Functional analysis of monocyte MHC class II compartments

Abstract

Circulating monocytes, as dendritic cell and macrophage precursors, exhibit several functions usually associated with antigen-presenting cells, such as phagocytosis and presence of endosomal/lysosomal degradative compartments particularly enriched in Lamp-1, MHC class II molecules, and other proteins related to antigen processing and MHC class II loading [MHC class II compartments (MIICs)]. Ultrastructural analysis of these organelles indicates that, differently from the multivesicular bodies present in dendritic cells, in monocytes the MIICs are characterized by a single perimetral membrane surrounding an electron-dense core. Analysis of their content reveals enrichment in myeloperoxidase, an enzyme classically associated with azurophilic granules in granulocytes and mast cell secretory lysosomes. Elevation in intracellular free calcium levels in monocytes induced secretion of beta-hexosaminidase, cathepsins, and myeloperoxidase in the extracellular milieu; surface up-regulation of MHC class II molecules; and appearance of lysosomal resident proteins. The Ca(2+)-regulated surface transport mechanism of MHC class II molecules observed in monocytes is different from the tubulovesicular organization of the multivesicular bodies previously reported in dendritic cells and macrophages. Hence, in monocytes, MHC class II-enriched organelles combine degradative functions typical of lysosomes and regulated secretion typical of secretory lysosomes. More important, Ca(2+)-mediated up-regulation of surface MHC class II molecules is accompanied by extracellular release of lysosomal resident enzymes.

Figure 1.

EDBs are the major Lamp-1⁺ compartments in human monocytes. A) Ultrathin cryosection of a monocyte. Endosomal compartments are visible following HRP endocytosis and 3,3′-diaminobenzidine staining. B, C) Images (×30,000) of a typical EDB (B) and a typical MVB (C) unlabeled (left panels) or immunogold labeled for MHC class II protein (right panels). D) Quantification of the number of EDBs vs. MVB late endosomal compartments in circulating CD14⁺ monocytes or monocytes treated with GM-CSF for 2 or 7 d. Monocyte preparations from 4 different subjects were analyzed.

Figure 2.

EDBs are HLA-DR⁺ compartments. Ultrathin cryosections of GM-CSF-treated monocytes. A) Ultrathin cryosection of GM-CSF-treated monocytes and immunogold labeling of EDBs with Lamp-1 (10 nm gold). B) Immunogold labeling with Lamp-1 (15 nm gold) and HLA-DR (10 nm gold). C) Immunogold labeling for empty HLA-DR (MEM-265; 15 nm gold) and total HLA-DR (10 nm gold). Monocyte preparations from 4 different subjects were analyzed.

Figure 3.

EDBs are myeloperoxidase positive and TGF-α negative. A–D) Ultrathin cryosections of monocytes and immunogold labeling: HLA-DR (10 nm gold) and myeloperoxidase (5 nm gold) (A–C); HLA-DR (10 nm gold) and TGF-α (5 nm gold) (D). E) Confocal immunostaining for MHC class II and cathepsin G (top panels) and myeloperoxidase (bottom panels). F) β-Hexosaminidase activity measured in each fraction (1 ml) of a 10/27% 2-step Percoll gradient to separate lysosomes and late endosomes. G) Western blot analysis of pulled fractions 6–7 of the 27% Percoll gradient (lysosomes, Ly), fractions 3–4 of the 10% Percoll gradient (late endosomes, LE) and total cell lysates (total). Membranes were probed using a rabbit serum recognizing both α and β subunits of HLA-DR1 or mAb specific for Lamp-1, cathepsin S, invariant chain, and myeloperoxidase. Monocyte preparations from 4 different subjects were analyzed.

Figure 4.

EDBs behave as secretory lysosomes. A) β-Hexosaminidase detection in the cell culture supernatant after treatment of monocytes with different concentrations of ionomycin for different times. B) Lactic-dehydrogenase detection in the cell culture supernatant after treatment of monocytes with different concentrations of ionomycin for different times. C) Western blot analysis for cathepsin S and myeloperoxidase secreted in the culture supernatant after ionomycin treatment of primary monocytes. D) Surface immunostaining of monocytes untreated or after ionomycin, phorbol-12-myristate-13-acetate (PMA), or ionomycin/PMA treatment. E) Western blot analysis of MHC class II proteins following ionomycin treatment, surface biotinilation, and streptavidin agarose pulldown. F) Western blot analysis of ubiquitinated surface biotinylated MHC class II proteins. G) Surface MHC class II protein staining of monocytes nontransfected (control) or transfected with single or duplex siRNA to the AP-2 adaptor complex. Data are reported as mean fluorescence intensity. H) Western blot analysis of monocytes nontransfected (control) or transfected with single or duplex siRNA. Membrane is probed with the μ2 mAb. Each reported experiment was performed between 3 and 5 times.

Figure 5.

Ionomycin-induced remodeling of the endocytic system. A) Overview of ionomycin-induced endosomal modification by whole-mount electron microscopy. B) β-Hexosaminidase detection in the cell culture supernatant of ionomycin-treated or untreated monocytes. β-Hexosaminidase detected in total cell lysate, after protein normalization between the different samples, is shown as control. Monocyte preparations from 3 different subjects were analyzed.

Figure 6.

Surface lysosomal exocytosis following ionomycin treatment. A) Surface scanning microscopy of control or ionomycin-treated monocytes. B) Visualization of HRP-positive endosomal compartment before (control) and after (Ca²⁺) calcium-induced degranulation. Monocyte preparations from 3 different subjects were analyzed.