Sputum eosinophils from asthmatics express ICAM-1 and HLA-DR.

Sputum from symptomatic asthmatics is a rich source of eosinophils from the respiratory tract. Following liquefaction of sputum with dithioerythritol (DTE), a cell suspension for indirect double immunofluorescence with flow cytometry was obtained. Eosinophils were identified using anti-CD9 fluorescein conjugate, and particular surface markers measured with the relevant mouse MoAb followed by goat anti-mouse immunoglobulin phycoerythrin conjugate. Blood and sputum eosinophil surface markers were determined in parallel from asthmatics not receiving steroid therapy. Sputum eosinophils were found to have considerably elevated levels of CD11b, a reflection of eosinophil activation. Sputum but not blood eosinophils were found to express ICAM-1 (nine out of 11 cases) and HLA-DR (eight out of 11 cases). Furthermore, following culture of normal blood eosinophils with pooled T cell supernatants, ICAM-1 and HLA-DR could be induced in vitro. The induction of eosinophil adhesion molecules such as ICAM-1 and HLA-DR may influence eosinophil localization and function in asthma.     

Eotaxin in induced sputum of asthmatics: relationship with eosinophils and eosinophil cationic protein in sputum

BACKGROUND: Eosinophilic inflammation is a crucial aspect of allergic diseases such as bronchial asthma. An eosinophil-active chemokine, eotaxin, may play a role in the pathogenesis of the tissue eosinophilia accompanying asthma. METHODS: Induced sputa were obtained from 53 patients with atopic asthma and six healthy subjects, and the concentration of eotaxin in the sputum was measured by ELISA. We investigated whether the sputum content of eotaxin is related to 1) asthma status or corticosteroid therapy, and 2) other sputum indices, including percentage of eosinophils and concentration of eosinophil cationic protein (ECP). RESULTS: The patients with stable or unstable asthma showed significantly higher concentrations of sputum eotaxin than the normal controls. The level of sputum eotaxin demonstrated a positive correlation with the percentage of eosinophils in stable asthmatics not receiving corticosteroid therapy, but not in stable patients treated with corticosteroids, or in unstable patients. Sputum eotaxin demonstrated a positive correlation with ECP in asthmatic patients who were either in a stable state or not receiving steroid therapy. CONCLUSIONS: The elevated level of eotaxin detected in association with increased eosinophils and ECP in the sputum of asthmatics suggests that eotaxin is involved in the pathogenesis of eosinophilic airway inflammation. The relationship of eotaxin to airway eosinophilia may be modified by the stability status of asthma and corticosteroid therapy.     

Sputum basophils are increased in eosinophilic asthma compared with non‐eosinophilic asthma phenotypes

Sputum basophil numbers are increased in allergic asthmatics, but it is unclear what role airway basophils play in “TH 2‐low” asthma phenotypes. Using flow cytometry, we found that basophils were significantly increased in all asthmatics (n=26) compared with healthy controls (n=8) (P =0.007) with highest levels observed in eosinophilic asthma (EA ); median 0.22%, IQR 0.11%‐0.47%; n=14) compared with non‐EA (NEA) (0.06%, 0.00%‐0.20%; n=12; P <0.05). In asthmatics, basophils were positively correlated with sputum eosinophils (r =0.54; P <0.005) and inversely with sputum neutrophils (r =?0.46: P <0.05), but not with FEV ₁ (% predicted), FEV ₁/FVC or bronchodilator reversibility. In a subgroup initially identified as inadequately controlled asthma (n=7), there was a trend (P =0.08) towards a reduction in sputum basophils following increased inhaled corticosteroid (ICS ) treatment. Our findings suggest that basophils may be particularly important in eosinophilic asthma and that sputum basophil assessment could be a useful additional indicator of “TH 2‐high” asthma.     

Integrin expression on neutrophils and mononuclear cells in blood and induced sputum in stable asthma

BACKGROUND: We speculated that the expression of integrins in the airway lumen of asthmatic subjects might be altered compared with normal subjects during cell recruitment from circulation. METHODS: To test this hypothesis, we investigated the expression of integrin alpha-chains (CD11a, CD11b, and CD11c) in hypertonic saline-induced sputum and peripheral blood leukocytes in mild to moderate stable asthmatic and control subjects. Immunoreactivity for integrin alpha-chains was assessed by immunocytology on cytospin preparations of sputum and blood. RESULTS: In comparison of the percentages of CD11a+, CD11b+ and CD11c+ mononuclear cells in sputum with their blood counterparts, no significant differences were observed in control subjects, whereas CD11a and CD11b in asthmatic subjects were less expressed on sputum cells. In both control and asthmatic subjects, sputum neutrophils tended to decrease their expression of integrin alpha-chains compared with circulating neutrophils. CONCLUSIONS: We showed that the sputum of asthmatics, unlike normal subjects, is characterized by decreased expression of integrins on mononuclear cells compared with their blood counterparts. The results suggest that downregulation of integrins occurs in asthmatic airways after cell recruitment from circulation.     

流式细胞术在外周血嗜酸性粒细胞及其相关分子检测中的应用

目的 :应用流式细胞术 ,建立一种准确、外周血嗜酸性细胞及其相关分子的快速测定方法。方法 :采集正常人外周血 ,用茶碱 (10 -4mol/L)、地塞米松 (10 -4mol/L)和rhIL 5 (10 -8mol/L)预处理 ,用抗CD16 PEmAb与FITC标记的抗相关细胞分子的进行双标记染色 ,并以CD16 FL2辅助设门 ,准确找到嗜酸性粒细胞群 ,然后对其相关分子进行分析。结果 :嗜酸性粒细胞定位准确 ,茶碱和地塞米松能够抑制IL 5引起的Eos的活化并使其表面的CD6 2L脱落。结论 :应用流式细胞术与二色荧光mAbCD16 PE阴性细胞法设门 ,可准确快速地检测外周血中嗜酸性粒细胞及分子的表达率 ,血液用样量小 ,人为影响因素少 ,是免疫学基础研究和临床检验较理想的测定方法。     

Expression of eotaxin in induced sputum of atopic and nonatopic asthmatics

BACKGROUND: The chemokine eotaxin has been implicated in airway eosinophilia in atopic asthma. We have compared airway eosinophils and eotaxin expression in induced sputum from well-matched atopic and nonatopic asthmatics. METHODS: Eosinophil numbers, eosinophil cationic protein (ECP), and the expression of eotaxin were examined in induced sputum from atopic asthmatics (AA = 11), nonatopic asthmatics (NAA = 11), and atopic (AC = 12) and normal (NC = 10) controls. Slides were prepared for differential cell counts by Romanowsky stain, and ECP levels were measured by RIA. Eotaxin expression was detected by in situ hybridization, with 35S-labelled riboprobes and immunocytochemistry. RESULTS: The numbers of eosinophils and ECP concentration were increased in the sputum of AA and NAA compared with AC and NC (P < 0.05). The numbers of eotaxin mRNA+ and immunoreactive cells were increased in NAA, but not AA, when compared with controls (P < 0.05). Eotaxin immunoreactive cells in NAA were significantly higher than in AA (P < 0.05). Eotaxin was expressed predominantly by macrophages, eosinophils, and epithelial cells. In NAA, but not AA, the numbers of eotaxin mRNA+ cells were correlated with histamine PC20 (r = -0.81, P < 0.01) and eosinophil numbers in sputum (r = 0.7, P < 0.05). CONCLUSIONS: Eotaxin production by macrophages, eosinophils, and epithelial cells may play a more pronounced role in airway eosinophilia in nonatopic than in atopic asthma.     

Increases in eotaxin‐positive cells in induced sputum from atopic asthmatic subjects after inhalational allergen challenge

BACKGROUND: Eosinophils are believed to be critical proinflammatory cells in airway mucosal damage in asthma. Eotaxin is a C-C chemokine with selective activity for eosinophils and basophils. Previous studies have shown increased expression of eotaxin in the airways of asthmatics at baseline. We aimed to investigate eotaxin expression during the late-phase reaction to allergen inhalation in atopic asthmatics. METHODS: Sputum induction was performed before and 24 h after inhalational allergen challenge in atopic asthmatics, and eotaxin protein was detected immunocytochemically. RESULTS: Thirteen patients with a mean decrease in forced expiratory volume in 1 s of 28% (+/-1.5) during the early asthmatic reaction, and 39% (+/-4.7) during the late asthmatic reaction produced sufficient sputum for study. The percentage of eosinophils in sputum was increased 24 h after allergen challenge (P<0.004), and eosinophil percentages in sputum after challenge correlated with the magnitude of the late-phase reaction (r=0.56, P=0.05). The percentage of eotaxin-positive cells increased from 12.6% (range 2-43.8) to 24.3% (8.1-47.1, P<0.005). Allergen-induced increases in eotaxin-positive cells correlated with increases in eosinophils (r=0.63, P<0.01). CONCLUSIONS: These findings suggest that eotaxin may contribute to allergen-induced recruitment of eosinophils to the airway in asthmatic subjects.     

Correlation between IgA antibody and eosinophil cationic protein levels in induced sputum from asthmatic patients

Background Eosinophils are known to be main effector cells in allergic inflammation and IgA antibody has been shown to be a potent stimulus for eosinophil degranulation in in vitro conditions. Objective To evaluate the possible role of IgA antibodies on eosinophil degranulation in lower respiratory mucosa of asthmatics, we tried to find a correlation between total IgA and eosinophil cationic protein (ECP) levels in induced sputum from asthmatics. Methods We measured total IgA and albumin levels by nephelometry, and eosinophil cationic protein levels by Pharmacia CAP system in induced sputum from 23 atopic asthmatics and 12 healthy controls. Results IgA and albumin levels in induced sputum from asthmatics with sputum eosinophilia (sputum eosinophil count 5% of 200 counted non-squamous cells) were significantly higher (P < 0.05) than those from controls. However, IgA and albumin levels in induced sputum from asthmatics without sputum eosinophilia were not significantly different with those from controls (P > 0.05). In induced sputum from asthmatics, ECP levels were significantly correlated with albumin (r= 0.44, P= 0.04) and IgA levels (r= 0.67, P= 0.002). ECP/albumin ratio was also significantly correlated with IgA/albumin ratio (r= 0.61, P= 0.004). Conclusion Our results support the hypothesis that IgA antibodies in tracheobronchial secretion may be involved in eosinophil degranulation in asthma, and further study is needed to prove this hypothesis.     

Nasal and pharyngeal eosinophil peroxidase levels in adults with poorly controlled asthma correlate with sputum eosinophilia

The objective of the study was to compare nasal, pharyngeal, and sputum eosinophil peroxidase (EPX) levels with induced sputum eosinophil percentage in 10 adults with poorly controlled asthma and 10 normal controls. EPX was measured using an ELISA and normalized for grams of protein for nasal and pharynx specimens and for mL‐gram of protein for sputum. Sputum EPX levels were statistically different between asthma and control subjects (P = 0.024). EPX levels measured in the nasal and pharyngeal swab samples derived from the same patients were also different between asthma and control subjects, each displaying a high degree of significance (P = 0.002). Spearman's correlation coefficients for nasal EPX and pharyngeal EPX levels compared to induced sputum eosinophil percentage were 0.81 (P = 0.0007) and 0.78 (P = 0.0017), respectively. Thus, there is a strong association in a given patient between both nasal and pharyngeal EPX levels and the eosinophil percentage of induced sputum.     

Down-regulated IL-5 receptor expression on peripheral blood eosinophils from budesonide-treated children with asthma

BACKGROUND: The expression and function of cytokine receptors on peripheral blood eosinophils (PBE) from healthy and asthmatic children are poorly characterized. METHODS: The PBE count and expression of IL-5 receptor (R) and GM-CSFR positive PBE was analyzed in nonsteroid-treated asthmatic children (n = 13), budesonide-treated asthmatic children (n = 24) and healthy children (n = 16) by flow cytometry. Alterations in intracellular EG2-epitope expression were used to measure the in vitro responsiveness of PBE to recombinant IL-5 and GM-CSF. RESULTS: The PBE count was increased (P < 0.05) in both asthmatic groups, independent of treatment, as compared to healthy children. The IL-5R expression on PBE, as well as the in vitro responsiveness of PBE to recombinant IL-5, was reduced (P < 0.05), in budesonide-treated asthmatic children compared to nonsteroid-treated asthmatic children and healthy children. The proportion of GM-CSFR positive PBE and in vitro responsiveness of PBE to recombinant GM-CSF were not different between the groups. In vitro treatment with budesonide did not down-regulate the proportion of IL-5R positive PBE. CONCLUSIONS: Budesonide-treatment of asthmatic children induces a selectively reduced IL-5R expression on PBE, concomitant with a reduced in vitro responsiveness of PBE to IL-5. We suggest that this budesonide-related down-regulation of the IL-5R might be a mechanism by which steroid treatment inhibits the action of IL-5 on eosinophil accumulation and activation in vivo.     

Expression of the apoptosis inhibitor Bcl-2 in sputum eosinophils from children with acute asthma

BACKGROUND: Apoptosis of eosinophils is of increasingly important value in modulating allergic airway inflammation in asthma. Our purpose was to investigate the degree of expression of the antiapoptotic B-cell lymphoma/leukaemia-2 (Bcl-2) protein in sputum eosinophils during acute asthma exacerbation and its relationship with exacerbation severity. METHODS: Sputum was obtained from 33 asthmatic children and 15 healthy children as a control group. Patients were studied during an acute asthma exacerbation. They were classified according to the severity of exacerbation into mild, moderate and severe (n=11 for each). Patients with severe exacerbation were followed up until remission and another sputum sample was obtained. Number of sputum eosinophils was expressed as percentage of leucocytes. Bcl-2 expression in sputum eosinophils was assessed by immunohistochemical staining techniques; the results were expressed as percentage of positively stained cells over total eosinophils. RESULTS: Sputum eosinophils and Bcl-2(+) eosinophils' percentages were significantly higher in patients with acute exacerbation than controls (P<0.01). Patients with severe exacerbation had significantly higher sputum Bcl-2(+) eosinophils' percentage than those with mild-to-moderate exacerbation (mean+/-SD=42.4+/-31.96% vs. 5.7+/-14.5%, P<0.01). A significant negative correlation was found between Bcl-2(+) eosinophils' percentage and peak expiratory flow rate % predicted (P<0.05). After remission, patients with severe exacerbation showed a significant decrease of Bcl-2(+) eosinophils' percentage (P<0.05). CONCLUSION: Our findings suggest that Bcl-2 prolongs survival and decreases apoptosis of airway eosinophils in asthma especially during exacerbation. Eosinophil apoptosis and Bcl-2 represent a target for new and effective therapeutic strategies of asthma.     

粘附分子CD11a、CD11b、CD62L在恶性淋巴增殖性疾病的表达

目的：观察恶性淋巴增殖性疾病肿瘤细胞表面β２整合素（ＣＤ１１ａ、ＣＤ１１ｂ）及Ｌ－选择素（ＣＤ６２１）的表达变化及其临床意义。方法：用流式细胞仪检测３５例初诊或复发急性淋巴白血病（ＡＬＬ）、４例慢性淋巴细胞白血病（ＣＬＬ）、３０例多发性骨髓瘤（ＭＭ）、１４例淋巴肉瘤白血症及２５例正常人骨髓单个核细胞粘附分子ＣＤ１１ａ、ＣＤ１１ｂ、ＣＤ６２Ｌ的表达。结果：（１）与正常造血细胞比较，ＣＤ１１ｂ、ＣＤ１     

Adrenomedullin Suppresses fMLP-Induced Upregulation of CD11b of Human Neutrophils

In this study we investigated the effect of adrenomedullin (AM) on fMLP-mediated activation of human neutrophils. AM partially, but significantly, suppressed fMLP-induced upregulation of CD11b expression. The inhibitory effects of AM upon fMLP-induced upregulation of CD11b expression were completely blocked by CGRP [8–37], a CGRP receptor antagonist. AM significantly increased cAMP content in neutrophils and SQ-22,536, an adenylate cyclase inhibitor, and KT-5720, a PKA inhibitor, significantly blocked the inhibitory effects of AM upon fMLP-induced upregulation of CD11b expression. This study indicates that binding of AM to the CGRP receptor suppresses fMLP-induced upregulation of CD11b expression of human neutrophils by increasing intracellular cAMP levels. AM may play an important role in the regulation of inflammatory processes, especially in the binding of neutrophils to vascular endothelial cells and subsequent neutrophil emigration evident in acute pulmonary inflammation.     

Differential expression of beta1 and beta2 integrins and L-selectin on CD4+ and CD8+ T lymphocytes in human blood: comparative analysis between isolated cells, whole blood samples and cryopreserved preparations

Flow cytometric analysis was used to compare the expression of adhesion molecules on human CD4+ and CD8+ T lymphocytes in isolated blood mononuclear cells (MNCs) in whole blood samples and in cryopreserved MNC preparations. Examination of MNCs revealed that the CD11b and CD11c components of the beta2 integrins were preferentially expressed on CD8+ T cells, whereas CD62L was present on more CD4+ T cells. All CD4+ and CD8+ T lymphocytes were positive for CD11a but the CD8+ population had a higher intensity of expression of CD11a and also CD11b. Virtually identical results were obtained with T cells in whole blood samples. In relation to the beta1 integrins, the only difference between isolated CD4+ and CD8+ T cells was that the latter subset had a greater proportion of cells bearing CD49d. The naive cell marker CD45RA was present on the majority of CD8+ T cells whereas CD45RA and the memory marker CD45RO were evenly distributed within the CD4+ T cell subset. Although cryopreservation of lymphocytes did not modify the expression of beta1 and beta2 integrins it produced a marked reduction in the percentage of CD4+ and CD8+ T cells bearing CD62L. With regard to endothelial interactions, it appears that cryopreserved lymphocytes are suitable for inclusion in studies of integrin-mediated adhesion but not for those relating to tethering or recognition of addressins on high endothelial venules. Differences in adhesion molecule expression between CD4+ and CD8+ T lymphocytes could underlie the selective extravasation of these subsets into sites of infection and inflammation.     

Metalloproteinase-9 is increased after toluene diisocyanate exposure in the induced sputum from patients with toluene diisocyanate-induced asthma

BACKGROUND AND OBJECTIVE: Persistent asthma symptoms are associated with airway inflammation and remodeling, which may be mediated through metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP). The aim of this study was to evaluate MMPs and TIMP involvement in toluene diisocyanate (TDI)-induced asthma. MATERIALS AND METHOD: Induced sputum was collected in eight newly diagnosed TDI-induced asthma subjects (group I) before and 7 h after the TDI and placebo challenges and in 12 subjects with TDI-induced occupational asthma diagnosed 5 years previously with persistent asthma symptoms (group II). Sera was collected in group I at diagnosis, and in group II, they were collected at the time of the study. 12 nonasthmatic healthy subjects were enrolled as controls. MMP-9, MMP-2 and TIMP-1 levels in both sputum and serum were measured by enzyme-linked immunosorbent assay (ELISA). Gelatinase activity in the sputum was confirmed by zymographic analysis. RESULTS: The serum TIMP-1 level was significantly higher in asthma patients than in healthy controls (P = 0.01), while MMP-9 level was significantly lower in asthmatic patients (P = 0.03). There was no significant difference in MMP-2 level (P = 0.27). MMP-9 level in the sputum was significantly increased after the TDI challenges (P = 0.01). TIMP-1 level in sputum tended to increase after TDI challenges, but no statistical significance was noted (P = 0.09). MMP-9 and MMP-9/TIMP-1 levels in the sputum were significantly higher in group II than in group I (P = 0.04, P = 0.02) with no significant difference in TIMP-1 level. Minimal amount of MMP-2 was found in sputum. Zymography demonstrated that MMP-9 level increased and active form of MMP-9 was generated after the TDI bronchoprovocation test. CONCLUSION: TDI exposure leads to overproduction of MMP-9, which may induce airway inflammation and remodeling, and then contribute to persistent asthmatic symptoms in TDI-induced asthma.     

Comparison between hypertonic and isotonic saline-induced sputum in the evaluation of airway inflammation in subjects with moderate asthma

Background Hypertonic saline-indueed sputum has recently been used for the evaluation of airway inflammation in asthma. Objective To assess the effect of hypertonicity on airway inflammation. Methods We compared the inflammatory cell composition of hypertonic saline-induced sputum with that of isotonic saline-induced sputum in 21 asthmatic subjects and, at baseline and 30min after each sputum induction, we measured bronchial hyper-responsiveness to methacholine as an indirect marker to detect increased airway inflammation. On two different days, the patients inhaled hypertonic saline (3–5% NaCl) or isotonic saline (0.9% NaCl) for 30 min via an ultrasonic nebulizer, while monitoring FEV₁. Sputum was collected for inflammatory cell analysis. Results There was no difference in inflammatory cell percentages obtained with the two methods. Eosinophils were >1% in 20 subjects after hypertonic saline and in 16 subjects after isotonic saline, but this difference was not statistically significant. Intraclass correlation coefficients for sputum inflammatory cells obtained with the two methods were +0.642 for eosinophils, +0.644 for neutrophils. +0.544 for lymphocytes and +0.505 for macrophages. Hypertonic saline induced bronchoconstriction in a significantly greater number of subjects than isotonic saline. Also, hypertonic saline increased bronchial responsiveness to methacholine. while isotonic saline did not. Conclusion We conclude that hypertonicity does not affect sputum cell composition, suggesting that inflammatory cells in hypertonic saline-induced sputum are probably preexisting and not acutely recruited in the airways by the hypertonic stimulus. However, the bronchoconstriction and the increase in bronchial hyper-responsiveness after hypertonic saline inhalation may imply the release of inflammatory mediators. This fact must be considered in the evaluation of soluble markers of inflammation in hypertonic salineinduced sputum.