Major histocompatibility complex class II molecules induce the formation of endocytic MIIC-like structures.

During biosynthesis, major histochompatibility complex class II molecules are transported to the cell surface through a late endocytic multilaminar structure with lysosomal characteristics. This structure did not resemble any of the previously described endosomal compartments and was termed MIIC. We show here that continuous protein synthesis is required for the maintenance of MIIC in B cells. Transfection of class II molecules in human embryonal kidney cells induces the formation of multilaminar endocytic structures that are morphologically analogous to MIIC in B cells. Two lysosomal proteins (CD63 and lamp-1), which are expressed in MIIC of B cells, are also present in the structures induced by expression of major histocompatibility complex class II molecules. Moreover, endocytosed HRP enters the induced structures defining them as endocytic compartments. Exchanging the transmembrane and cytoplasmic tail of the class II alpha and beta chains for that of HLA-B27 does not result in the induction of multilaminar structures, and the chimeric class II molecules are now located in multivesicular structures. This suggests that expression of class II molecules is sufficient to induce the formation of characteristic MIIC-like multilaminar structures.

Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface.

Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway.

Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments.

The HLA-DM genes encode an unconventional HLA (human leukocyte antigen) class II molecule that is required for appropriate binding of peptide to classical HLA class II products. In the absence of DM, other class II molecules are unstable upon electrophoresis in sodium dodecyl sulfate and are largely associated with a nested set of peptides derived from the invariant chain called CLIP, for class II-associated invariant chain peptides. DMA and DMB associated and accumulated in multilaminar, intracellular compartments with classical class II molecules, but were found infrequently, if at all, at the cell surface. Thus, DM may facilitate peptide binding to class II molecules within these intracellular compartments.

HLA-DO is a negative modulator of HLA-DM-mediated MHC class II peptide loading.

BACKGROUND: Class II molecules of the major histocompatibility complex become loaded with antigenic peptides after dissociation of invariant chainderived peptides (CLIP) from the peptide-binding groove. The human leukocyte antigen (HLA)-DM is a prerequisite for this process, which takes place in specialised intracellular compartments. HLA-DM catalyses the peptide-exchange process, simultaneously functioning as a peptide 'editor', favouring the presentation of stably binding peptides. Recently, HLA-DO, an unconventional class II molecule, has been found associated with HLA-DM in B cells, yet its function has remained elusive. RESULTS: The function of the HLA-DO complex was investigated by expression of both chains of the HLA-DO heterodimer (either alone or fused to green fluorescent protein) in human Mel JuSo cells. Expression of HLA-DO resulted in greatly enhanced surface expression of CLIP via HLA-DR3, the conversion of class II complexes to the SDS-unstable phenotype and reduced antigen presentation to T-cell clones. Analysis of peptides eluted from HLA-DR3 demonstrated that CLIP was the major peptide bound to class II in the HLA-DO transfectants. Peptide exchange assays in vitro revealed that HLA-DO functions directly at the level of class II peptide loading by inhibiting the catalytic action of HLA-DM. CONCLUSIONS: HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.

Interactions between lymphoid tumor cells and isolated liver endothelial cells.

Interactions were studied between highly metastatic murine MB6A lymphosarcoma cells and rat liver endothelial cells that had been isolated by collagenase perfusion and purified by unit gravity sedimentation. Experiments were performed on the day of isolation. MB6A cells were observed to adhere to the endothelial cells. Addition of rat serum had a striking effect: The endothelial cells spread over the MB6A cell surface, engulfing the tumor cells. The factor involved was nondialyzable and also was present in rat plasma. Similar interactions were seen with highly metastatic ESb and MDAY-D2 lymphoma cells and with nonmetastatic Eb cells. Low-metastatic GRSL 34 leukemia and TA3/Ha ascites mammary carcinoma cells did not adhere to the endothelial cells. With this in vitro model the molecular mechanisms of adhesion to liver endothelium were studied. As a first step, univalent antibodies against MB6A cells were found to inhibit adhesion, indicating involvement of specific cell surface molecules.

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity.

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity. The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes. By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [3H]thymidine injection it was found that the label moved largely into the tetraploid compartment. Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation. The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.

A separation chamber to sort cells and cell organelles by weak physical forces. V. A sector-shaped chamber and its application to the separation of peripheral blood cells.

A separation chamber of 90 ml effective volume is described, that incorporates a sector shape (to prevent wall sedimentation) and anti-vortex lamellae (to prevent swirling upon acceleration and deceleration) as well as a flow-diverter for undisturbed layering and fractionation. The chamber is run in an ordinary table centrifuge and possesses a resolving power for equilibrium density centrifugation that seems unsurpassed. Peripheral human blood cells were separated in a Percoll gradient yielding in 1 single run the concurrent isolation of 99% pure monocytes, 99% pure lymphocytes and 99% pure granulocytes. Recoveries varied from 94% to 102%. By modifying the shape of the density gradient, either 99% of all monocytes collected were more than 92.5% pure and at the same time separation of small versus large lymphocytes was obtained, or substantial purification of neutrophils and eosinophils was achieved. The potential for simultaneous separation of monocytes, B, T cells and granulocytes was established. Good separations were obtained with buffy coats from 1 U of blood. The chamber also functioned well in the standard Böyum method for mononuclear cell preparation. In addition, a method for the rapid removal of erythrocytes (within 9 min) at unit gravity is described with nearly quantitative yield of white cells.

Binding and repair of 2-acetylaminofluorene adducts in distinct liver cell populations.

The study of the binding of the liver carcinogen, N-acetyl-2-aminofluorene, to the DNA of the target organ-as the probable initial step in the process of carcinogenesis-has shown that three modes of interaction occur. N-Acetyl-2-aminofluorene is covalently bound with the nitrogen to the carbon 8 of guanine (I) and with the 3-position to the free NH(2)-group of guanine (II). The third mode of interaction is formed by a covalent bond between the nitrogen of 2-aminofluorene and the carbon 8 of guanine (III). In this study the different modes of interaction were measured separately in stromal and parenchymal cells of the rat liver, after a single intraperitoneal dose. The DNA was isolated from nuclei that had been separated by 1g sedimentation. In parenchymal DNA the types of interaction I and III occur in the same amounts one day after application. In stromal cells the amount of interaction I is relatively small and interaction III predominates (ratio III:I = 5). The amount of interaction III in tetraploid hepatocytes (the largest cell population in the studied rats) per mg DNA is about two times higher than in the stromal cells. While the removal of the total amount of DNA-bound carcinogen takes place at the same rate in the two cell types, a difference in rate and efficiency of repair is observed for the different types of interaction. In tetraploid hepatocytes, interaction I is almost completely removed from the DNA 2 weeks after application, while interaction III diminishes to about 1/3 during the first week but the remaining part disappears very slowly. As shown in earlier studies, interaction II remains in the DNA at a constant level.

Binding of the chemical carcinogen N-hydroxy-acetyl-aminofluorene to ploidy classes of rat liver nuclei as separated by velocity sedimentation at unit gravity.

A large sedimentation device was developed that allows separation of 5 X 10(8) rat liver nuclei by velocity sedimentation at unit gravity. Using the apparatus isolated rat liver nuclei were separated into classes of diploid stromal (Von Kuppfer, sinusoidal lining) nuclei, diploid parenchymal nuclei and tetraploid parenchymal nuclei respectively. DNA content and volume of the nuclei were measured. Diploid nuclei were 100% pure; tetraploid nuclei 98%. The in vivo binding of the liver carcinogen [3H]-N-hydroxy-AAF to these classes of nuclei was determined (total binding to protein, DNA and RNA). Binding and the subsequent removal of the fluorene derivatives was registered as a function of time. At all stages diploid stromal nuclei bound 2.6--5 times less carcinogen than did diploid parenchymal nuclei. Tetraploid parenchymal nuclei bound more than twice (2.3--3.95) the amount, that was present in their diploid counterpart. This effect became more pronounced 11 days after application of N-hydroxy-N-acetyl-2-aminofluorene. DNA was enzymatically purified from pooled classes of the various nuclear types. For purified DNA also it was found that DNA derived from diploid stromal nuclei bound 2.6--2.8 times less carcinogen than did DNA derived from diploid parenchymal nuclei.

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

Analysis of members of the Ig-gene superfamily by thermal gradient polyacrylamide gel electrophoresis.

A solid aluminum block, connected with a warm and cold thermostated waterbath, provided for a linear transversal temperature gradient (TG) during polyacrylamide gel electrophoresis (PAGE). Noncovalently bound heavy chain dimers as well as heavy-light chain dimers, derived from human monoclonal IgG, could be melted into monomers using a 40-75 degrees C TG under conditions of sodium dodecyl sulfate-PAGE. Using native PAGE, major histocompatibility complex (MHC) class I molecules, preloaded with the iodinated peptide FAPGNYPAL could be melted in a 4-40 degrees C TG to release the peptide. The method is in general applicable to thermal stability analysis of noncovalently bound hetero-oligomers if the product after melting possess different electrophoretic mobilities.

Thermostability analysis of major histocompatibility complex class I molecules by temperature gradient gel electrophoresis.

Empty major histocompatibility complex (MHC) class I molecules present on the surface of RMA-S (26 degrees C) cells were loaded with the iodinated peptides APGNYPAL, FAPGNYPAL (SEV-9) and RGYVYQGL (VSV-8), respectively. The thermostability of these peptide-loaded MHC class I molecules was assessed using temperature gradient native polyacrylamide gel electrophoresis. A linear temperature gradient perpendicular to the direction of electrophoresis yielded a graphical representation of the melting of MHC class I molecules. The class I signal disappeared when the peptide melted out of the groove, and gave rise to a second signal due to released peptide. APGNYPAL-loaded class I molecules melted at 11 degrees C with considerable release even at 0 degrees C. VSV-8-loaded class I molecules melted first at 36 degrees C, whereas SEV-9-loaded molecules melted at about 22 degrees C. A discrimination between the binding of SEV-9 to Kb and Db molecules was seen in the melting patterns. Results are discussed in correlation with known crystallographic structures of class I molecules containing peptides in the binding groove.

Application of an improved density gradient electrophoresis apparatus to the separation of proteins, cells and subcellular organelles.

A DGE apparatus, made of Perspex, consisting of a separation column (5 x 2.2 cm) and containing a 0-4% linear Ficoll density gradient, was constructed. Only 2.5 cm of the column were used for high resolution separations. A specially designed removable top cone permitted precise gradient introduction, thin sample layering (0.3-1 mm) and precise fractionation after electrophoresis. A bottom circular palladium anode (nongassing) was separated hydrodynamically but not electrically from the density gradient by a cation-permeable membrane. A top circular platinum cathode caused negatively charged particles to migrate upwards (levitation). Thin sample layering permitted short separation times (30-60 min) at only 3 V/cm (10 mA). As for proteins, glycoforms of a1-antitrypsin were separated as well as isoenzymes of beta-hexoseaminidase. Furthermore, separation of transferrin (Tf) from the putative Tf-receptor complex was effectuated. The device was equally suitable for the separation of Megadalton proteins (mucins). Artificial mixtures of intact erythrocytes (rat, rabbit, human) were separated with high resolution. About 10(7) cells (of 100 microns3 cell volume) could be loaded onto the device. Crude microsomes from the human melanoma cell line Mel JuSo were separated after brief trypsin treatment within 38 min at 10 mA. Ratios of the migration velocities of the constituent organelles were: late endosomes (LE):lysosomes (L):Golgi (G):early endosomes (EE) = 1:0.94:0.77:0.55 and under slightly different conditions LE:L:G:endoplasmatic reticulum (ER):plasma membrane (PM) = 1:0.87:0.64:0.58:0.49.

Lectin-induced retardation of subcellular organelles during preparative density gradient electrophoresis: selective purification of plasma membranes.

Plasma membranes (PM) are difficult to separate by conventional means from other cellular compartments. Using a density gradient electrophoresis (DGE) apparatus (7 cm, x 2.2 cm), mammalian subcellular organelles were separated from a total postnuclear supernatant. The sialic acid-binding lectin wheat germ agglutinin (WGA) permitted 1.5-fold electrophoretic retardation of plasma membranes lagging far behind endoplasmic reticulum, endosomes, Golgi and lysosomes (in order of increasing electrophoretic mobility). Mobilities of the latter organelles were not affected by wheat germ agglutinin. The retarded plasma membrane was monitored by surface iodination, the presence of Ca(++)- and Na+/K(+)-ATPases and by the presence of clathrin-coated pits using Western immunoblotting. In the presence of WGA two clathrin-containing compartments were detected; in the absence of WGA three clathrin populations were seen in the electropherogram: clathrin-coated vesicles, clathrin-coated pits (on the PM) and clathrin-coated structures on the trans-Golgi network (TGN). Both in the presence and absence of WGA, plasma membrane domains of different electrophoretic mobilities were detected.

Electromigration for separations of protein complexes.

This paper describes electromigration of complexes, consisting of two or more proteins and non-covalently associated peptides. Relatively small complexes (Mr < 1000000) can be resolved in sieving matrices. Large complexes are separated in free liquid systems. Examples of separation are given using native gels, denaturing gels and special formats thereof: blue native PAGE and gels incorporating a transversal temperature gradient. Both preparative and analytical applications are discussed as well as separations leading to mechanistic models of protein interaction. Carrier-free electrophoresis is represented by capillary zone electrophoresis, free-flow electrophoresis and density gradient electrophoresis. Emphasis is put on the free liquid separation of clathrin-coated vesicles and proteasomes.

Use of a unit gravity sedimentation chamber for the purification of Mycobacterium leprae.

The present paper summarizes results concerning a mild isolation method of Mycobacterium lepraemurium and M. bovis-BCG from host tissues, and describes the application of such a method in purifying M. leprae (M1) from either infected armadillo tissues or human skin biopsies. This isolation method consists of homogenization, two-phase partition in dextranpolyethylene glycol and finally sedimentation in sucrose gradient using a unit gravity chamber. Such a purified M1 preparation appears to be devoid of host-tissue contaminants as examined by light and electron microscopy as well as by a radioimmuno spot test. The results indicate that the present method is mild enough to allow the purification of M1 from infected host tissues with in vivo conservation of antigenicity and viability of the bacilli.