Accumulation of major histocompatibility complex class II molecules in mast cell secretory granules and their release upon degranulation

Abstract

To investigate the relationship between major histocompatibility complex (MHC) class II compartments, secretory granules, and secretory lysosomes, we analyzed the localization and fate of MHC class II molecules in mast cells. In bone marrow-derived mast cells, the bulk of MHC class II molecules is contained in two distinct compartments, with features of both lysosomal compartments and secretory granules defined by their protein content and their accessibility to endocytic tracers. Type I granules display internal membrane vesicles and are accessed by exogenous molecules after a time lag of 20 min; type II granules are reached by the endocytic tracer later and possess a serotonin-rich electron-dense core surrounded by a multivesicular domain. In these type I and type II granules, MHC class II molecules, mannose-6-phosphate receptors and lysosomal membrane proteins (lamp1 and lamp2) localize to small intralumenal vesicles. These 60-80-nm vesicles are released along with inflammatory mediators during mast cell degranulation triggered by IgE-antigen complexes. These observations emphasize the intimate connection between the endocytic and secretory pathways in cells of the hematopoietic lineage which allows regulated secretion of the contents of secretory lysosomes, including membrane proteins associated with small vesicles.

Figure 1

Metabolic labeling and immunoprecipitation of newly synthesized MHC class II molecules in BMMCs and B cells. BMMCs and B cells were metabolically labeled with [³⁵S]methionine for 16 h (left panel). Right panel, mast cells were pulsed for 30 min and then chased for 4 h. After cell lysis, MHC class II molecules were immunoprecipitated with the Y3P antibody recognizing mature α-β dimers or with the IN1 antibody directed against the cytoplasmic domain of the invariant chain. Before running on 12% SDS gels, samples were incubated in sample buffer at 20°C for 30 min (nonboiled conditions, nb) or at 95°C for 10 min (boiled conditions, b). The SDS-stable mature α-β dimer, named compact form, is indicated “C.”

Figure 2

Immunofluorescence localization of MHC class II, H2-M, and lamp1. MHC class II molecules were detected after cell permeabilization with rabbit polyclonal antibodies specific for the cytoplasmic domain of the I-A molecule or with the rat monoclonal M5114 (anti-I-Ab,d). Upper left and lower right panels show that, despite some heterogeneity, MHC class II molecules are localized in H-2 M and lamp1 positive compartments.

Figure 3

Ultrastructure of BMMCs. (A) Low magnification bar, 1 μm. (B) High magnification bar, 0.5 μm. The cytoplasm of BMMCs shows three types of morphologically distinct granules. Granules displaying internal vesicles (type I), granules with an electron-dense core surrounded by membrane vesicles (type II), and electron-dense granules (type III).

Figure 4

Immunogold localization of MHC class II, serotonin, and MPR. (A) Ultrathin cryosections were single-immunogold labeled with anti-class II antibody M5.114 and PAG 10. MHC class II molecules were detected in compartments displaying intralumenal membrane vesicles (type I) and in the multivesicular domain surrounding the electron-dense core of a secretory granule (type II). (B) Ultrathin cryosections were double-immunogold labeled with anti-class II antibodies (PAG 10) and antiserotonin antibodies (PAG 5). Serotonin was present in the electron-dense core whereas MHC class II labeling was restricted to the surrounding vesicles. (C) Ultrathin cryosections were double-immunogold labeled with anti-class II (PAG 10) and anti-MPR antibodies (PAG 15). MHC class II and MPR were both detected in multivesicular compartments (type I granules) and in granules displaying an electron-dense core (type II). Bars, 200 nm.

Figure 5

Immunogold localization of MHC class II, lamp1, and serotonin. (A) Ultrathin cryosections were double-immunogold labeled with anti-class II (PAG 10) and anti-lamp1 (PAG 15) antibodies. Both MHC class II molecules and lamp1 are associated with the internal membrane vesicles surrounding the electron-dense core of type II granules. (B) Ultrathin cryosections were double-immunogold labeled with anti-lamp1 (PAG 10) and antiserotonin antibodies (PAG 15). Serotonin was only detected in the electron-dense core of type II granules. lamp1 was present in the internal vesicles of multivesicular granules (type I) and intermediate granules (type II). Bars, 200 nm.

Figure 6

Kinetics of accessibility of mast cell granules to BSAG. BMMCs were pulsed for 10 min with BSA coupled to 5 nm of gold (BSAG) and then chased for 20 min (A) or 80 min (B). Ultrathin cryosections were labeled with the anti-class II antibody M5114 (PAG 10). After 20 min of chase, BSAG was detected in multivesicular compartments (type I granules) intensely labeled with anti-class II antibodies. The majority of type II granules as well as type III granules were not reached by the tracer. After 80 min of chase, the majority of class II-positive type II granules contained BSAG. Only a small percentage of type I granules still contained BSAG. Bars, 200 nm.

Figure 7

Release of membrane-associated MHC class II molecules by BMMCs during degranulation. BMMCs were incubated overnight with IgE anti-DNP antibodies, and degranulation was induced for 60 min with or without the addition of DNP-BSA. Ultrathin cryosections were immunogold labeled with anti-class II antibodies and PAG 10 (A and B). Upon degranulation, small vesicles (60–80 nm) highly labeled with anti-class II antibodies were detected at the cell surface between plasma membrane extensions (arrows). PM, plasma membrane. Bars, 200 nm.

Figure 8

BMMCs were incubated overnight with IgE anti-DNP antibodies and then degranulation was induced by addition of DNP-BSA in the presence of 1 mM CaCl₂ or 1 mM EGTA for 2 min, 5 min, or for 60 min in calcium-containing medium (Lower panels). Western blot with anti-I-A and anti-lamp1 antibodies on the 70,000-g pellets of supernatants (S-P) of mast cells degranulated with or without calcium. (Upper and middle panels) Western blot with anti-I-A and anti-li antibodies of mast cell lysates and of the 70,000-g pellets of supernatants (S-P). MHC class II molecules were recovered in S-P only from degranulated cells whereas li was not detected.