Monocyte-derived inhibitor of interleukin 1 induced by human cytomegalovirus.

It has previously been shown that human cytomegalovirus (HCMV) can exert immunosuppressive effects, and it has been suggested that these may be mediated by monocytes, although the mechanism is unclear. We showed that infection of human monocytes with the AD169 strain of HCMV abrogates their production of interleukin 1 (IL-1) activity. This was associated with the release from infected monocytes of an inhibitor of IL-1 activity which was also released after HCMV infection of the U937 macrophage-like cell line. The inhibitor of IL-1 activity is a protein with an apparent molecular weight of ca. 95,000. This action of HCMV strain AD169 was virus specific and required infectious virus but occurred without virus replication or detectable expression of viral proteins. This effect may account, at least in part, for the previously observed immunosuppressive properties of HCMV.     

Appearance of IgG (Fc) receptor(s) on cultured human fibroblasts infected with human cytomegalovirus.

Cultured human diploid fibroblasts (WI-38) after infection with human cytomegalovirus (CMV) but not when uninfected, could hemadsorb sheep red blood cells (SRBC) coated with rabbit anti-SRBC IgG. The adsorption of IgG-coated SRBC to virus-infected cells was completely abolished if the tests were carried out in the simultaneous presence of rabbit antiserum elicited against CMV. Normal sera of rabbit or human origin as well as purified human IgG but not Fab fragment of human IgG could also abolish the binding of sensitized SRBC to CMV-infected fibroblasts. Active metabolism on the part of CMV-infected fibroblasts proved to be an important requisite for demonstrating binding of sensitized SRBC to their surfaces. By using an indicator Staphylococcus aureus to which rabbit antiserum against normal human IgG, IgM, or IgA was bound via Fc fragments, evidence has been obtained which suggests the existence of receptor(s) on CMV-infected WI-38 cells that react specifically with Fc region of human IgG.     

高效价人巨细胞病毒免疫球蛋白的研制

目的 制备人源性高效价人巨细胞病毒免疫球蛋白(HCMV-IgC)。方法 用ELISA方法从检验合格的献血员血浆中筛选HCMV-IgC滴度≥1:5000的血浆,采用低温乙醇法制备HCMV-IgC。结果 按《中国生物制品规程2000版》肌肉注射人血球蛋白检定标准,各项检定均合格,且HCMV-IgC滴度为1:150000。结论成功地制备出高效价HCMV-IgC。     

The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein.

A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor.     

Nonspecific human lymphocyte-mediated cytotoxicity induced by immobilized IgG aggregate

Normal human lymphocytes were induced to lyse nonsensitized erythrocytes when concomitantly incubated on immobilized IgG aggregate with various erythrocyte target cells. These included ox, sheep, chicken, and human red blood cells. Only immobilized aggregate would initiate cytolysis. The IgG aggregate was prepared from normal, healthy adult donors and did not possess target cell specificity (e.g., human erythrocyte lysis was initiated by autologous IgG). Normal human lymphocytes could be induced to lyse the red blood cell targets only after a preincubation with adherent mononuclear cells; however, freshly prepared lymphocytes depleted of IgG-FcR^? cells were cytolytic. Cytolysis induced by immobilized IgG-aggregate can be distinguished from NCMC and ADCC by its requirement for immobilized IgG aggregate and the absence of target cell specificity in the IgG-aggregate preparation.     

Characteristics of MNNG induced repair synthesis and DNA synthesis induced by human cytomegalovirus

DNA synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine in mouse embryo and human embryo cells was compared with DNA synthesis induced in these cells by human cytomegalovirus. In virus infected human embryo cells grown in the medium depleted of arginine DNA synthesis showed resistance to hydroxyurea and arabinofuranosylcytosine, similarly as repair synthesis induced by MNNG. DNA synthesis induced by the virus in mouse embryo cells was partially sensitive to both inhibitors.     

The human cytomegalovirus receptor on fibroblasts is a 30-kilodalton membrane protein.

Previous studies have demonstrated that human cytomegalovirus (HCMV) binding to human foreskin fibroblasts (HFF) is mediated by a single type of molecule, likely a glycoprotein, which serves as a specific receptor for the virus. In the present experiments, HCMV was found to bind to an HFF membrane protein with an approximate molecular mass of 30 kilodaltons (kDa); weak binding to 28- and 92-kDa membrane components was also observed. Binding was specific, as it was inhibited by excess unlabeled HCMV. Radiolabeled HCMV also bound selectively to Raji and Daudi lymphoblastoid cell membrane proteins of the same molecular masses. The 30-kDa radiolabeled HFF membrane protein bound to HCMV in solution; this binding was also specific, as it was blocked by an excess of HCMV. These data suggest that a membrane protein with a molecular mass of approximately 30 kDa mediates HCMV binding to several cell types.     

Isolation and characterization of phosphonoacetic acid-resistant mutants of human cytomegalovirus.

Mutants of the human cytomegalovirus (HCMV) that were 6- to 13-fold more resistant to phosphonoacetic acid than the wild-type HCMV (Towne) were isolated. Extracts from mycoplasma-free, mutant-infected cells had phosphonoacetate-resistant DNA polymerase activity in vitro. This strongly suggests that the selected mutations are in the HCMV DNA polymerase genes of these viruses.     

The receptor for IgG on human normal and chronic myeloid leukemic granulocytes: functional and biochemical characterization

Receptor mediated endocytosis of fluorescein isothiocyanate-conjugated heat-aggregated IgG (Fl-HAIgG) via the receptor for IgG (Fc gamma R) was studied using granulocytes from normal donors and patients suffering from chronic myeloid leukemia (CML). Within 15 min of incubation at 37 degrees C, 75% of the normal granulocytes internalized the ligand, while only 13% of the CML granulocytes could internalize the ligand in the same time. This functional defect was seen in all the CML patients analyzed. To elucidate the reason for this defective endocytosis, the Fc gamma R was isolated from normal and leukemic granulocytes and biochemically characterized, by gel electrophoresis, high pressure liquid chromatography, and one- and two-dimensional peptide mapping. Our results show that the molecule from the two cell types is very similar. The defective endocytosis must therefore be due to events which occur after ligand-receptor binding.     

Autoimmunity induced by human cytomegalovirus in patients with systemic lupus erythematosus

Human cytomegalovirus is a common herpesvirus that is linked to autoimmunity, especially in genetically predisposed persons. The article by Hsieh and colleagues in a previous issue of Arthritis Research & Therapy suggests that a C-terminal peptide of the human cytomegalovirus protein pp65 is highly immunogenic in patients with systemic lupus erythematosus and that antibodies against this peptide cross-react with nuclear proteins and double-stranded DNA, which are highly frequent autoantibodies in systemic lupus erythematosus patients. These observations highlight the fact that immunization with one small cytomegalovirus-specific peptide results in multiple autoreactive antibodies, probably through molecular mimicry and epitope spreading, in genetically predisposed persons.     

Expression of a human cytomegalovirus receptor correlates with infectibility of cells.

Previous studies have demonstrated that human cytomegalovirus (HCMV) specifically binds to a fibroblast membrane glycoprotein(s) with a molecular mass from 30 to 34 kDa. In this study, the distribution of the putative receptor proteins was analyzed in a variety of cell types, including cell types representative of those that are infected in vivo. Using a sensitive microbinding assay (to score virus attachment) and an indirect detection method (to score HCMV-binding proteins), we found that the 34- and 32-kDa HCMV binding proteins are ubiquitous molecules, broadly distributed among diverse cell types. In addition, the level of virus attachment was found to correlate with the abundance of the 34- and 32-kDa cellular proteins, while the ability of the virus to penetrate cells and initiate infection did not. The results support the hypothesis that the 34- and 32-kDa cellular proteins represent the HCMV (attachment) receptor. The data also support the notion that additional cellular components are required for virus entry and fusion.     

Thermally induced aggregation of human transferrin receptor studied by light-scattering techniques.

The thermal stability of transferrin receptor isolated from human placenta in detergent-free solution has been investigated by static light-scattering and photon correlation spectroscopy. In detergent-free solution at 293.2 K, human transferrin receptor (hTfR) forms stable associates with a hydrodynamic radius of 16 nm. With increasing temperature the particles get more compact, above 340 K a phase transition takes, place and spontaneous aggregation of the receptor occurs. Under these conditions large clusters are formed that lead to fractal aggregates, coexisting with dendritic crystalline structures. The experimental findings are compatible with a model, which involves a reaction limited cluster-cluster aggregation mechanism in conjunction with a nucleation process. The molar enthalpy change associated with the phase transition was determined to be (1860 +/- 150) kJ/mol(-1) at a transition temperature of (341.3 +/- 0.2) K.     

Graves' IgG recognizes linear epitopes in the human thyrotropin receptor.

Twenty-nine peptides covering the full extracellular domain of the human thyrotropin receptor have been synthesized and tested for reactivity with Graves' patients' and normal sera in ELISA. Two peptides, amino acids 331-350 and the second extracellular loop of the transmembrane segment, bound IgG-s from 5 and 4 of 10 Graves' disease patients' sera, respectively. Neither of these two peptides showed enhanced binding to normal IgG. There were no apparent differences between the Graves' disease and normal group with respect to the other 27 peptides. We conclude that peptide 331-350 and the second extracellular loop carry important linear epitopes which may contribute to the disease process in selected Graves' patients.     

Lymphocyte mitogenesis induced by monoclonal antibodies to the T3 complex. Differential modulation by human IgG

Murine monoclonal antibodies OKT3 (IgG2), 64.1 (IgG2), and Leu 4 (IgG1) react with a common membrane antigen on human T cells and induce potent mitogenesis at concentrations of 1 ng/ml, 10 ng/ml, and 100 ng/ml, respectively. Human serum inhibits the mitogenic effect of antibodies OKT3 and 64.1, but not that of Leu 4. The inhibitor in serum has been identified as immunoglobulin G (IgG) as evidenced by the ability of anti-human IgG-Sepharose affinity columns to retain the inhibitory activity. Various immunoglobulin classes and subclasses obtained from human myelomas differ in their ability to inhibit the OKT3-induced activation. The best inhibition is obtained with the IgG subclasses IgG1 and IgG3, followed by IgG2; IgG4, IgM, and IgA have little if any effect. None of the IgG subclasses inhibit the Leu 4-induced mitogenesis. Indomethacin as well as supernatants containing interleukin 2 (IL-2) can reverse the inhibitory effects of IgG. Prostaglandins (PGE1 and PGE2) inhibit both the OKT3- and Leu 4-induced mitogenesis, thus lacking the selectivity seen with IgG. Since stimulation by the monoclonal antibodies requires the participation of monocytes, an interpretation consistent with the present data is that IgG stimulates monocytes via its Fc portion to release prostaglandins and/or other suppressor factors via an indomethacin-sensitive pathway. The inability of IgG to inhibit Leu 4-induced mitogenesis may therefore relate to an inability of the monocyte subpopulation, which mediates the Leu 4 response, to secrete suppressor factors. These data suggest a potential value of the mitogenic monoclonal antibodies as probes in studying monocyte heterogeneity and T-cell-monocyte interactions.     

Endocytosis of human IgG:Fc receptor complexes by transfected BHK cells

We have analyzed the mode of uptake of human beta FcRII molecules expressed in BHK cells (clone 2/14). When challenged with aggregated human IgG (ahIgG), these cells bind the ligand at 4 degrees C and endocytose the IgG: receptor complexes rapidly upon warming to 37 degrees C, as seen by fluorescence microscopy with antibodies directed against human IgG. Using 125I-labeled ahIgG, we found that 40% of the bound ligand was internalized within 15 min, and approximately 60% within 2 h. Surface replication and thin sectioning combined with immunogold labeling revealed that the ligand was taken up by coated vesicles and was transferred to the endosomal/lysosomal compartment. This was confirmed by confocal laser microscopy of cells double labeled for clathrin and ahIgG. After modulation of the coated vesicle pattern by hypertonic medium, ahIgG transport was impaired. These data show that a single isoform of human FcRII, expressed in an animal cell negative for Fc receptors, can use the coated vesicle based endocytic pathway of the host cell. Reincubation of cycloheximide-treated cells with a second batch of ligand showed that approximately 20% of the beta FcRII was recycled. This finding is in apparent contrast to the fate of the endogenous Fc receptors expressed on mouse macrophages.     

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.     

Overexpression and characterization of the human mineralocorticoid receptor.

The full-length human renal mineralocorticoid receptor (hMR) has been overproduced in Spodoptera frugiperda (Sf9) insect cells using baculovirus-mediated expression. The overproduced hMR binds aldosterone with high affinity (Kd = 1.36 nM) and has high affinity for cortisol, cortexolone, and progesterone. Immunoprecipitation and immunoblot analysis of the recombinant hMR with MR-specific antibodies reveal three major protein bands with molecular masses of 115, 119, and 125 kDa. hMR isoforms show maximal accumulation at 48 h post-infection with the recombinant baculovirus. Maximal aldosterone binding was detected at 24 h rather than at 48 h post-infection, suggesting that the assembly of hMR monomers into the nonactivated steroid-binding receptor complexes and/or their stability deteriorates after 24 h post-infection. It is estimated by specific aldosterone binding that 1.2 x 10(6) hMR molecules are expressed per Sf9 cell (equivalent to 7 pmol/mg of cytosolic protein) at 24 h post-infection. 5-Fold more receptor molecules/cell are expressed but not detected by steroid binding at 48 h post-infection as determined by immunoblot analysis. Using the MR-specific H10E anti-idiotypic monoclonal antibody, immunoprecipitation of cytosol from recombinant baculovirus-infected Sf9 cells pulse-labeled with 32Pi demonstrated for the first time that the recombinant hMR is highly phosphorylated. The hMR is expressed as 9-10 S oligomeric complexes (Stokes radii approximately 67-85 A) that are slightly heavier than the unactivated glucocorticoid receptor and can be converted to smaller 4 S receptor monomers (Stokes radii approximately 25-55 A) by elevated temperature, pH, and ionic strength. Unlike the glucocorticoid receptor, the oligomeric hMR complex can bind DNA-cellulose without prior activation. Finally, indirect immunofluorescence demonstrated that the hMR is expressed primarily as a cytoplasmic protein that can be induced to translocate to the nucleus upon treatment with hormone.