Abstract
Astrocytes are the key homeostatic cells in the central nervous system; initiation of reactive astrogliosis contributes to neuroinflammation. Pro-inflammatory cytokine interferon γ (IFNγ) induces the expression of the major histocompatibility complex class II (MHCII) molecules, involved in antigen presentation in reactive astrocytes. The pathway for MHCII delivery to the astrocyte plasma membrane, where MHCII present antigens, is unknown. Rat astrocytes in culture and in organotypic slices were exposed to IFNγ to induce reactive astrogliosis. Astrocytes were probed with optophysiologic tools to investigate subcellular localization of immunolabeled MHCII, and with electrophysiology to characterize interactions of single vesicles with the plasmalemma. In culture and in organotypic slices, IFNγ augmented the astrocytic expression of MHCII, which prominently co-localized with lysosomal marker LAMP1-EGFP, modestly co-localized with Rab7, and did not co-localize with endosomal markers Rab4A, EEA1, and TPC1. MHCII lysosomal localization was corroborated by treatment with the lysosomolytic agent glycyl-l-phenylalanine-β-naphthylamide, which reduced the number of MHCII-positive vesicles. The surface presence of MHCII was revealed by immunolabeling of live non-permeabilized cells. In IFNγ-treated astrocytes, an increased fraction of large-diameter exocytotic vesicles (lysosome-like vesicles) with prolonged fusion pore dwell time and larger pore conductance was recorded, whereas the rate of endocytosis was decreased. Stimulation with ATP, which triggers cytosolic calcium signaling, increased the frequency of exocytotic events, whereas the frequency of full endocytosis was further reduced. In IFNγ-treated astrocytes, MHCII-linked antigen surface presentation is mediated by increased lysosomal exocytosis, whereas surface retention of antigens is prolonged by concomitant inhibition of endocytosis.
Highlights
Astrocytes, a class of morphologically and functionally heterogeneous neuroglial cells maintain homeostasis at all levels of organization of the central nervous system (CNS).1 3 Vol.:(0123456789)Astroglial cells regulate transport of water and ions, provide metabolic support, participate in neurotransmission and synaptic connectivity, regulate microcirculation, and help preserve the integrity of the blood–brain barrier [1]
We directly demonstrated that cell treatment with interferon γ (IFNγ) alters the fusion pore geometry and kinetics in vesicles reversibly interacting with the plasmalemma and favors reversible exocytosis of larger, lysosome-like vesicles accompanied by a simultaneous decrease of full vesicle endocytosis
Our study reveals that astroglial major histocompatibility class II (MHCII) localizes only negligibly to early and recycling endosomes carrying Rab4A, EEA1, and TPC1 [24, 25] and modestly to compartments carrying Rab7, which regulates transport from early to late endolysosomes and plays a role in autophagosome maturation [24]
Summary
Astrocytes, a class of morphologically and functionally heterogeneous neuroglial cells maintain homeostasis at all levels of organization of the central nervous system (CNS).1 3 Vol.:(0123456789)Astroglial cells regulate transport of water and ions, provide metabolic support, participate in neurotransmission and synaptic connectivity, regulate microcirculation, and help preserve the integrity of the blood–brain barrier [1]. Exposure to IFNγ induces expression of MHCII molecules in non-professional antigen-presenting cells, such as astrocytes that do not constitutively express MHCII [10,11,12] This process was demonstrated in primary cultured astrocytes from rodents [13,14,15,16] and humans [17, 18]. While astrocytes are capable of activating T cells during EAE, astrocytic MHCII molecules are not required for disease induction, but most likely play a complex (destructive) role in potentiation and exacerbation of ongoing disease [9, 10] It is still unknown how MHCII molecules are relocated to the cell surface, where they perform their function. The detailed subcellular localization and specific mechanism(s) by which MHCII molecules translocate to and are retained at the cell surface have never been studied
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