Glycosphingolipid-induced relocation of Lyn and Syk into detergent-resistant membranes results in mast cell activation.

Sphingosine, sphingosine-1-phosphate, and the more complex sphingolipid ceramide exert strong immunomodulatory effects on a variety of leukocytes. However, little is known regarding such a potential of glycosphingolipids, a class of sugar derivatives of sphingosine. Here we demonstrate that galactosylsphingosine, one of the smallest representatives of this group, accumulates in the detergent-resistant membranes resulting in the relocation of the tyrosine kinases Lyn and Syk into this compartment. The result of this is an enhanced tyrosine phosphorylation and kinase activity leading to priming and activation of mast cells by conveying a weak yet significant activation of the mitogen-activated protein kinase pathway(s). In comparison to IgE/Ag triggering, galactosylsphingosine stimulates the mitogen-activated protein kinase pathway more rapidly and favors c-Jun NH2-terminal kinase 1 activation over extracellular signal-regulatory kinase 1 and 2. At the transcription factor level, this "ultratransient signaling event" results in an activation of JunD as the predominant AP-1 component. In this respect, the effects of galactosylsphingosine are clearly distinct from the signaling elicited by other sphingolipids without the sugar moiety, such as sphingosine-1-phosphate.

The balance between sphingosine and sphingosine-1-phosphate is decisive for mast cell activation after Fc epsilon receptor I triggering.

Over the last few years, sphingolipids have been identified as potent second messenger molecules modulating cell growth and activation. A newly emerging facet to this class of lipids suggests a picture where the balance between two counterregulatory lipids (as shown in the particular example of ceramide and sphingosine-1-phosphate in T lymphocyte apoptosis) determines the cell fate by setting the stage for various protein signaling cascades. Here, we provide a further example of such a decisive balance composed of the two lipids sphingosine and sphingosine-1-phosphate that determines the allergic responsiveness of mast cells. High intracellular concentrations of sphingosine act as a potent inhibitor of the immunoglobulin (Ig)E plus antigen-mediated leukotriene synthesis and cytokine production by preventing activation of the mitogen-activated protein kinase pathway. In contrast, high intracellular levels of sphingosine-1-phosphate, also secreted by allergically stimulated mast cells, activate the mitogen-activated protein kinase pathway, resulting in hexosaminidase and leukotriene release, or in combination with ionomycin, give cytokine production. Equivalent high concentrations of sphingosine-1-phosphate are dominant over sphingosine as they counteract its inhibitory potential. Therefore, it might be inferred that sphingosine-kinase is pivotal to the activation of signaling cascades initiated at the Fc epsilon receptor I by modulating the balance of the counterregulatory lipids.

Development of amphotropic murine retrovirus vectors resistant to inactivation by human serum.

Replication-deficient amphotropic retrovirus vectors (RV) or RV-producer cells are being developed for a variety of human gene therapy strategies. One of the hurdles to in vivo use of these agents is their inactivation by components of human serum. Murine leukemia viruses (MLV), from which most current RV are derived, are known to be inactivated by human serum via activation of the classical complement cascade. Other type C retroviruses, e.g., RD114 and BaEV, are resistant to inactivation by human serum when derived from infection of human and mink cells but not murine cells. We hypothesized that amphotropic RV could be made resistant to human serum inactivation if a more appropriate producer cell could be found. To test this hypothesis, RV were made using a variety of human (293, HOS, TE671) and murine (NIH-3T3) cell types as the producer cell. The parental cell lines, RV-producer cells, and RV themselves were evaluated for sensitivity to inactivation by human serum. Results showed that the murine NIH-3T3 cell line, the NIH-3T3-derived PA317 producer cell line, and RV derived from it were all sensitive to human serum inactivation. In contrast, all human cell lines tested were resistant to lysis. RV and RV-producer cells derived from 293 cells were also resistant; RV derived from HOS cells were resistant. Surprisingly, while TE671 cells were resistant, TE671-derived RV were sensitive to inactivation. To test whether expression of the amphotropic envelope protein was responsible for conferring this serum sensitivity to the RV, env was expressed in the absence of gag and pol in TE671 cells. However, TE671 cells expressing env were resistant to human serum inactivation. These observations have important implications for use of RV and RV-producer cells for human gene therapy.

Secretion of a soluble, chimeric gamma delta T-cell receptor-immunoglobulin heterodimer.

Soluble derivatives of T-cell antigen receptors (TCRs) should prove invaluable for studying the interaction of these receptors with antigens and major histocompatibility complex molecules, for structural studies, and for the identification of unknown ligands. We have engineered chimeric proteins, containing the extracellular domains of the mouse V gamma 1.1-C gamma 4 and V delta 6.2-C delta (V, variable; C, constant) TCR chains fused to the hinge region, CH2 (H, heavy), and CH3 domains of human IgG1 heavy chain, and expressed them by transient transfection in COS cells. We show here that TCR gamma-IgH and TCR delta-IgH chimeric chains are produced intracellularly in significant amounts, that the two chains can assemble correctly to form disulfide-linked, glycosylated heterodimers, and that a selective mechanism allows secretion of correctly paired receptor chains into the medium. Identity of the chimeric secreted TCR gamma delta-IgH heterodimer was confirmed by immunoblot analysis using V gamma 1-specific anti-peptide antiserum and immunoprecipitation analysis using the monoclonal antibody UC7, which is shown to be specific for the TCR delta chain. In addition, the soluble TCR gamma delta-IgH heterodimer can be immunoprecipitated with the anti-clonotypic monoclonal antibody F10/56, which suggests that the fusion protein likely has a structural conformation similar to that of the native TCR. The COS cell expression system may prove useful for the production of additional TCR-IgH fusion proteins.

Gene transfer demonstrates that the V gamma 1.1C gamma 4V delta 6C delta T cell receptor is essential for autoreactivity.

Murine T cell lines and hybridomas derived from the epidermis that express the V gamma 1.1C gamma 4V delta 6C delta TCR and may, therefore, recognize an autoantigen, secrete cytokines spontaneously in culture. In addition, activation of these cells requires engagement of the vitronectin receptor (VNR) by extracellular matrix proteins. To further evaluate the role of the TCR, the VNR, and the putative autoantigen in the activation of this T cell subset, we cloned complete cDNA encoding the V gamma 1.1C gamma 4 and V delta 6C delta TCR and transfected the cDNA constructs into a TCR- murine hybridoma and into a TCR- variant of the human Jurkat line. The murine transfectant spontaneously produced IL-2 in culture and IL-2 production could be inhibited by anti-CD3, anticlonotypic mAb to the transfected TCR, and anti-VNR mAb, as well as by RGDS. These results demonstrate that transfection of the gamma delta TCR confers to recipient T cells the phenotype of constitutive activation, as well as dependence on engagement of the VNR as an accessory molecule. In contrast, the Jurkat gamma delta transfectant failed to produce cytokines spontaneously, although the transfected TCR was capable of signal transduction after stimulation by anti-TCR mAb. Surprisingly, neither the murine transfectant nor the human transfectant could be induced to respond to autoantigen bearing cells in coculture assays. One interpretation of these results is that coexpression on the surface of the same cell of the V gamma 1.1 V delta 6 TCR, the VNR, and a putative autoantigen are necessary for T cell activation in this system.

Cross-linking studies show that herpes simplex virus type 1 glycoprotein C molecules are clustered in the membrane of infected cells.

Chemical cross-linking using ethylene glycol succinimidyl succinate (EGS) and dithiobispropionimidate (DTBP) was performed to determine the association of herpes simplex virus type 1 glycoprotein C (HSV-1 gC) with its nearest neighbours. Human embryonic lung (HEL) cells were infected with HSV-1 strain KOS, treated with EGS, lysed with Nonidet P40, immunoprecipitated with monoclonal antibodies specific for gC, and analysed by SDS-PAGE. These analyses demonstrated the presence of cross-linked complexes that migrated with an apparent Mr in the range 150,000 to 260,000. Two-dimensional SDS-PAGE (non-reduced and then reduced) analyses of HSV-1-infected HEL cells treated with the cleavable cross-linker DTBP demonstrated that molecules that comigrated with gC were the only components of these high Mr complexes. Immunoelectroblot (Western blot) analyses using polyclonal rabbit antiserum specific for gC verified that the high Mr complexes contained gC. These results indicated that gC molecules may be localized in the infected cell membrane as dimers.

Delta-chains of dendritic epidermal T cell receptors are diverse but pair with gamma-chains in a restricted manner.

To assess the diversity of TCR delta-gene expression in dendritic epidermal T cells (DETC), we characterized the delta-gene used by a panel of cell lines that express the gamma delta TCR on the cell surface. Northern hybridization analyses with a panel of V delta probes representing each of the six known V delta families showed that each of these lines expressed either V delta 1 or V delta 6 containing transcripts. Southern hybridization analysis with the V delta probes gave rearrangement patterns consistent with Northern hybridization results. The correlation of V delta expression with V gamma expression revealed that not only may V delta usage be restricted in DETC cells, but that the pairing of gamma- and delta-chains may not be random. In the DETC cells examined, V delta 1 chains paired exclusively with V gamma 3C gamma 1 chains and V delta 6 chains paired with C gamma 2 and C gamma 4 chains. Sequence analysis of delta-cDNA clones corresponding to the expressed delta-chains from one of the V delta 1 expressing cell lines and two of the V delta 6 expressing cell lines revealed that, although the V delta 1 gene segment sequence and one of the V delta 6 gene segment sequences were identical to previously published V delta sequences, considerable variable region diversity was generated by complex V-D-J rearrangement patterns that utilized various combinations of N-additions, D delta 2 and possibly D delta 1 segments, and J delta 1 or J delta 2 segments. These complex rearrangement patterns distinguish these DETC lines from early thymocytes and suggest that, if DETC are thymic derived, they originate from relatively mature thymic cells.

Purification and structural characterization of herpes simplex virus glycoprotein C.

Purification of herpes simplex virus glycoprotein C (gC) in microgram amounts yielded sufficient material for an analysis of its secondary structure. Purification was facilitated by using the mutant virus gC-3, which bears a point mutation that interrupts the putative hydrophobic membrane anchor sequence, causing the secretion of gC-3 protein into the cell culture medium. gC-3 protein was purified by size fractionation of concentrated culture medium from infected cells on a gel filtration column of Sephacryl S-200, followed by immunoaffinity chromatography on a column constructed of gC-specific monoclonal antibodies cross-linked to a protein A-Sepharose CL-4B matrix. Purified gC-3 had a molecular weight of 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size expected for gC, was reactive with gC-specific monoclonal antibodies in protein immunoblots, and contained amino acid sequences characteristic of gC as determined by radiochemical amino acid microsequence analyses. Polyclonal antisera obtained from a rabbit immunized with gC-3 reacted with wild-type gC in immunoprecipitation, enzyme immunoassay, and immunoelectroblot (western blot) assays. Deglycosylation by treatment with trifluoromethanesulfonic acid reduced the molecular weight of gC-3 by approximately 35%. Analyses of both native and deglycosylated gC-3 by Raman spectroscopy showed that the native molecule consists of about 17% alpha-helix, 24% beta-sheet, and 60% disordered secondary structures, whereas deglycosylated gC-3 consists of about 8% alpha-helix, 10% beta-sheet, and 81% disordered structures. These data were in good agreement with the 11% alpha-helix, 18% beta-sheet, 61% beta-turn, and 9% disordered structures calculated from Chou-Fasman analysis of the primary sequence of gC-3.

Biochemical characterization of peptides from herpes simplex virus glycoprotein gC: loss of CNBr fragments from the carboxy terminus of truncated, secreted gC molecules.

A biochemical characterization of peptides from herpes simplex virus type 1 glycoprotein gC was carried out. We utilized simple micromethods, based on immunological isolation of biosynthetically radiolabeled gC, to obtain gC in pure form for biochemical study. CNBr fragments of gC were prepared, isolated, and characterized. These CNBr fragments were resolved into six peaks by chromatography on Sephacryl S-200 in 6 M guanidine hydrochloride. Only three of the CNBr fragments contained carbohydrate side chains, as judged from the incorporation of [14C]glucosamine. Radiochemical microsequence analyses were carried out on the gC molecule and on each of the CNBr fragments of gC. A comparison of this amino acid sequence data with the amino acid sequence predicted from the DNA sequence of the gC gene showed that the first 25 residues of the predicted sequence are not present in the gC molecule isolated from infected cells and allowed alignment of the CNBr fragments in the gC molecule. Glycoprotein gC was also examined from three gC mutants, synLD70, gC-8, and gC-49. These mutants lack an immunoreactive envelope form of gC but produce a secreted, truncated gC gene product. Glycoprotein gC from cells infected with any of these gC- mutants was shown to have lost more than one CNBr fragment present in the wild-type gC molecule. The missing fragments included the one containing the putative transmembrane anchor sequence. Glycoprotein gC from the gC-8 mutant was also shown, by tryptic peptide map analysis, to have lost more than five major arginine-labeled tryptic peptides arginine-labeled tryptic peptides present in the wild-type gC molecule and to have gained a lysine-labeled tryptic peptide not present in wild-type gC.