MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

Abstract

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-b-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD41 T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.Keywords: eosinophils; antigen presentation; HLA-DR; CD9; immunoelectron microscopy Eosinophils have been increasingly recognized to have important immunoregulatory functions that extend beyond their traditional characterization as end-stage mediators of antiparasitic activity and of allergic inflammation (1). One such function is the ability of eosinophils to act as antigen-presenting cells (APCs). When exposed to the proper stimuli, human and murine eosinophils have been demonstrated to express MHC Class II and the requisite costimulatory molecules necessary to act as professional APCs (2-4). Moreover, murine eosinophils have been shown to act as professional APCs, as defined by their ability to stimulate naive antigen-specific T cells in in vitro experiments and in murine models (5-7).Detergent-resistant membrane microdomains (DRMs), often referred to as lipid rafts, have important roles in the organization and activity of MHC Class II molecules in a variety of APCs (8). Cross-linking HLA-DR results in colocalization with markers of DRMs microscopically in a myelomonocytic cell line (9). MHC Class II in murine B cells has been observed in DRMs, with DRM disruption inhibiting their ability to present antigen (10). In human monocyte-derived dendritic cells (DCs), HLA-DR similarly coaggregates with DRM markers when cross-linked, and biochemical disruption of DRMs inhibits T-cell activation by DCs (11). The ...

Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes

“…1 and 5b; ref. 24), as well as cytoplasmic distribution of varied proteins, such as CD63 (ref. 17; Fig.…”

“…17,24,26) and protein disulfide isomerase (PDI) 27 in leukocytes. We demonstrated that CD9 is abundant on the surface of eosinophil leukocytes, presenting the first EM data of the ultrastructural immunolocalization of CD9 in human eosinophils, which represents a novel insight into the organization of the antigen presentation complex of these cells 24 . The technique has also been applied to the study of molecules involved in the endocytic pathway such as Rab7 and LC3, labeled in phagosomes within macrophages infected with mycobacteria (R.C.N.M., unpublished observations).…”

“…Our methodology, a combination of different strategies for optimal labeling and morphology preservation, has been used for almost 10 years (refs. 17,20–24,27). Figure 2 presents a summary of all the stages of the protocol.…”

Exosome secretion by eosinophils: A possible role in asthma pathogenesis

“…CD9 is an intracellular and cell surface protein abundant in eosinophils to such an extent as to be considered an eosinophil marker. 49 In fact, CD9 serves as a molecule that delivers stimulation signals on eosinophils. 50 Several authors have found decreasing CD9 expression in eosinophils on activation, a phenomenon probably caused by release/shedding of soluble forms of CD9 and not caused by intracellular degradation.…”

Cholesterol Selectively Regulates IL-5 Induced Mitogen Activated Protein Kinase Signaling in Human Eosinophils

“…The potential for CRMM disruption to provide separation of distinct intracellular responses to the same extracellular signal is highlighted by recent studies demonstrating that while the IL-5 receptors localize to both CRMMs and detergent-soluble membrane regions in eosinophils, the localization determines which intracellular signaling proteins are directly associated with the receptor [42], [43]. Lei and colleagues found that IL-5Rs localized to CRMMs co-immunoprecipitate with the membrane-anchored MAPK signaling cascade initiator, Lyn tyrosine kinase.…”