Biomedical subjects
K Zeibecoglou
Publications and source records attributed to K Zeibecoglou.
Induced sputum in asthma.
This is a short review of the use of induced sputum in bronchial asthma. The clinical applications, the procedure of this method as well as the safety validity and reproducibility of the results are discussed in detail. It is shown that sputum induction is a practical and safe procedure and an important research tool of local inflammation of bronchial asthma.
Basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin.
BACKGROUND: Previous studies used indirect methods to identify basophils in the bronchi in asthma, and the numbers were not compared with eosinophils and mast cells. Furthermore, differences in basophil numbers between atopic and nonatopic asthma at baseline and between late-phase skin and asthmatic reactions have not been previously documented. OBJECTIVE: The basophil granule-specific mAb BB1 was used to identify basophils in (1) bronchial biopsy specimens from atopic asthmatic subjects and nonatopic asthmatic subjects and control subjects, (2) biopsy specimens from atopic asthmatic subjects before and after inhalational allergen challenge, and (3) late-phase skin reactions. Basophil numbers were compared with EG2(+) eosinophils and tryptase(+) mast cells. METHODS: Cells were enumerated in bronchial and skin biopsy specimens by means of immunohistochemistry with the alkaline phosphatase-antialkaline phosphatase method. RESULTS: There were elevated numbers of basophils in baseline biopsy specimens in atopic asthmatic subjects compared with atopic control subjects or normal control subjects, although eosinophils and mast cells were 10-fold higher. There was an intermediate number of basophils in nonatopic asthmatic subjects. Basophils increased after allergen inhalation, but again basophils were less than 10% of eosinophils. In contrast, basophils in cutaneous late-phase reactions were approximately 40% of infiltrating eosinophils. The peak of basophil accumulation was at 24 hours, whereas maximal eosinophil infiltration occurred at 6 hours. One third of cutaneous basophils had morphologic appearances suggestive of degranulation. CONCLUSION: Numerous basophils infiltrated cutaneous late-phase reactions in atopic subjects. However, this cell was not prominent in bronchial biopsy specimens of asthmatic subjects, either at baseline or after allergen challenge.
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.
Macrophage subpopulations and macrophage-derived cytokines in sputum of atopic and nonatopic asthmatic subjects and atopic and normal control subjects.
BACKGROUND: Previous studies have shown a prominent macrophage signal in the bronchial mucosa from nonatopic (intrinsic) compared with atopic (extrinsic) asthmatic subjects. This observation might have represented an expansion of a proinflammatory macrophage population or a homeostatic mechanism to decrease T(H)2-type inflammation. OBJECTIVE: The aim of the study was to investigate the numbers of macrophages and macrophage subpopulations and the expression of IL-10 and IL-12 in sputum from asthmatic and control subjects. METHODS: Eight atopic asthmatic (AA) subjects, 10 nonatopic asthmatic (NAA) subjects, 6 atopic control (AC) subjects, and 7 normal control (NC) subjects underwent sputum induction. Macrophages were enumerated by using Romanowsky stain and immunocytochemistry (CD68). RFD1 (interdigitating cell marker) and RFD7 (mature phagocyte marker) mAbs were used for immunocytochemical phenotyping, whereas IL-10 and IL-12 messenger (m)RNA was examined with in situ hybridization by using (35)S-labeled riboprobes. The phenotype of cells expressing IL-10 or IL-12 mRNA was examined by simultaneous in situ hybridization and immunostaining. RESULTS: No differences in the numbers of CD68(+) macrophages and RFD1(+), RFD7(+), and RFD1(+)/RFD7(+) subpopulations were found between AA, NAA, AC, and NC subjects. However, the numbers of IL-10 and IL-12 mRNA(+) cells were increased in AA subjects compared with NAA, AC, and NC subjects (P <.05). No other differences were found among the groups. Most of the IL-10 and IL-12 mRNA(+) cells in sputum from asthmatic subjects were macrophages (>80%), with less than 10% of mRNA colocalizing to epithelial cells. CONCLUSIONS: Sputum macrophage numbers, unlike tissue macrophages, as previously reported, were not elevated in NAA subjects. Increased IL-10 and IL-12 expression in atopic asthma may indicate the existence of a homeostatic mechanism to decrease lung inflammation. The lack of such cytokines in intrinsic asthma may predispose to bronchial inflammation in these subjects.
Eosinophil chemotactic chemokines (eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), and MCP-4), and C-C chemokine receptor 3 expression in bronchial biopsies from atopic and nonatopic (Intrinsic) asthmatics.
Atopic (AA) and nonatopic (NAA) asthma are characterized by chronic inflammation and local tissue eosinophilia. Many C-C chemokines are potent eosinophil chemoattractants and act predominantly via the CCR3. We examined the expression of eotaxin, eotaxin-2, RANTES, monocyte chemoattractant protein-3 (MCP-3), MCP-4, and CCR3 in the bronchial mucosa from atopic (AA) and nonatopic (intrinsic; NAA) asthmatics and compared our findings with atopic (AC) and nonatopic nonasthmatic controls (NC). Cryostat sections were processed for immunohistochemistry (IHC), in situ hybridization (ISH), and double IHC/ISH. Compared with AC and NC, the numbers of EG2+ cells and the cells expressing mRNA for eotaxin, eotaxin-2, RANTES, MCP-3, MCP-4, and CCR3 were significantly increased in AA and NAA (p < 0.01). Nonsignificant differences in these variants were observed between AA and NAA and between AC and NC. Significant correlations between the cells expressing eotaxin or CCR3 and EG2+ eosinophils in the bronchial tissue were also observed for both AA (p < 0.01) and NAA (p = 0.01). Moreover, in the total asthmatic group (AA + NAA) there was a significant inverse correlation between the expression of eotaxin and that of the histamine PC20 (p < 0.05). Sequential IHC/ISH showed that cytokeratin+ epithelial cells, CD31+ endothelial cells, and CD68+ macrophages were the major sources of eotaxin, eotaxin-2, RANTES, MCP-3, and MCP-4. There was no significantly different distribution of cells expressing mRNA for these chemokines between atopic and nonatopic asthma. These findings suggest that multiple C-C chemokines, acting at least in part via CCR3, contribute to bronchial eosinophilia in both atopic and nonatopic asthma.
Increased mature and immature CCR3 messenger RNA+ eosinophils in bone marrow from patients with atopic asthma compared with atopic and nonatopic control subjects.
BACKGROUND: Eosinophil infiltration of the bronchial mucosa is characteristic of asthma. Eosinophils differentiate from CD34(+) progenitors. Animal models suggest cooperation between IL-5 and eotaxin to allow rapid mobilization of a pool of bone marrow eosinophils followed by recruitment to the airway mucosa. OBJECTIVE: The purpose of this study was to enumerate CD34(+) cell numbers in blood and bone marrow from atopic asthmatics and control subjects and to test the hypothesis that there is an increased bone marrow pool of CCR3(+) eosinophils in patients with atopic asthma, as compared with control subjects. METHODS: Bone marrow aspirates and peripheral blood were obtained from volunteers with asthma and control volunteers. CD34(+) cell numbers were evaluated by flow cytometry, and eosinophil colony-forming activity was evaluated by methylcellulose cultures. Mature eosinophils, eosinophil myelocytes, metamyelocytes, and band forms (immature eosinophils) were enumerated by morphologic findings and immunocytochemistry for eosinophil cationic protein. CCR3 and eotaxin mRNA expression was examined by in situ hybridization, and protein expression was examined by immunocytochemistry. CCR3(+) cells were further identified with Chromotrope 2R staining. RESULTS: CD34(+) cell numbers in bone marrow were increased in atopic subjects. Numbers of eosinophil colony-forming units in blood and bone marrow did not differ between groups. Percentages of both mature and immature eosinophils were increased in bone marrow from patients with atopic asthma, but not atopic patients with no asthma or normal control subjects. CCR3 was expressed by immature and mature bone marrow eosinophils. Eotaxin was expressed by bone marrow cells from all 3 groups, but there was no increase in subjects with asthma. CONCLUSION: These findings suggest that in humans there is an increased bone marrow pool of CCR3(+) mature and immature eosinophils available for rapid mobilization in subjects with asthma but not in atopic subjects with no 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.
CD34(+)/interleukin-5Ralpha messenger RNA+ cells in the bronchial mucosa in asthma: potential airway eosinophil progenitors.
Eosinophil differentiation is thought to occur by the action of interleukin (IL)-5 on CD34(+) progenitor cells. The allergen-induced increase in eosinophil numbers in isolated airway preparations in vitro, and detection of increased numbers of circulating CD34(+) cells in atopic subjects, led us to the hypothesis that the eosinophil infiltration of the airway in asthma may result from local mucosal differentiation, in addition to recruitment from the bone marrow. We examined CD34(+) cell numbers by immunohistochemistry and IL-5 receptor alpha (IL-5Ralpha) messenger RNA (mRNA) expression by in situ hybridization in bronchial biopsies from atopic asthmatic patients, and from atopic and nonatopic control subjects. CD34(+) cell numbers were increased in the airway in atopic asthmatic and atopic nonasthmatic subjects. In contrast, CD34(+)/ IL-5Ralpha mRNA+ cells were increased in asthmatic subjects when compared with both atopic and nonatopic control subjects. Airway numbers of CD34(+)/IL-5Ralpha mRNA+ cells were correlated to airway caliber in asthmatic subjects and to eosinophil numbers. These findings support the concept that eosinophils may differentiate locally in the airway in asthma.
IL-3, IL-5, granulocyte-macrophage colony-stimulating factor receptor alpha-subunit, and common beta-subunit expression by peripheral leukocytes and blood dendritic cells.
BACKGROUND: IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors consist of cytokine-specific alpha-subunits, which associate with a shared signalling common beta-subunit (beta(c)) to form a high-affinity complex. The expression of IL-3, IL-5, and GM-CSF is upregulated in atopic inflammation, and these cytokines are thought to contribute to pathology through mechanisms that include eosinophil activation. OBJECTIVE: We sought to examine the distribution of receptor expression between cells relevant to allergic inflammation from individual subjects and to compare atopic and nonatopic individuals. METHODS: Peripheral blood was obtained from atopic and nonatopic volunteers. Cytokine-receptor expression was examined by flow cytometry with monoclonal antibodies specific for alpha-subunits and beta(c) in combination with phenotypic markers for eosinophils, basophils, neutrophils, dendritic cells, monocytes, and T cells. RESULTS: Using a ligand-independent system, we confirmed the cellular distribution of IL-5Ralpha, IL-3Ralpha, and GM-CSFRalpha. IL-3Ralpha and GM-CSFRalpha were detected on high-affinity IgE receptor blood dendritic cells. Beta(c) expression was detected on basophils, eosinophils, neutrophils, and, at low levels, on monocytes and dendritic cells. There was intense staining of basophils for IL-3Ralpha relative to IL-5Ralpha, GM-CSFRalpha, and beta(c), whereas eosinophil-staining intensity was similar for IL-3Ralpha, IL-5Ralpha, GM-CSFRalpha, and beta(c). There were no significant differences between atopic and nonatopic subjects in cytokine-receptor staining. CONCLUSION: IL-3Ralpha and GM-CSRalpha are shown on a newly defined population of Fc(epsilon)RI-high dendritic cells. The intense staining of basophils for IL-3Ralpha, relative to that of IL-5Ralpha and GM-CSFRalpha, is in contrast to eosinophils from the same subjects and may explain the higher sensitivity of basophils to IL-3 compared with IL-5 and GM-CSF. We found no evidence for downregulation of receptor expression in atopic compared with nonatopic subjects, suggesting that these receptors remain accessible as potential targets for therapeutic intervention in atopic allergic disease.
Prevalence of asthma and asthma-like symptoms in Athens, Greece.
Asthma is a common chronic condition and there is evidence that its prevalence may be rising. The European Respiratory Health Survey (ECRHS) was planned to produce comparable data on asthma epidemiology in Europe. In Greece, in particular, a similar study has not been conducted previously and no epidemiological data are available on adult asthma prevalence. Furthermore, the role of air pollution in the pathogenesis of asthma is currently an issue of debate. Athens is a city with high air pollution, and a study of asthma epidemiology in this city may confirm or refute a possible link with the expression of asthma. A questionnaire developed by the ECRHS was sent to a random sample of 3533 households in Peristeri, an industrialized borough of Athens. Responses were received from 2774 households (response rate 78%). Of all the data on individual subjects, only those from 3325 adults aged 20-44 years were considered, according to the study protocol. The self-reported current prevalence of asthmatic attacks and asthma-like symptoms were as follows: asthma attack 2.4%, use of asthma medication 2.1%, awakening by shortness of breath 5.6%, awakening by cough 17.8%, wheezing 15.8% and nasal allergies 18.4%. It is concluded that the prevalence of asthma and related symptoms in this region of Athens is rather low, despite the high air-pollution levels in the city.
Eosinophil development and bone marrow and tissue eosinophils in atopic asthma.
BACKGROUND: Eosinophils develop from bone marrow (BM) progenitors, and interleukin-5 (IL-5) and eotaxin may act in expansion and mobilisation of BM eosinophils in asthma. METHODS: We have examined phenotypic changes as CD34+ cells develop to the eosinophil lineage in vitro, and have evaluated BM eosinophils from asthmatic and control subjects for expression of the eotaxin receptor, CCR3. RESULTS: Acquisition of receptors for IL-5 and CCR3 was an early event in eosinophil development. There were increased CD34+ cells, and mature and immature CCR3+ eosinophils in BM from asthmatics. CONCLUSION: These data suggest that IL-5 may act early in eosinophil development, and that eotaxin has the capacity to mobilise a BM eosinophil pool in asthma.