Abstract: Fecal calprotectin (FC) is used as a marker for intestinal inflammation in inflammatory bowel disease (IBD) but there is no reliable marker for collagenous colitis (CC). We have previously demonstrated that the mucosal inflammation in CC is characterized by eosinophil activation, which is restored during budesonide treatment, but there is no enhanced neutrophil activity. The aim of this study was to evaluate the use of fecal eosinophil cationic protein (F-ECP) and eosinophil protein X (F-EPX) compared with the neutrophil-derived myeloperoxidase (F-MPO) and FC in patients treated for active CC.
Abstract: Selective release of individual eosinophil granule proteins has been demonstrated in eosinophilic conditions and in vitro using different stimuli. The aim of this study was to investigate if selective release of eosinophil cationic protein (ECP), eosinophil protein X/eosinophil derived-neurotoxin (EPX/EDN) and eosinophil peroxidase (EPO) could be due to the involvement of different signal transduction pathways. Peripheral blood granulocytes from healthy donors were incubated with Wortmannin, LY294002, Genistein, Staurosporine, GÃ6976 or PD98059 prior to the induction of degranulation by C3b. The released amounts of ECP, EPO and EPX/EDN were determined by immunoassays, and related to the total cell content of respective protein. Wortmannin caused a significant, dose-dependent inhibition of all three granule proteins. LY294002 (10â»â¶ M) also inhibited the release of all proteins. Genistein (10â»â¶ M) inhibited the release of ECP, whereas the release of EPO was increased. However, there was a tendency towards similar concentration-dependent patterns of release of all three proteins. Staurosporine (10â»â· M), GÃ6976 (10â»â¶ M) and PD98059 (10â»âµ M) caused an increased release of the three proteins. PI3-kinases play an important role in the C3b-induced release of ECP, EPO and EPX/EDN, whereas protein kinase C seems to have inhibitory effects on C3b-induced degranulation.
Abstract: The PI3K pathway plays a major role in many vital cell processes. Our primary aim was to investigate signalling through PI3K for in vitro degranulation from allergen-primed eosinophils and neutrophils in allergic rhinitis and allergic asthma after seasonal and experimental allergen challenge. Nine patients with allergic rhinitis, eight with allergic asthma and four controls were studied during birch pollen season and after nasal and bronchial allergen challenge. Primed blood eosinophils and neutrophils were stimulated for in vitro degranulation with C3b-coated Sephadex particles, after prior incubation with Wortmannin, a PI3K inhibitor. The released amounts of eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and myeloperoxidase (MPO) were measured by radioimmunoassay. Wortmannin (10(-6) to 10(-9)Â M) inhibited ECP, EPO and MPO release in a dose-dependent manner in allergic rhinitis and allergic asthma in all three allergen challenge models. Inhibition of ECP release tended to be lower in the asthmatics in all allergen challenge models, statistically significant compared to the controls during season for 10(-8)Â M Wortmannin (pâ=â0.01). A clear propensity towards less inhibition in the rhinitic patients was seen after nasal and bronchial challenge compared to seasonal exposure, significant for ECP (10(-8)Â M Wortmannin; pâ=â0.034 and 0.002, respectively). Signalling through PI3K is clearly involved in ECP, EPO and MPO release in allergic rhinitis and allergic asthma irrespective of allergen challenge model. Allergic asthma demonstrated less inhibition of ECP release via PI3K during pollen season, indicating that other pathways play a greater role in eosinophil degranulation in allergic asthma than allergic rhinitis.
Abstract: Altered intestinal barrier function has been implicated in the pathophysiology of ulcerative colitis (UC) in genetic, functional, and epidemiological studies. Mast cells and corticotropin-releasing factor (CRF) regulate the mucosal barrier in human colon. Because eosinophils are often increased in colon tissues of patients with UC, we assessed interactions among mast cells, CRF, and eosinophils in the mucosal barrier of these patients.
Abstract: The aim of this study was to assess the activity of eosinophils, neutrophils, and CD4+ as well as CD8+ T-cells in 11 patients with active collagenous colitis (CC) before and after 8 weeks of budesonide treatment (9 mg once daily) compared to 10 healthy individuals.
Abstract: The aim of this investigation was to study the involvement of eosinophil and neutrophil granulocytes in different stages of Crohn's disease (CD) and ulcerative colitis (UC). Biopsy samples were taken from the right flexure of the colon and from the rectum in patients with active (n=12) and inactive colonic CD (n=7), patients with active (n=33) and inactive UC (n=24), and from control subjects (n=11). Cell suspensions from biopsies and blood were analyzed by flow cytometry with regards to activation markers and viability. Immunohistochemistry was used to evaluate cell number and degranulation. Blood eosinophils were cultured with Th1 and Th2 cytokines, and the expression of activity markers was assessed by flow cytometry. Eosinophil number, viability, and activity were increased during active CD and UC compared with controls. The activity, assessed as CD44 expression, tended to diminish during inactive CD but was increased further in quiescent UC. Neutrophil number and activity were increased only during inflammation in both diseases. Culture of blood eosinophils with IL-5 and IL-13 caused increased CD44 expression, whereas IL-5 and IFN-gamma induced elevated CD69 expression. We observed different patterns of eosinophil activation in CD and UC, with the highest CD44 expression during quiescent UC. Our in vitro experiments with recombinant cytokines suggest that the diverse mechanisms of eosinophil activation in CD and UC are a result of different cytokine milieus (Th1 vs. Th2). In contrast, neutrophil activation reflects the disease activity in CD and UC, irrespective of Th cell skewing.
Abstract: Neutrophil granulocytes infiltrate the intestinal mucosa in active ulcerative colitis (UC), and may contribute to tissue damage and inflammation. The aim of this investigation was to study the importance of locally produced factors and the impact of steroid treatment on neutrophil functions in UC.
Abstract: Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.
Abstract: Albumin is known to induce chemokinesis and facilitate chemotaxis of human granulocytes in the Boyden chamber assay, but its mechanisms of action remain obscure. We have previously found that IL-2 inhibits albumin-stimulated eosinophil migration. The aim of this study was to identify the mechanisms behind the effects of albumin and IL-2 on the migration of human eosinophils.
Abstract: The idiopathic hypereosinophilic syndrome (HES) is characterised by blood eosinophilia associated with organ involvement. Elevated numbers of blood neutrophils have been observed during episodes of active HES. However, an increased responsiveness of eosinophils to chemotactic and chemokinetic stimuli may explain the selective eosinophil infiltration of the tissue. We have studied the migratory responses of blood eosinophils and neutrophils from 9 patients with HES and from 13 healthy control subjects. Chemokinetic and chemotactic responses to factors acting on both cell types were analysed by means of a modification of the Boyden chamber technique. We found increased migratory responses of the eosinophils, but not of the neutrophils, from the patients with HES. Increased blood neutrophil counts in three of the patients did not coincide with alterations of the neutrophil migratory responses. Our finding of increased migratory responses of eosinophils from patients with HES towards non-specific chemoattractants suggests selective priming of eosinophils in this disease. Interleukin (IL)-5 has previously been shown to prime eosinophils for migratory responses, and successful anti-IL-5 therapy of patients with HES indicates an important role for this cytokine in the development of hypereosinophilia.
Abstract: The aim of this study was to establish a method of investigating intestinal eosinophil and neutrophil granulocytes by flow cytometry, and to compare the distribution and activity of these cells in different stages of ulcerative colitis (UC).
Abstract: The role of cytokines in the accumulation of eosinophil granulocytes in inflamed tissue has been studied extensively during recent years, and these molecules have been found to participate throughout the whole process of eosinophil recruitment. Haematopoietic cytokines such as IL-3, IL-5 and GM-CSF stimulate the proliferation and differentiation of eosinophils in the bone marrow, and the release of mature eosinophils from the bone marrow into the blood is probably promoted by IL-5. Priming of eosinophils in the blood following, for example, allergen challenge is performed mainly by IL-3, IL-5 and GM-CSF. An important step in the extravasation of eosinophils is their adhesion to the vascular endothelium. Adhesion molecules are upregulated by, e.g. IL-1, IL-4, TNF-alpha and IFN-gamma and the same cytokines may also increase the affinity of adhesion molecules both on eosinophils and endothelial cells. Finally, a number of cytokines have been shown to act as eosinophil chemotactic factors, attracting the cells to the inflammatory focus in the tissue. Some of the most important eosinophil chemoattractant cytokines are IL-5, IL-8, RANTES, eotaxin, eotaxin-2, eotaxin-3, MCP-3, MCP-4 and TNF-alpha. Th2 cells, mast cells and epithelial cells are important sources of proinflammatory cytokines, but in recent years, the eosinophils have also been recognized as cytokine-producing and thereby immunoregulatory cells. The aim of this paper is to review the role of cytokines in the process of eosinophil recruitment in asthma, allergy and ulcerative colitis.
Abstract: There is an increased influx of activated eosinophils to the intestinal mucosa in active ulcerative colitis, and an increased release of eosinophil-derived proteins, such as ECP, has also been observed. These findings indicate that eosinophils may contribute to tissue damage and intestinal inflammation in this disease. The relative importance of different chemotactic factors and the impact of steroid treatment on their effect in active ulcerative colitis are not known. We measured the eosinophil chemotactic activity in perfusion fluids from 11 patients with ulcerative colitis before and after steroid treatment and from 7 control patients. The effect of neutralizing antibodies to IL-5 and -8, RANTES, eotaxin, MCP-3, TNF-alpha, GM-CSF was investigated. The chemotactic activity was higher in perfusion fluids from patients than from controls (P = 0.0043). Anti-IL-5 (P = 0.005) and -TNF-alpha (P = 0.017) inhibited the activity in perfusion fluids obtained before treatment. Steroid treatment prevented the effect of all antibodies but had no significant effect on the chemotactic activity. The chemotactic activity correlated with the levels of eosinophil granule proteins in the perfusion fluids. In conclusion, in ulcerative colitis, eosinophils are attracted to the intestinal tissue by chemotactic factors, of which IL-5 and TNF-alpha may be the most prominent steroid-sensitive ones. The steroid-insensitive chemotactic activities remain unidentified.
Abstract: Conflicting data on the role of interleukin-2 in the recruitment of eosinophil granulocytes (EOS) to sites of inflammation have been presented. The objective of the present study was to investigate the effect of recombinant human IL-2 and anti-IL-2 on the migration of purified blood EOS. Neutralizing antibodies to IL-2 were added to a cytokine mixture with significant eosinophil chemotactic activity (ECA), and afterwards the ECA was tested on EOS from both normal and allergic donors. EOS migration was measured by a modification of the Boyden technique, using a 48-well microchemotaxis chamber. Recombinant human IL-2 was either added to the lower compartment of the chemotaxis chamber, or to the EOS for a pre-incubation period of 20 min, before migration assays towards the chemotaxins were performed. Anti-IL-2 caused a significant increase of EOS migration towards the cytokine mixture. Pre-incubation of the EOS with rhIL-2 inhibited the chemotaxis towards RANTES, PAF, IL-8 and eotaxin, and EOS migration towards IL-2 was lower than that towards buffer. These effects were more pronounced on EOS from normal than from allergic donors. Priming of the EOS with IL-5 prevented the inhibitory effect of IL-2. We hypothesize that IL-2 acts as an autocrine regulator of EOS migration, and that this inhibitory effect may be downregulated in allergy, allowing an increased migration of EOS towards chemotactic factors.
Abstract: Bronchoalveolar lavage (BAL) fluid from patients with birch-pollen allergy lavaged during the season showed an elevated chemotactic activity for eosinophils compared with BAL fluid from the same patients before the start of the season.
Abstract: The study was carried out to identify those molecules that are important in vivo in the attraction of eosinophil granulocytes to the lungs of patients with asthma. Asthmatic patients with birch pollen allergy had lavages performed before and during the pollen season, and the chemotactic activity of the bronchoalveolar lavage fluid was tested against normal eosinophils. The activity was significantly increased during the pollen season as compared with the activity before the pollen season (p less than 0.01). Neutralizing antibodies to IL-2, IL-5 and IL-8, leukemia inhibitory factor, and to RANTES were added to the bronchoalveolar lavage fluid. Antibodies to IL-5 and RANTES, but not to IL-2 and IL-8 or leukemia inhibitory factor, significantly inhibited the chemotactic activity for eosinophils (p less than 0.001). It is concluded that IL-5 and RANTES are important chemoattractants in the lungs of patients with allergic asthma. The effect of IL-5 may be that of a cofactor to the chemotactic molecules, of which RANTES may be one of the most important in allergic asthma.
