Enhanced adhesion to laminin by apoptotic eosinophils

Apoptotic cells are regarded as inert bodies that turn off intracellular processes and functional capabilities. The objective was to study adhesion by eosinophils in relation to the apoptotic process. Eosinophils were cultured for up to 72 h. The living cells were separated from the apoptotic cells, and their adhesion to transfected cell lines expressing vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), E-selectin and laminin was measured. To relate the functional studies with cell structure, the surface receptor expression of beta1- and beta2-integrins was investigated by flow cytometry. Apoptotic eosinophils evidenced an increased expression of the alpha-chain of the laminin receptor and CD49f and an increased ability to adhere to a laminin-coated surface. Adhesion to the endothelial cell adhesion receptors E-selectin, VCAM-1 and ICAM-1 was absent in apoptotic eosinophils and was paralleled by a low expression of CD11b, CD29, CD49d and CD66b. The specifically increased adhesion to laminin and expression of the laminin receptor alpha-chain is a unique feature of apoptotic eosinophils. When an eosinophil goes into apoptosis, it still possesses the ability to interact with its environment. Our results point to new ideas as to how the apoptotic eosinophil behaves in apoptosis.     

Gastrointestinal eosinophils

Summary: The gut‐associated lymphoid tissue (GALT) is composed of lymphocytes residing in Peyer's patches, lamina propria, and intraepithelial compartments. In addition to these features which distinguish GALT from other peripheral sites of the immune system, the gastrointestinal immune system is also composed of resident eosinophils. Eosinophils are generally considered to be peripheral blood leukocytes that have an important pro‐inflammatory role in various immune disorders. Although most research concerning this cell has focused on understanding its trafficking and function in the blood and lung, recent studies have also started to elucidate its regulation and function in the gastrointestinal tract. Interestingly, eosinophil numbers in the gastrointestinal tract are substantially higher than in other tissues. At baseline (healthy conditions), most eosinophils reside in the lamina propria in the stomach and intestine. Eosinophil homing to these sites occurs during embryonic development and their levels in perinatal mice are comparable to those in adults, indicating that their homing is not dependent upon the presence of intestinal flora. Furthermore, eosinophil localization to the lamina propria at baseline is critically regulated by eotaxin, a chemokine constitutively expressed throughout the gastrointestinal tract. Although eotaxin is required for eosinophil homing, its expression in the esophagus is not sufficient for eosinophil accumulation, since this organ is devoid of eosinophils at baseline. During Th2‐associated inflammatory conditions (e.g. interleukin (IL)‐5 overexpression or oral allergen challenge), marked increases of eosinophils occur not only in the lamina propria but also in Peyer's patches. The accumulation of Peyer's patch eosinophils, which mainly occurs in the outer cortex and interfollicular regions, is critically regulated by IL‐5 and less significantly by eotaxin, suggesting the involvement of other eosinophil chemokines in this lymphoid compartment. Preliminary investigations have shown that gastrointestinal eosinophils express the α4β7 integrin and that this molecule is responsible, in part, for eosinophil homing. In summary, eosinophils are resident cells of the gastrointestinal immune system whose levels can be induced by antigen exposure under Th2 conditions, in a manner that is critically regulated by eotaxin and IL‐5. We propose that eosinophils are integral members of the gastrointestinal immune system and are likely to be important in innate, regulatory and inflammatory immune responses. This work was supported in part by the National Health Medical Research Council (Australia) C.J. Martin Post‐doctoral Fellowship (S.P.H.), the Jaffe Family Fund of the American Academy of Allergy, Asthma, and Immunology (S.P.H.), NIH grant R01 AI45898 (M.E.R.) and the Human Frontier Science Program (M.E.R.). The authors wish to thank Drs. K. Frank Austen, Mitchell Cohen, Paul Foster, Glenn Furuta, and Nives Zimmermann for helpful discussions, as well as numerous other colleagues.     

GM-CSF regulation in eosinophils

Allergic asthma is characterized by a temporally and quantitatively inappropriate immunologic response. One of the hallmarks of this response is the accumulation of eosinophils in the airway and lung parenchyma, which results in broncho-constriction, lung damage and, ultimately, fibrosis. Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a pivotal role in this process by modulating eosinophil function and survival. In this review, we discuss the effects and molecular regulation of GM-CSF secretion by eosinophils. Recent data demonstrate that activated eosinophils release small amounts of anti-apoptotic GM-CSF by stabilizing its coding mRNA.     

DNA probes bind non-specifically to eosinophils during in situ hybridization: carbol chromotrope blocks binding to eosinophils but does not inhibit hybridization to specific nucleotide sequences

Eosinophils were found to bind radiolabelled DNA probes non-specifically during in situ hybridization. Pretreatment of cells with carbol chromotrope blocked non-specific binding without interfering with the recognition of specific nucleotide sequences.     

A method to study apoptosis in eosinophils by flow cytometry

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.     

Hypodense eosinophils are much labeled with 32P than normodense eosinophils]

Peripheral blood eosinophils from the patients with atopic dermatitis were isolated on a Percoll gradient and incubated with H3(32)PO4. After stopping the reaction, SDS/PAG electrophoresis was performed and autoradiographs were prepared to determine the incorporation of 32P into proteins. Eosinophils developed at least 14 protein bands below 66.2 K by SDS/PAG electrophoresis and the differences of the staining patterns between hypodense and normodense eosinophils were observed. In the autoradiographs 5 distinct radioactive bands were observed below 31 K. 32P incorporation into the bands of hypodense eosinophils were stronger than that of normodense eosinophils, suggesting possible involvement of protein phosphorylation in the activation process of eosinophils.     

Degranulation of eosinophils by IgG antibody to Candida antigen]

The pathophysiological role of IgG antibody to fungi antigen widely distributed in environment such as Candida albicans in bronchial asthma has not been clarified. Wells of microtiter plate were coated with the extract of Candida albicans and then IgG antibody was immobilized on the wells by incubation with patient's serum. After cultivation of eosinophils on the well, degranulation of eosinophils, as assessed by quantitation of EPX in the supernatant, has been observed. Degranulation was completely abrogated after depletion of IgG in the serum and also decreased by incubation of the cells with anti-CD32 antibody, or anti-CD18 antibody, but not anti-CD23 antibody. Immune complex, which had been prepared by incubation of the extract of Candida albicans with patient's serum, also evoked degranulation of eosinophils. We have examined whether degranulation can be induced by two purified antigens of Candida albicans, i.e., mannan A and acid protease. IgG antibody to acid protease was detected at no or minimal levels in most sera and the antigen did not induce degranulation. On the other hand, mannan A induced degranulation. This observation may be due to response for the presence of IgG antibody to mannan A in the sera. These results suggest that immobilized IgG induced degranulation of eosinophils through Fc gamma RII (CD 32) on eosinophils and mannan A is a major allergen associated with IgG-induced eosinophil degranulation.