Nasal polyposis is an inflammatory condition of the nose and the sinuses characterised by a marked infiltration of eosinophils in addition to lymphocytes, mast cells and macrophages. The selective recruitment of eosinophils to inflammatory sites is mediated by CC chemokines such as Eotaxin and Eotaxin-2. In the present study histology, immunohistochemistry and ELISA were performed. The levels of Eotaxin and Eotaxin-2 and for comparison other chemokines RANTES and IL-8 were measured in nasal polyp tissue and in control nasal tissue. On histological examination 6 polyps showed an oedematous structure, one was glandular and one had a fibromatous pattern, while all showed a marked eosinophil infiltration. Immunohistochemistry of the polyps showed that epithelial cells were strongly positive for Eotaxin and IL-8, whereas endothelial cells stained positive for Eotaxin-2. Significantly higher amounts of Eotaxin, Eotaxin-2 and IL-8 were detected in polyp tissue when compared with control middle turbinates. The increased levels of eosinophil-stimulating chemokines, such as Eotaxin and Eotaxin-2 in nasal polyps suggest that they may be important regulators of eosinophil recruitment in this inflammatory disease.
BACKGROUND: Bronchial mucosal inflammation and epithelial damage are characteristic features of asthma. Activation of T helper lymphocytes may contribute to this process by mechanisms including the release of cytokines promoting eosinophil infiltration and activation. METHODS: Bronchial washings and bronchoalveolar lavage fluid were obtained from 29 atopic asthmatic patients (19 with current symptoms and 10 symptom free) and 13 normal volunteers. Flow cytometry was used to assess T cell phenotype and activation status in bronchoalveolar lavage fluid and peripheral blood, and differential cell counts were made on bronchial washings and bronchoalveolar lavage fluid. Findings were related to severity of disease as reflected by symptom scores, baseline lung function, and airway responsiveness. RESULTS: CD4 T lymphocytes in bronchoalveolar lavage fluid and blood from asthmatic patients were activated by comparison with controls (CD4 CD25, median 16.8% v 8.7% for bronchoalveolar lavage fluid, and 15.3% v 8.7% in blood). Bronchoalveolar lavage fluid CD4 T cells from both asthmatic patients and controls were of memory phenotype (95.8% and 96.8% CD45RO and 1.7% and 0.4% CD45RA respectively), whereas both CD45RO and CD45RA T cells were present in blood. Patients with asthma and current symptoms showed increased bronchoalveolar T cell activation compared with patients without symptoms (CD4 CD25 18.7% v 12.3%). Within the asthmatic group there was a significant association between CD4 CD25 lymphocytes and asthma symptom scores (rs = 0.75), airway methacholine responsiveness (log PC20, rs = -0.43) and baseline FEV1 (rs = -0.39). A correlation was also found between CD4 CD25 lymphocytes and eosinophils in bronchoalveolar lavage fluid (rs = 0.48). Eosinophils in bronchoalveolar lavage fluid were increased in asthmatic patients compared with controls and the percentage of eosinophils in bronchoalveolar lavage fluid correlated with asthma symptom score. A relation was found between percentage of epithelial cells in bronchoalveolar lavage fluid and FEV1 and methacholine PC20. CONCLUSION: These results support the hypothesis that selective activation of memory CD4 T cells contributes to eosinophil accumulation, bronchial hyperresponsiveness, and symptoms in asthma.
Our studies indicate that human eosinophils can be activated in terms of up-regulation of surface receptors, increased membrane lipid generation, and enhanced cytotoxicity by a variety of pharmacological mediators, through IgE-, IgG-, and complement-dependent pathways. PAF is an impressive activator of eosinophil function and is the most potent chemotactic mediator for eosinophils so far described. It is not selective in that neutrophils are also attracted by PAF. In our hands, IL-1, IL-2, granulocyte/macrophage-colony stimulating factor, tumor necrosis factor, interferon-gamma, and IL-5 tested over a wide concentration range gave very weak chemotaxis for eosinophils in vitro. Nevertheless, in vivo infiltration of eosinophils into the site of allergic tissue reactions is closely associated with the local accumulation of activated (CD4+, IL-2 rec+) T cells. We suggest, therefore, that the selective eosinophil infiltration seen in allergic inflammation may result from the initial recruitment and activation of granulocytes by lipid mediators and the subsequent selective activation of eosinophils by cytokines.
Abstract Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-α and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-α nor IL-5, a known eosinophil-priming agent. Priming caused activation of αMβ2 integrin; mAb against either the common β2 integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against β1 or α5, but not α4 integrin subunit, together with anti-β2 integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking α5β1 integrin increased αMβ2 integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of α5β1 integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for α5β1 integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-β2 integrin mAb, reduced adhesion to basal levels. In conclusion, α5β1 integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-α, potentiated C5a activation of αMβ2 integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for α5β1 integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.
The microanatomical structure of human and rat splenic white pulp is compared, with special emphasis on the localization of the marginal zone occupied by immunoglobulin M (IgM) + IgD −/dull B lymphocytes and its specialized macrophages. Our study reveals that in contrast to rats, the marginal zone of humans primarily exists in the vicinity of primary and secondary splenic follicles and that it is almost absent around the periarteriolar T‐cell zones. We demonstrate that in humans there is an additional compartment, the perifollicular zone, located between the marginal zone and the red pulp. The perifollicular zone is a dynamic region of variable cellular and phenotypic composition, which can be regarded either as a part of the red pulp or of the follicles. In most cases the perifollicular zone appears as a compartment of the red pulp containing erythrocyte‐filled spaces which differ from the typical red pulp sinusoids. Similar to the splenic cords, the perifollicular zone mostly harbours scattered B and T lymphocytes. However, sometimes B lymphocytes clearly predominate in the perifollicular area. In addition, strongly sialoadhesin‐positive macrophages form sheaths around capillaries in the perifollicular zone. Such capillary sheaths are not observed in rats. In humans weakly sialoadhesin‐positive macrophages are also present in the perifollicular zone and in the red pulp. In some specimens sialoadhesin is, however, strongly expressed by a large number of dispersed perifollicular macrophages. Interestingly, in striking contrast to rats, the human marginal zone does not contain sialoadhesin‐positive macrophages and marginal metallophilic macrophages are also absent in humans. Thus, sialoadhesin‐positive macrophages and IgM + IgD − memory B lymphocytes both share the marginal zone as a common compartment in rats, while they occupy different compartments in humans. We show that the human splenic marginal zone does not contain a marginal sinus and assume that in humans the perifollicular region is the compartment where antigen and recirculating lymphocytes enter the organ.
Mast cells are tissue-resident cells with important functions in allergy and inflammation. Pluripotential hematopoietic stem cells in the bone marrow give rise to committed mast cell progenitors that transit via the blood to tissues throughout the body, where they mature. Knowledge is limited about the factors that release mast cell progenitors from the bone marrow or recruit them to remote tissues. Mouse femoral bone marrow cells were cultured with IL-3 for 2 wk and a range of chemotactic agents were tested on the c-kit+ population. Cells were remarkably refractory and no chemotaxis was induced by any chemokines tested. However, supernatants from activated mature mast cells induced pronounced chemotaxis, with the active principle identified as leukotriene (LT) B4. Other activation products were inactive. LTB4 was highly chemotactic for 2-wk-old cells, but not mature cells, correlating with a loss of mRNA for the LTB4 receptor, BLT1. Immature cells also accumulated in vivo in response to intradermally injected LTB4. Furthermore, LTB4 was highly potent in attracting mast cell progenitors from freshly isolated bone marrow cell suspensions. Finally, LTB4 was a potent chemoattractant for human cord blood–derived immature, but not mature, mast cells. These results suggest an autocrine role for LTB4 in regulating tissue mast cell numbers.
In an attempt to explain the preferential accumulation of eosinophils at sites of allergic tissue reactions, we have studied the effects of interleukin-5 (IL-5) on the adherence of human eosinophils and neutrophils to plasma-coated glass (PCG) or human microvascular endothelial cells (HMVEC). IL-5 was compared with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet-activating factor (PAF), since all these agents have biological properties associated with eosinophil activation and/or survival in vitro. IL-5, IL-3 and GM-CSF induced a time-dependent increase in adherence of normal density eosinophils to PCG optimal at 60 min, whereas the effect of PAF was greater at 15 min. Similar results were obtained with neutrophils, with the exception that IL-5 had minimal and non-significant effects on this cell type. Unstimulated eosinophils and neutrophils also adhered to PCG or HMVEC, but in low numbers. Preincubation of eosinophils with IL-5, GM-CSF or PAF resulted in dose-dependent increases in the numbers of adherent cells to PCG. IL-3 had a smaller but significant effect on enhanced eosinophil adhesion to PCG, while IL-2 and lyso-PAF were ineffective. Neutrophils gave similar levels of baseline and stimulated adhesion to PCG as eosinophils, IL-5 again had no significant stimulatory effect. IL-5 also increased eosinophil, but not neutrophil, adherence to HMVEC in a concentration-dependent manner. Preincubation with the protein synthesis inhibitor cycloheximide had no effect on IL-5-, GM-CSF- or PAF-stimulated eosinophil adhesion. The contribution of the CD11/18 leucocyte integrins to IL-5- and PAF-induced eosinophil hyperadherence was investigated by inhibition experiments utilizing monoclonal antibodies (mAb). Enhanced adhesion to PCG (by PAF) or HMVEC (by IL-5) was inhibited by (ranked in order of potency) anti-CR3 alpha = common beta-chain greater than LFA-1 alpha. Anti-p150,95 alpha had no measurable effect. Baseline adhesion by unstimulated eosinophils was not significantly influenced by prior incubation with these mAb. Using flow cytometry, IL-5 and IL-3 were found to up-regulate cosinophil but not neutrophil CR3 expression. These findings demonstrate that IL-5 enhances cosinophil, but not neutrophil, adherence reactions, by a mechanism dependent, at least in part, on the CD11/18 family of adhesion glycoproteins.
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