Coimmunoprecipitation studies with subsequent MALDI-TOF-TOF or western blot analysis addressed L-plastin phosphorylation and protein-protein interactions. Chemotaxis to suboptimal concentrations of eotaxin (Boyden chamber) and expression of CD11b/CD18 (flow cytometry) was determined to assess eosinophil priming by GM-CSF. RESULTS: Phosphoproteomic analysis revealed constitutive phosphorylation of L-plastin on Ser7 which was further upregulated by GM-CSF. Coimmunoprecipitation studies showed interaction of L-plastin with the a chain of the GM-CSF receptor, PKCb II , vimentin and cophilin. Pharmacological inhibition of PKCb II blocked GM-CSF-inducible phosphorylation of L-plastin. Flow cytometric analysis showed upregulation of constitutively expressed aMb2 integrin which was sensitive to PKCb II inhibitor. Similarly, GM-CSF allowed eosinophils to respond to suboptimal concentrations of eotaxin in chemotaxis assay and this effect was abrogated by PKCb II inhibitor. On the other hand, internalization of L-plastin-derived phosphorylated peptide encompassing Ser7 upregulated aMb2 integrin expression and increased eosinophil chemotaxis to eotaxin independently on GM-CSF stimulation. CONCLUSIONS: Our data establish a novel role of L-plastin in linking GM-CSF signaling to eosnophil priming and suggest that signaling events implicated in integrin function act via induction of PKCb II -mediated L-plastin phosphorylation.
Background: In steady state, hemopoietic progenitors constantly egress from the bone marrow (BM) ... more Background: In steady state, hemopoietic progenitors constantly egress from the bone marrow (BM) into the blood and circulate through the peripheral tissues. In allergic diseases, the BM releases increased numbers of CD34 1 progenitor cells that migrate to the site of allergic inflammation, where they differentiate into tissue-dwelling and classic effector cells of allergy, such as mast cells, eosinophils, and basophils. Objective: To examine whether peripheral blood CD34 1 cells in addition to being progenitors may also directly function as inflammatory effector cells. Methods: Highly purified neonatal or adult blood CD34 1 cells were examined for the expression of thymic stromal lymphopoietin (TSLP) and IL-33 receptors and for their response to these cytokines as well as to supernatants of primary small airway epithelial cells and nasal explants from rhinosinusitis and control subjects. Sputum of patients with asthma was examined before and after allergen inhalation for the presence of IL-5 and IL-13-containing CD34 1 cells. Results: Circulating CD34 1 cells expressed receptors for TSLP and IL-33 and responded to these cytokines by rapidly releasing high levels of proinflammatory T H 2-like cytokines and chemokines. These cells were activated in a TSLP-dependent manner by the supernatant fluids from activated primary human small airway epithelial cells and from nasal explants of patients with chronic rhinosinusitis. Moreover, activated CD34 1 cells containing IL-5 and IL-13 could be detected in the sputum of individuals with allergic asthma, with numbers increasing in response to specific allergen inhalation challenge. Conclusion: Blood CD34 1 cells, in addition to being progenitors, may act as proinflammatory effector cells by themselves and directly contribute to the allergic inflammation. (J Allergy Clin Immunol 2009;123:472-8.)
Mobilization of hemopoietic progenitor cells from the bone marrow (BM) is a feature of inflammato... more Mobilization of hemopoietic progenitor cells from the bone marrow (BM) is a feature of inflammatory asthmatic responses. Understanding the mechanisms regulating progenitor cell mobilization and trafficking to the peripheral circulation might be important for the development of effective asthma therapies. We investigated the role of adhesion molecules in the mobilization of hemopoietic progenitor cells from the BM during an allergen-induced asthmatic response. BM and peripheral blood samples were obtained from dual-responders with mild asthma before and at several time points after allergen challenge. Fluctuations in expression and adhesive properties of beta1- and beta2-integrins on CD34(+)CD45(+) progenitor cells were assessed by using flow cytometry and adhesion to protein-coated wells, respectively. On BM-derived CD34(+)CD45(+) cells, expression of very late antigen (VLA) 4, but not VLA-5 or Mac-1, decreased significantly 24 hours after allergen challenge and had begun to recover by 48 hours after challenge. In peripheral blood allergen challenge induced a significant decrease in VLA-4 levels after 6 hours, which had not recovered by 96 hours after challenge. Similarly, VLA-5 expression decreased, most prominently at 72 to 96 hours after allergen challenge. In contrast, Mac-1 levels did not change. Chemokine-stimulated adhesion of BM-derived CD34(+)CD45(+) cells to fibronectin was significantly attenuated 24 hours after challenge. Furthermore, adhesion to fibronectin and vascular cell adhesion molecule 1 was greatly reduced by anti-VLA-4 or anti-VLA-5 antibodies. Preferential downregulation of beta1-integrin expression on progenitor cells can reduce the tethering forces to BM components, thus facilitating their egress to the peripheral circulation during an allergic inflammatory response.
Background: IL-5 plays a central role in eosinophil and basophil differentiation, exerting its ef... more Background: IL-5 plays a central role in eosinophil and basophil differentiation, exerting its effects through the IL-5 receptor (IL-5Rα). Currently, little is known concerning regulation of IL-5Rα expression in the context of commitment of hemopoietic progenitor cells to the eosinophil and basophil lineages. Objective: Because all-trans retinoic acid (ATRA) is known to modulate some aspects of hemopoietic differentiation, we examined the effects of ATRA on eosinophil-basophil differentiation and IL-5Rα expression. Methods: Progenitor cells were obtained from bone marrow aspirates and cord blood samples. Enriched populations of CD34 + cells were isolated by means of positive immunomagnetic selection with MACS beads. Results: In semisolid methylcellulose cultures of normal human bone marrow, ATRA (10 -6 mol/L) selectively suppressed eosinophil-basophil colony-forming units but had no effect on granulocyte-macrophage colony-forming units. Similarly, ATRA (10 -6 mol/L) inhibited eosinophil-basophil differentiation of cord blood CD34 + cells in liquid culture, whereas neutrophil differentiation proceeded without impediment. Most importantly, these effects of ATRA (10 -8 to 10 -6 mol/L) on CD34 + cells were associated with a dose-dependent inhibition of IL-5Rα but no change in GM-CSF receptor expression, as detected with flow cytometry. Conclusions: These findings indicate that retinoids can differentially regulate expression of IL-5Rα, but not GM-CSF receptor, and that these effects have functional consequences in vitro on eosinophil and basophil differentiation. (J Allergy Clin Immunol 2002;109:307-13.)
The role of in¯ammatory effector cells in the pathogenesis of airway allergy has been the subject... more The role of in¯ammatory effector cells in the pathogenesis of airway allergy has been the subject of much investigation. However, whether systemic factors are involved in the development of local responses in both upper and lower airways has not been fully clari®ed. The present study was performed to investigate aspects of the pathogenesis of isolated allergic rhinitis in a murine model sensitized to ovalbumin (OVA). Both upper-and lower-airway physiological responsiveness and in¯ammatory changes were assessed, as well as bone marrow progenitor responses, by culture and immunohistological methods. Signi®cant nasal symptoms and hyper-responsiveness appeared after intranasal OVA challenge (P<0 . 0001 and P<0 . 01, respectively), accompanied with signi®cant nasal mucosal changes in CD4 + cells (P<0 . 001), interleukin (IL)-4 + cells (P<0 . 01), IL-5 + cells (P<0 . 01), basophilic cells (P<0 . 02) and eosinophils (P<0 . 001), in the complete absence of hyper-responsiveness or in¯ammatory changes in the lower airway. In the bone marrow, there were signi®cant increases in CD34 + cells, as well as in eosinophils and basophilic cells. In the presence in vitro of mouse recombinant IL-5, IL-3 or granulocyte±macrophage colony-stimulating factor (GM-CSF), the level of bone marrow eosinophil/basophil (Eo/Baso) colony-forming cells increased signi®cantly in the OVA-sensitized group. We conclude that, in this murine model of allergic rhinitis, haemopoietic progenitors are upregulated, which is consistent with the involvement of bone marrow in the pathogenesis of nasal mucosal in¯ammation. Both local and systemic events, initiated in response to allergen provocation, may be required for the pathogenesis of allergic rhinitis. Understanding these events and their regulation could provide new therapeutic targets for rhinitis and asthma.
American Journal of Respiratory Cell and Molecular Biology, 1998
Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of at... more Higher numbers of eosinophil/basophil colony-forming units (Eo/B CFU) are observed in blood of atopic individuals, and can be enhanced in atopic asthmatics by allergen-inhalation challenge. It is known that mature basophils and eosinophils synthesize cytokines relevant to allergic inflammation. To investigate the potential role of growth factors in allergic disease we examined the expression of the hemopoietic cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-5, in differentiating Eo/B colony cells from normal and atopic individuals, and from atopic asthmatics before and after allergen-inhalation challenge. Peripheral blood was collected from two normal and 12 atopic individuals, and also from 25 atopic asthmatics before and 24 h after allergen challenge. Nonadherent mononuclear cells were isolated and grown in semisolid growth medium. Eo/B colonies were selected and cytospins were prepared for immunocytochemical analysis of colony cells. Eo/B colonies, especially carbol chromotrope 2R+ cells, selected at Days 10, 14, and 18 from atopic donors contained messenger RNA for GM-CSF by combined in situ reverse transcription-polymerase chain reaction and cytochemistry, and demonstrated time-dependent expression of GM-CSF by immunocytochemistry (P = 0.007). Atopic individuals demonstrated a higher percentage of cells expressing GM-CSF than did normal subjects under all growth conditions when examined at Day 14 (P = 0. 04). Atopic asthmatics challenged with inhaled allergen who demonstrated a dual airway response, an increase in the number of blood eosinophils (P = 0.0001), and an increase in the number of Eo/B CFU (P = 0.02) also demonstrated a significant increase in the percentage of colony cells expressing immunostainable GM-CSF (P = 0. 0009), but only a variable effect on those expressing IL-5, 24 h after allergen. These results suggest that GM-CSF expression by differentiating Eo/Bs may provide an additional stimulus in vivo to enhance Eo/B progenitor differentiation in atopic and asthmatic individuals, especially after allergen challenge. The concept of microenvironmental differentiation, where blood progenitor cells may aid in their own differentiation, is supported by these ex vivo findings.
American Journal of Respiratory and Critical Care Medicine, 2008
The drug product TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides desig... more The drug product TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides designed to inhibit allergic inflammation by down-regulating human CCR3 and the common beta chain (beta(c)) of IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors. This study examined the effects of inhaled TPI ASM8 on sputum cellular influx, CCR3 and beta(c) mRNA and protein levels, and the airway physiologic response after inhaled allergen. Seventeen subjects with mild atopic asthma were randomized in a crossover study to inhale 1,500 microg TPI ASM8 or placebo by nebulizer, once daily for 4 days. On Day 3, subjects underwent allergen inhalation challenge. Sputum samples were collected before and after allergen. CCR3 and beta(c) protein levels were measured by flow cytometry, mRNA was measured using real-time quantitative polymerase chain reaction, and the FEV1 was measured over 7 hours after challenge. Compared with placebo, TPI ASM8 inhibited sputum eosinophil influx by 46% (P = 0.02) and blunted the increase in total cells (63%) after allergen challenge. TPI ASM8 significantly reduced the early asthmatic response (P = 0.04) with a trend for the late asthmatic response (P = 0.08). The allergen-induced (Day 2 to Day 3) levels of beta(c) mRNA and CCR3 mRNA in sputum-derived cells were inhibited by TPI ASM8 (P = 0.039 and P = 0.054, respectively), with no significant effects on the cell surface protein expression of CCR3 and beta(c) (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt; 0.05). No serious adverse events were reported. TPI ASM8 attenuates the allergen-induced increase in target gene mRNA and airway responses in subjects with mild asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 00264966).
Peroxisome proliferator-activated receptor (PPAR) agonists have been suggested as novel therapeut... more Peroxisome proliferator-activated receptor (PPAR) agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists has been shown to inhibit airway eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including attenuated generation of chemoattractants (e.g. eotaxin) and decreased eosinophil migrational responses. In addition, studies report that PPAR agonists can inhibit the differentiation of several cell types. To date, no studies have examined the effects of PPAR agonists on interleukin-5 (IL-5) -induced eosinophil differentiation from haemopoietic progenitor cells. Non-adherent mononuclear cells or CD34 + cells isolated from the peripheral blood of allergic subjects were grown for 2 weeks in Methocult â cultures with IL-5 (10 ng/ml) and IL-3 (25 ng/ml) in the presence of 1-1000 nM PPARa agonist (GW9578), PPARb/d agonist (GW501516), PPARc agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony-forming units (Eo/B CFU) was quantified by light microscopy. The signalling mechanism involved was assessed by phosphoflow. Blood-extracted CD34 + cells cultured with IL-5 or IL-5 + IL-3 formed Eo/B CFU, which were significantly inhibited by rosiglitazone (100 nM, P < 0Á01) but not GW9578 or GW501516. In addition, rosglitazone significantly inhibited IL-5-induced phosphorylation of extracellular signal-regulated kinase 1/2. We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, mediated by attenuation of the extracellular signal-regulated kinase 1/2 signalling pathway. These findings indicate that the PPARc agonist can attenuate tissue eosinophilia by interfering with local differentiative responses.
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