Phosphorylation of class I MHC molecules in the absence of phorbol esters is an intracellular event and may be characteristic of trafficking molecules

Class I Major Histocompatibility Complex (MHC) molecules are displayed at the cell surface where they present antigenic peptides to T lymphocytes. Class I MHC molecules undergo cytoplasmic domain phosphorylation on a serine residue late in their biosynthesis. Here we show that phosphorylation occurs on mature, beta(2)-microglobulin-associated class I MHC molecules in a mouse lymphoid cell line. Both recently synthesized class I MHC molecules and molecules which are at least 3 h old become phosphorylated. Approximately 14% of phosphorylated class I MHC molecules occur at the cell surface. Density gradient analysis indicates that phosphorylated class I MHC molecules also occur in lamp(+) intracellular compartments and in fractions containing rab4, a GTP-binding protein associated with recycling endosomes. Class I MHC molecules are endocytosed and recycled to the cell surface in these cells. Furthermore, the lysosomotropic drug, primaquine, inhibits both class I MHC phosphorylation and its recycling back to the cell surface, suggesting that phosphorylation is related to class I MHC recycling. These observations are intriguing since several studies have shown that class I MHC molecules can acquire antigenic peptides in NH(4)Cl-sensitive compartments. Hence, class I MHC phosphorylation may play a role in regulating intracellular sorting through these compartments.     

Cell surface expression of immature H glycoprotein in measles virus-infected cells

Two forms of hemagglutinin (H) protein, one with an apparent molecular mass of 78 kDa (78K H protein) and the other with that of 74 kDa (74K H protein), are present in cells infected with measles virus (MV). We previously observed that only the mature 78K H protein, a completely glycosylated form of the 74K H protein, was expressed on the cell surface of the infected cells. In the present study, we detected transient expression of the 74K H protein on the cell surface of infected cells by pulse-chase studies, although the level of this expression was much lower than that of the 78K H protein. On the cell surface the 74K H protein was present as dimers and sensitive to endo-beta-N-acetylglucosaminidase H digestion. Treatment with brefeldin A, which blocks the transport of membrane and secretory proteins from the endoplasmic reticulum to the Golgi apparatus, inhibited the cell surface expression of the 78K H protein, but not that of the 74K H protein. These data suggest that a part of the MV 74K H proteins could be transported directly to the cell surface - probably via an alternative pathway - without processing to the complex form in the Golgi apparatus.     

Competitive adsorption of proteins: key of the relationship between substratum surface properties and adhesion of epithelial cells

The adhesion of Hep G2 cells was investigated using different substrata (commercial substrata, polystyrene modified by oxygen or ammonia plasma discharge), the surface properties of which were characterized (surface chemical composition, water contact angle, zeta potential). Some substrata were pre-conditioned with solutions of extracellular matrix (ECM) protein (collagen, laminin, fibronectin), solutions of albumin or polylysin, fetal calf serum or culture medium. The culture medium contained the surfactant Pluronic F68; cycloheximide was added in certain tests to inhibit protein synthesis. Cells spread within 1.5 h provided ECM proteins were present at the surface. Adsorption of ECM proteins was subject to competition with adsorption of Pluronic F68. When the substratum was exposed simultaneously to ECM protein and Pluronic F68, either by pre-conditioning or through protein cell secretion, a weaker substratum hydrophobicity favored adsorption of the proteins and subsequent cell adhesion. On the other hand, when ECM proteins were pre-adsorbed, they were not displaced by Pluronic F68 and cell adhesion was not influenced by substratum hydrophobicity. When ECM proteins were present, no difference was observed between substrata of similar hydrophobicity carrying positive or negative charges, respectively. In absence of ECM proteins, the presence of cationic sites at the substratum surface (NH3 plasma treatment, adsorption of polylysine) allowed cell attachment but no spreading within 1.5 h.     

Regulatory role of tryptophan degradation pathway in HLA-G expression by human monocyte-derived dendritic cells

Dendritic cells (DC) are strong inducers of immunity but they can also be tolerogenic. During monocyte differentiation to DC the immunosuppressive indoleamine-2,3-dioxygenase (IDO) is induced. IDO degrades Trp to kynurenine, which is further metabolized to 3-hydroxyanthranilic acid. DC can also express mRNA and protein of the tolerogenic molecule HLA-G, but there is no surface expression. We studied the effect of the Trp degrading pathway on HLA-G expression by DC. When monocytes were differentiated to immature DC in presence of either Trp or its metabolites kynurenine or 3-hydroxyanthranilic acid they expressed cell surface HLA-G, and Trp also increased shedding of HLA-G1. Trp induced HLA-G cell surface expression when present during maturation with IFN-γ + LPS, but not with TNF-. Kynurenine increased HLA-G expression in both TNF- and IFN-γ + LPS matured DC, and 3-hydroxyanthranilic acid had a very weak effect on HLA-G cell surface expression when present during maturation. Shedding of HLA-G1 was more pronounced in IFN-γ + LPS-matured DC than in immatured DC. Maturation with IFN-γ + LPS in presence of kynurenine also increased HLA-G5 secretion. The mechanism involved seems to be post-translational as mRNA and cellular HLA-G protein content was not increased with Trp, kynurenine or 3-hydroxyanthranilic acid treatments. Finally, immature DC preincubated with Trp, kynurenine and 3-hydroxyanthranilic acid have after a decreased capacity to stimulate T cells in mixed lymphocyte reaction. In IFN-γ + LPS-matured DC this decreased capacity was obtained with kynurenine and 3-hydroxyanthranilic acid. These results suggest that IDO can induce HLA-G cell surface expression in DC, and that these two molecules can cooperate in the immune suppression.     

Special organization of the HLA-G protein on the cell surface

The human leukocyte antigen G (HLA-G) molecule possesses unique properties such as low polymorphism and restricted distribution mainly to the extravillous cytotrophoblast (EVT) cells. The EVT cells vigorously penetrate into the maternal decidual tissues and are found in contact with maternal lymphocytes, mainly with natural killer (NK) cells. The HLA-G molecule inhibits the effector function of maternal NK cells via interaction with the KIR2DL4 and the ILT-2 inhibitory NK receptors. Previously, we have demonstrated that complexes of the HLA-G protein are expressed on the cell surface. We reported that these complexes are formed due to the presence of two unique cysteine residues located at positions 42 and 147. Finally, we demonstrated that efficient binding and function of ILT-2 is dependent on the presence of HLA-G complexes on the cell surface. Here we expand the significance of these observations by revealing that complexes of HLA-G are present on the cell surface using different assays and cell lines and further demonstrate that complexes of HLA-G might be present in a soluble form after interaction with ILT-2. Therefore, the HLA-G molecule has developed a special mechanism to increase the avidity of NK receptors to the HLA-G molecule, which provides better protection for the fetus from maternal NK rejection.     

The murine heat-stable antigen: a differentiation antigen expressed in both the hematolymphoid and neural cell lineages

The murine heat-stable antigen (HSA) and the p31 antigen are cell surface glycoconjugates which are transiently expressed during the development and differentiation of the hematolymphoid and neural cell lineages, respectively. We show here that monoclonal antibodies which react with these two species recognize a common antigenic determinant which is expressed on both HSA and p31, and the HSA and p31 share a common protein core. Differences in the molecular weights of the antigens most likely reflect variations in the extent of post-translational modifications. From these studies we conclude that these antigens are members of the same family of heat-stable antigens. Our results lead us to speculate on how these molecules are related, their function, and what role they play in cellular differentiation in hematolymphoid and neural cell development.     

Protein σ1 is the reovirus cell attachment protein

The minor outer capsid shell component σ1 is the reovirus cell attachment protein because (a) IgGs directed against σ1 prevent reovirus particle adsorption, (b) free protein σ1 present in lysates of infected cells is capable of adsorbing to cells, and (c) σ1 competes with reovirus particles for cell surface receptors. Competition experiments indicate that reovirus particles of all three serotypes attach to the same cell surface receptor. It is also shown that σ1 is located on the surface of reovirus particles in close juxtaposition to λ2, the major, if not the only, component of the 12 icosahedrally distributed reovirus core projections or spikes, which penetrate through the outer capsid shell to the reovirus particle surface.     

Peptides related to the antigenic determinant block T cell recognition of the native protein as processed by antigen-presenting cells

A mouse T cell hybrid specific for pigeon cytochrome c in the context of I-Ek responds by secreting interleukin 2 when co-cultured with the native antigen and the B cell lymphoma, LK-35.2, or naive splenic B cells as antigen-presenting cells (APC). Cytochromes c and their corresponding C-terminal fragments which are not capable of stimulating the TPc9.1 cells, including the autologous mouse cytochrome c, block the T cells' response to pigeon cytochrome c. In contrast, nonstimulatory N-terminal peptides of cytochrome c, which share no homology with the antigenic peptide, do not block. Blocking is observed when the nonstimulatory cytochromes c or peptides are present in culture with the live APC and nonsaturating concentrations of pigeon cytochrome c. With tobacco hornworm moth cytochrome c as antigen, a protein for which the T cell has a higher functional affinity, the response of TPc9.1 cannot be blocked by the nonstimulatory cytochromes c or by peptides, even when limiting concentrations of the tobacco hornworm moth cytochrome c are used. When paraformaldehyde-fixed APC are employed, no native cytochrome c can stimulate the T cells, including the tobacco hornworm moth protein which with the live APC is effective at 50 to 100-fold lower concentrations than pigeon cytochrome c. However, with fixed APC the T cells are stimulated by the C-terminal fragments containing residues 81-104 of the pigeon protein or residues 81-103 of the tobacco hornworm moth protein as readily and with the same relative efficiencies as the native protein, presented by live APC. The nonstimulatory peptides, but not the native cytochromes c, block T cell activation by pigeon cytochrome c pulsed-fixed APC, indicating that the nonstimulatory peptides compete with the stimulatory pigeon cytochrome c peptides produced by the APC. This competition appears to be due to nonstimulatory peptides which associate at the APC surface and not to those acting from solution because the APC which have been incubated with pigeon cytochrome c and nonstimulatory peptides and washed free of excess antigen and peptides are not stimulatory to the T cell hybrid. It was concluded that the activation of a pigeon cytochrome c-specific T cell, which recognizes a peptide fragment of the native protein on the surface of an APC, can be blocked by an excess of nonstimulatory homologous peptides when these are also associated on the surface of the APC.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cellular organization of fibroblastic cells and macrophages at regions of uncalcified cartilage resorption in the embryonic chick femur as revealed by alkaline acid phosphatase histochemistry

Resorption of uncalcified cartilage in the embryonic chick femur appears to be mediated by two types of mononuclear cells. One cell type lies flattened and adherent along the surface of the cartilage matrix into which it extends cellular processes. Cytological characteristics of a large, euchromatic nucleus containing a nucleolus, and cytoplasm containing moderate to extensive amounts of rough endoplasmic reticulum indicate that these are protein synthetic cells. Macrophages, characterized by a pleomorphic shape and cytoplasm containing numerous mitochondria and vesicles, comprise the second cell type. These may be seen lying in contact with cartilage matrix, but are more likely located in the nonhematopoietic marrow adjacent to resorbing cartilage, where they establish close cellular associations with protein synthetic cells. Alkaline and acid phosphatase histochemical studies differentiate these two cellular types. Marrow alkaline phosphatase activity is restricted to the cartilagemarrow interface from which it diffuses a short distance into cartilage matrix, but does not diffuse into nearby marrow. Intracellular alkaline phosphatase is present only in protein synthetic cells that line the surface of cartilage, and thus appears to be produced by these cells. Acid phosphatase positive macrophages are scattered throughout the marrow, but are found in greatest concentrations in the region of cartilage resorption. They are rarely in direct contact with cartilage, and there is no evidence that acid phosphatase is released from these cells. The relative localizations and the presence of cellular interactions of these two cell types suggests that protein synthetic cells may be of fibroblastic origin, and may play a primary role in cartilage degradation, while macrophages, in keeping with biochemical evidence, play an adjunct or possibly a regulative role.     

Polyreactive antigen-binding B (PAB-) cells are widely distributed and the PAB population consists of both B-1+ and B-1- phenotypes

B cells that make polyreactive antibodies (PAB+ cells) express polyreactive Ig receptors on their surface and can bind a variety of different antigens. The present study shows that PAB+ cells are widely distributed, are present in varying numbers in different lymphoid organs and that their phenotype varies depending on the organs from which they are isolated. Up to 10 times more cells in PAB+ enriched populations bind antigens as compared to PAB- populations. Comparison of PAB+ with B-1+ cells showed that a high percentage of PAB+ cells are B-1+, but that many PAB+ cells do not express B-1 cell surface markers and, in fact, are B-1-. It is concluded that the B cell population consists of PAB+/B-1+, PAB+/B-1-, PAB-/B-1+, and PAB-/B-1- cells. The presence of PAB+ cells in the thymus points to the possibility that PAB+ cells may carry endogenous host antigens from peripheral tissues to the thymus where they may contribute to immunological tolerance.     

Mouse mammary tumor virus and murine leukemia virus cell surface antigens on virus producer and nonproducer mammary epithelial tumor cells.

Mouse mammary tumor virus (MMTV)- and murine leukemia virus (MuLV)- specific cell surface antigens (CSA) on virus producer and nonproducer mammary epithelial tumor cells were studied using the techniques of lactoperoxidase catalyzed iodination of cell surface proteins followed by radioimmune precipitation with monospecific antisera to the major MMTV proteins gp52, gp36, p27, and p10 and to the major MuLV proteins gp70 and p30. The incorporation of iodinated CSA into extracellular virus was determined by analyzing labeled proteins in purified virus. On cells producing only MMTV both gp52 and gp70 were present on the cell surface. Furthermore, gp52 was the only labeled protein in extracellular MMTV produced by these cells. On cells producing both MMTV and MuLV, both gp52 and gp70 were present on the cell surface, and were the only labeled proteins present in their respective extracellular viruses indicating that gp70 and gp52 are present on mutually exclusive cellular viral budding sites. In addition, MuLV anti-p30 serum precipitated two iodinated proteins with molecular weights of 85,000 and 95,000 daltons, analogous to the Gross cell surface antigen (GCSA). Labeled gp52 and gp70 represent true CSA as demonstrated by the fact that they were also present on the surface of cells producing no virus, but producing large amounts of MMTV glycoproteins and nonglyco-proteins. These results further demonstrate that the precursor to the MMTV glycoproteins (gPr75-MMTV env) is cleaved prior to the appearance of gp52 on the cell surface.     

Biochemical and immunological differences between hydrophobic and hydrophilic strains of Streptococcus mutans.

Hydrophobic strains of Streptococcus mutans were compared with paired variants showing reduced hydrophobicity. Extracts of hydrophobic cells contained a number of high-molecular-weight proteins which were not present on cells with decreased hydrophobicity. The proteins were found in purified cell walls, suggesting that they are located on the bacterial surface. Trypsin treatment of whole cells destroyed the proteins and reduced the hydrophobicity. Chemical analysis did not reveal any marked differences in the proportion of cell wall constituents. The amino acid compositions and lipoteichoic acid contents of hydrophobic and hydrophilic cell walls were similar. Culture supernatants from the hydrophilic variants contained high-molecular-weight proteins similar to those extracted from the cell walls of the hydrophobic parent strains, indicating that the variants were impaired in their ability to incorporate the hydrophobicity-associated proteins into the cell wall. The dominant protein had a molecular weight of 190,000, similar to that of antigen I/II (B) of S. mutans.     

Characterization of the cord-like structures emerging from the surface of influenza C virus-infected cells

When HMV-II cells (a human malignant melanoma cell line) infected with a newly isolated influenza C strain (Yamagata/1/88) were examined by simple light microscopy, it was found that a large number of cord-like structures which had lengths up to about 500 microns or greater were emerging from the cell surface. The existence of viral glycoproteins (hemagglutinin-esterase, HE) on the surface of these huge structures was confirmed by hemadsorption experiments with erythrocytes from a variety of species as well as by immunofluorescent staining with anti-HE monoclonal antibody. Furthermore, electron microscopy revealed that numerous filamentous particles in the process of budding, each covered with a layer of surface projections approximately 13 nm in length, aggregated with their long axes parallel to form a cord-like structure visible under a light microscope. An electron-dense layer, which presumably consists of membrane protein (M), was seen in cross-sections of all filamentous virions whereas internal nucleocapsids were rarely seen. SDS-polyacrylamide gel electrophoresis of the purified cords also showed that they contained HE and M polypeptides but not nucleoprotein, confirming that long filamentous particles are mostly devoid of nucleocapsids. The emergence of cords on the cell surface was observed in various cell cultures infected with C/Yamagata/1/88 though their number and length varied markedly depending on cell type. The production of cord-like structures was also evident in HMV-II cells infected with any of several different influenza C strains, which suggests that the cord formation is a common feature of influenza C virus group.     

Immune blot analysis of viral surface proteins in serum and liver of patients with chronic hepatitis B virus infection

The small and the middle surface proteins of hepatitis virus form either the virion or the 22 nm particle both of which are secreted. The large surface protein by itself remains cell bound in artificially transfected cell culture unless it is accompanied by an excess of the smaller protens. Its behavior in vivo is not yet well studied. Using specific monoclonal antibodies for immunoblotting, we found an abundance of small surface protein in the serum of chronic virus carriers and moderate amounts in the liver irrespective of viremia. The large surface protein was present in the serum and the liver of viremic carriers. In nonviremic carriers, the large protein was absent from serum, but in the liver a shorter form of the large protein was readily detectable. These findings suggest a complex regulatory mechanism of the viral surface protein depending on the expression of other viral gene products.     

Inducible differentiation and apoptosis of the pre-B cell receptor-positive pre-B cell line

The function of the pre-B cell receptor (pre-BCR) during B cell differentiation is not precisely defined. To investigate the pre-BCR receptor activity, we have established pre-BCR-positive pre-B cell lines that are able to differentiate into immature B cells in vitro. Antibody cross-linking of the pre-BCR induced apoptosis and differentiation accompanied with tyrosine phosphorylation. A specific tyrosine-phosphorylated 43 kDa protein (p43) was found down-stream of the pre-BCR. The results demonstrated the receptor function of pre-BCR, which indicates that a ligand-like molecule or a cross-linking structure on the cell surface is possibly present.     

Comparative studies of hepatitis B virus precore and core particles

Hepatitis B virus core antigen gene expresses two cocarboxy-terminal proteins, termed precore and core proteins. Both precore and core proteins can form nucleocapsid-like particles. In order to understand the mechanism that leads to the formation of the nucleocapsid, we have expressed precore and core protein sequences in COS cells, a monkey kidney cell line, and compared the properties of these two particles. Our results show that core protein can form particles with various densities and they are present mostly in the cytosol. Precore protein, on the other hand, forms particles with one predominant density, and a majority of these particles are present in the lumen of the endoplasmic reticulum (ER). Furthermore, our results show that, when coexpressed in the same cells, core protein and the ER-associated surface antigens (envelope protein) show colocalization, indicating interaction between these two viral structural proteins.     

Changes in cell surface proteins and glycoproteins during the encystation of Entamoeba invadens

Changes in cell surface components of axenically grown trophozoites of Entamoeba invadens which occur during encystation were followed. Protein patterns of trophozoites, immature and mature cyst forms, were analyzed by sodium dodecyl sulphate gel electrophoresis. Total protein profiles of trophozoites and cyst forms stained by Coomassie blue gave similar patterns. In contrast, a number of different bands were observed in gels stained with the carbohydrate-specific Schiff's reagent as well as when nitrocellulose blottings were treated with 125I-radiolabelled wheat germ or soybean agglutinins. The most notable differences were bands at 250 and 95-105 kDa present in the cyst forms and absent in the trophozoites, and two bands at 70 and 75 kDa present in the latter and missing in the cysts. Labelling of trophozoites and cyst cell surfaces by iodination with lactoperoxidase revealed a number of protein bands which were exposed on the trophozoite surface and missing in the cysts. Moreover, gel electrophoresis patterns of non-reduced or reduced samples also differed considerably, indicating that a number of proteins are linked by disulphide bonds. This study shows that specific glycoproteins are produced during cyst formation.     

Presence of parasite antigen on the surface of P388D1 cells infected with Ehrlichia risticii.

Indirect immunofluorescence staining of macrophages infected with Ehrlichia risticii by anti-E. risticii serum revealed a punctate staining pattern on the surface of the host cell. This pattern was distinguishable by fluorescence microscopy from E. risticii bound to the surface of the macrophage and from intracellular E. risticii. The surface localization of ehrlichial antigen on infected macrophages was confirmed by electron microscopy with immunoferritin labeling. As the intracellular ehrlichial burden increased, the amount of ehrlichial antigen on the host cell surface increased. Prokaryotic protein synthesis was necessary for the maintenance of ehrlichial antigen on the host cell surface, as demonstrated by disappearance of the surface antigen following treatment with oxytetracycline. However, host cell protein synthesis was not required, as demonstrated by the continued presence of ehrlichial antigen on the surface of host cells after cycloheximide treatment. Pronase treatment abolished the ehrlichial antigen present on the cell surface, indicating that this antigen is a protein. Anti-E. risticii serum or immunoglobulin G-mediated antibody-dependent cellular cytotoxicity of infected cells was demonstrated in a chromium release assay. These results imply that the parasite antigen on the host cell surface has a role in the pathogenesis of ehrlichiosis.     

Expression of complement factor H on the cell surface of the human monocytic cell line U937

Binding assays and immunocytochemical staining with monoclonal antibodies against the human serum complement protein factor H indicate that factor H antigen is present on the surface of more than 95% of the cells of the human monocytic cell line U937. The antigen is uniformly distributed and there are 10 000-15 000 copies/cell. Factor H antigen is strongly associated with the cell surface and is not removed by hypotonic or hypertonic washes. Factor H antigen has been isolated from surface radioiodinated and 35S biosynthetically labeled cells using polyclonal anti-factor H-Sepharose columns. The antigen is indistinguishable from serum factor H in molecular weight. Secretion of factor H by U937 cells was not detected using sensitive tests in which factor H secretion by monocytes was apparent. Phorbol myristate acetate stimulation of the cells had no effect on the average number of factor H molecules expressed. We conclude that factor H is synthesized by U937 cells, but is not secreted, and remains strongly associated with the cell surface. The surface-bound factor H may function as a C3b receptor.     

Microscopy of internal structures of Sendai virus associated with the cytoplasmic surface of host membranes

The cytoplasmic surface (PS) of the plasma membrane of cells infected with Sendai virus was studied by immunofluorescence microscopy and freeze-drying electron microscopy. After cells had been attached to glass coverslips, they were subjected to a jet stream of physiological buffer which sheared off the upper portion of each cell, leaving the attached membrane with the PS exposed. This uncapping maneuver permitted direct examination of internal virus-specific elements associated with the inner surface of the host cell. At a stage of infection at which viral budding occurs, strands of nucleoprotein (RNP) were observed to be attached to the PS of plasma membranes. The sites at which RNP was adherent to the membrane were modified by virus-specific particles arranged in orthogonal patterns. The presence of the same crystalline structures in the hydrophobic domain of freeze-fractured membranes indicated that they were inserted into the inner lipid leaflet. The spatial association of the surface glycoprotein spikes and the internal RNP with this crystalline structure suggests its special relationship if not identity with the internal viral matrix (M) protein. The possible significance of the localization and crystalline nature of this structural element with respect to viral morphogenesis, hemolytic and cell-fusing activities is discussed. In contrast to the foregoing changes observed in the infected cell, no detectable viral antigens were found on the PS of normal cells to which exogenous virions had been fused. Absence of internalized antigen from the PS under these circumstances could indicate that infectious viral components are processed by the potential host cell in a manner which differs from what is observed with human erythrocytes. In the latter instance internalized antigens after fusion of virus to the cell remain associated with the PS.