Salahaddin Mahmudi-Azer

Eosinophil granules function extracellularly as receptor-mediated secretory organelles

Intracellular granules in several types of leukocytes contain preformed proteins whose secretions contribute to immune and inflammatory functions of leukocytes, including eosinophils, cells notably associated with asthma, allergic inflammation, and helminthic infections. Cytokines and chemokines typically elicit extracellular secretion of granule proteins by engaging receptors expressed externally on the plasma membranes of cells, including eosinophils. Eosinophil granules, in addition to being intracellular organelles, are found as intact membrane-bound structures extracellularly in tissue sites of eosinophil-associated diseases. Neither the secretory capacities of cell-free eosinophil granules nor the presence of functional cytokine and chemokine receptors on membranes of leukocyte granules have been recognized. Here, we show that granules of human eosinophils express membrane receptors for a cytokine, IFN-γ, and G protein–coupled membrane receptors for a chemokine, eotaxin, and that these receptors function by activating signal-transducing pathways within granules to elicit secretion from within granules. Capacities of intracellular granule organelles to function autonomously outside of eosinophils as independent, ligand-responsive, secretion-competent structures constitute a novel postcytolytic mechanism for regulated secretion of eosinophil granule proteins that may contribute to eosinophil-mediated inflammation and immunomodulation.

Mast cells express connexins on their cytoplasmic membrane

BACKGROUND Because of the close association between mast cells and fibroblasts in the microenvironment and the importance of connexins (Cxs) in fibroblast communication with other cells, we hypothesized that mast cells also express Cxs, allowing them to similarly communicate with other cells through gap junctions. OBJECTIVES We sought to identify the expression of Cxs (particularly Cx43, Cx32, and Cx26) by murine mast cells. METHODS The expression of Cxs was studied by RT-PCR, Northern blot analysis, Western blot analysis, flow cytometry, and confocal laser scanning microscopy. RESULTS In this report we demonstrate that murine bone marrow cultured mast cells and the growth factor-independent murine mast cell line C57, express Cx43 and Cx32 as assessed by RT-PCR, Northern blot analysis, Western blot analysis, and flow cytometry, but do not express Cx26. We also show, by confocal laser scanning microscopy, that Cx43 localizes to the cytoplasmic membrane of mast cells in a pattern similar to that seen in fibroblasts. CONCLUSIONS Mast cells express Cx43 and Cx32, and Cx43 is associated with the cytoplasmic membrane, suggesting that mast cells have the potential to communicate with other cells in their microenvironment in part through gap junctions.

Inhibition of nonspecific binding of fluorescent-labelled antibodies to human eosinophils.

Eosinophils and their products play a major role in inflammatory reactions associated with asthma and allergic diseases. There is a growing body of evidence that eosinophils synthesize, store, and release bioactive cytokines and chemokines with the potential to contribute to local inflammatory changes. Fluorescein isothiocyanate (FITC) has been widely used as an immunofluorescent conjugate for antibodies specific for detection of these molecules. However, FITC is an ionic fluorochrome (negatively charged) which binds strongly to positively charged eosinophil granule proteins. We developed new methods to prevent charge-based interactions of ionic fluorochromes with granule proteins, and optimised immunofluorescent staining techniques for eosinophils. An antibody to interleukin-6 (IL-6) was used to optimise this procedure for eosinophil-derived granule proteins. We attempted to block nonspecific binding of FITC-labelled anti-IL-6 using normal human IgG, foetal calf serum (FCS), bovine serum albumin (BSA), and goat, horse, and normal human sera at concentrations ranging between 1-10%. Only human IgG (2%; 20 mg/ml) was able to reduce background fluorescence. These results were confirmed using Texas Red conjugates. We also used antibodies conjugated to a nonionic fluorochrome, BODIPY FL, to detect IL-6 in eosinophils. Unlike FITC, BODIPY FL-conjugated antibodies did not require strong blocking conditions (2% BSA). We recommend that a neutral fluorochrome (BODIPY FL) should be used for immunofluorescence studies in eosinophils. Alternatively, strong blocking conditions may be used to decrease background binding of FITC-conjugated antibodies.

Interleukin-4 and RANTES expression in maturing eosinophils derived from human cord blood CD34+ progenitors

Eosinophils elaborate a number of proinflammatory mediators, including immunoregulatory cytokines and chemokines. Interleukin (IL)‐4 and RANTES are important cytokines that have previously been shown to be expressed by mature eosinophils. We hypothesized that de novo synthesis of IL‐4 and RANTES occurs in nascent eosinophils, leading to storage of newly produced proteins in crystalloid granule‐like structures. Cytokine mRNA and protein expression were examined in cultured eosinophil colonies, which were derived from purified cord blood CD34+ cells and generated in semisolid media (methylcellulose) in the presence of recombinant human (rh)IL‐3 and rhIL‐5. Cytokine mRNA profiles were analysed by the reverse transcription–polymerase chain reaction (RT–PCR) to determine transcription of IL‐4 and RANTES in cells on days 0, 7, 14, 21 and 28 of culture. The expression of translated cytokine products and granule major basic protein (MBP) was confirmed, from day 23 onwards, for colonies cultured in semisolid media, by immunofluorescent labelling and confocal laser‐scanning microscopy (CLSM). We found that mRNA sequences encoding IL‐4 and RANTES were expressed in freshly prepared, non‐differentiated CD34+ cells. Furthermore, RANTES mRNA localized to carbol chromotrope 2R‐positive colony cells, as assessed using in situ RT–PCR on day 21 of culture in semisolid media, and was found to gradually decrease (relative to β2‐microglobulin) in rhIL‐3‐ and rhIL‐5‐treated colony cells (comprising > 90% eosinophil‐like cells) up to day 28. Immunoreactivity for IL‐4 and RANTES co‐localized with MBP in maturing colony eosinophils on day 23 of culture in semisolid media, as judged by CLSM. These results suggest that synthesis and storage of immunoregulatory cytokines, essential for processes associated with adaptive immunity, occurs in nascent eosinophils during their growth and differentiation.

Connexin 43 expression on peripheral blood eosinophils: role of gap junctions in transendothelial migration.

Eosinophils circulate in the blood and are recruited in tissues during allergic inflammation. Gap junctions mediate direct communication between adjacent cells and may represent a new way of communication between immune cells distinct from communication through cytokines and chemokines. We characterized the expression of connexin (Cx)43 by eosinophils isolated from atopic individuals using RT-PCR, Western blotting, and confocal microscopy and studied the biological functions of gap junctions on eosinophils. The formation of functional gap junctions was evaluated measuring dye transfer using flow cytometry. The role of gap junctions on eosinophil transendothelial migration was studied using the inhibitor 18-a-glycyrrhetinic acid. Peripheral blood eosinophils express Cx43 mRNA and protein. Cx43 is localized not only in the cytoplasm but also on the plasma membrane. The membrane impermeable dye BCECF transferred from eosinophils to epithelial or endothelial cells following coculture in a dose and time dependent fashion. The gap junction inhibitors 18-a-glycyrrhetinic acid and octanol did not have a significant effect on dye transfer but reduced dye exit from eosinophils. The gap junction inhibitor 18-a-glycyrrhetinic acid inhibited eosinophil transendothelial migration in a dose dependent manner. Thus, eosinophils from atopic individuals express Cx43 constitutively and Cx43 may play an important role in eosinophil transendothelial migration and function in sites of inflammation.

Effects of Interferon–γ on Mobilization and Release of Eosinophil–Derived RANTES

Eosinophils synthesize and release a number of cytokines and chemokines, including RANTES, a potent chemoattractant particularly for memory T cells and eosinophils. Long–term (>12 h) incubation with interferon–γ (IFN–γ) has been shown to activate eosinophils and induce expression of membrane receptors. We hypothesized that IFN–γ mobilizes intracellular RANTES in eosinophils in advance of mediator release. Highly purified peripheral blood eosinophils were obtained from asthmatics and stimulated with IFN–γ at 500 U/ml for time course analysis up to 2h. By specific ELISA, RANTES was detected in supernatants (80±15 pg per 2×106 cells) following 120min of stimulation. Immunoreactive RANTES in resting cells (5×107 eosinophils) was detected in two intracellular compartments in studies of subcellular fractionation by density gradient centrifugation. After 10 min IFN–γ stimulation, RANTES immunoreactivity was confined to crystalloid granules. RANTES was redistributed from secretory granules to light–membrane fractions after 60 min of IFN–γ incubation. Our data suggest that rapid mobilization and release of RANTES occurs from stimulated eosinophils. These findings may have important implications for the role of IFN–γ in activating human eosinophils, particularly in severe chronic asthma or viral exacerbation of asthmatic inflammation, where this cytokine may play a role.

Tracing Intracellular Mediator Storage and Mobilization in Eosinophils

In this chapter, we will describe two different techniques used to trace storage and mobilization of intracellular granule-derived mediator proteins in eosinophils. The first is confocal laser scanning microscopy (CLSM) used to investigate immunofluorescence labeling in cytospins, and the second is subcellular fractionation, leading to the generation of fractions that may be analyzed for their organelle elution profiles using appropriate protein, enzyme, and organelle marker assays.