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Murine spienie T lymphocytes display maximal cellular myc gene (c-myc) expression already 3 h after concanavalin A timulation and sub equent down-regulation before the onset of DNA syntbesis. Stimulation by leucoagglulinin in the prcsence or absence of interleukin 2 Ieads to only low initiaJ Ievels of c-myc-specific RNA which, however, increase later on. A similar pattero of c-myc expression is shown by the Lyt- 2+ T cell subpopulation stimuiated with eilher concanavalin A or leucoagglutinin in the prescncc of interleukin 2. Although eH]thyn1idine incorporation was identical, the leucoagglutinin-stimulated Lyt-2+ T cells werc void of any demon. trable c-mycspeci. fic RNA at 3 h post-stimulation. Thus, the kinetics of c-myc expression in mause T lymphocytes arenot at all uniform, but depend on the mitogen and the subpopulation. [n contrast, lcvel8 of c-rasH•-spccific R A wcre always low at early times, always increased towards tbe onset ofDNA synthesis and down-regulationwas not observed.
Two cellular proteins, membrane cofactor protein (MCP) and moesin, were reported recently to be functionally associated with the initiation of a measles virus infection. We bave analyzed the interaction of measles virus with cell surface proteins, using an overlay binding assay with cellular proteins immobilized on nitrocellulose. Among surface-biotinylated proteins from a human rectal tumor cellline (HRT), measles virus, was able to bind only to a 67-kDa proteinthat was identified as MCP. The virus recognized dift'erent isoforms of MCP expressed from human (HRT and HeLa) and simian (Vero) celllines. The binding of measles virus to MCP was abolished after cleavage of the disulfide bonds by reducing agents as weil as after enzymatic release of N-linked oligosaccharides. By contrast, removal of sialic acid or 0-linked oligosaccharides did not aft'ect the recognition of MCP by measles virus. These data indicate that the receptor determinant of MCP is dependent on a conformation of the protein that is maintained by disulfide bonds and N-glycans present in tbe complement binding domains. Our results are consistent with a roJe of MCP as primary attacbment site for measles virus in the initial stage of an infection. The functional relationship between MCP and moesin in a measles virus infection is discussed.
To probe into the functional properties of the major peripheral myelin cell surface glycoprotein P 0 , its ability to confer adhesion and neurite outgrowth-promoting properfies was studied in cell culture. Tothis aim, Po was expressed as integral membrane glycoprotein at the surface of CV -1 cells with the help of a recombinant vaccinia virus expression system. Furthermore, the immunoglobulin-like extracellular domain of P0 (P0 -ED) was expressed as soluble profein in a bacterial expression system and used as substrafe coated to plastic dishes or as competitor in cell adhesion and neurite outgrowth-promoting assays. The adhesion of P0 -expressing CV-1 cells to P0 -ED substrafe was specifically inhibitable by polyclonal Po antibodies (54% :t 6% ). In addition, the specific interaction between Po molecules could be reduced ( 49% ± 8%) by adding soluble P0 -ED to the culture medium, demonstrating that the homophilic inter~ction between recombinant Po molecules can be mediated, at least on one partner of interacting molecules, by the unglycosylated Ig-like domain. Substrate-coated p -ED also conferred adhesion and neurite outgrowth ability to dorsal root ganglion neurons with neurites of a mean length of about 150 ,_..m. This neurite outgrowth was specifically inhibitable by soluble P" (74% ± 14%) and P 0 antibodies (65% ± 9% ). These observations indicate that Po is capable of displaying two different types of functional roles in the myelination process of . peripheral nerves: The heterophilic interaction with neurons may be responsible for the recognition between axon and myelinating Schwann cell at the onset of myelination, whereas the homophilic interacton may indicate its roJe in the selfrecognition of the apposing loops of Schwann cell surface membranes during the myelination process and in the mature compact myelin sheath.
Quantification of the peripheral nerve myelin glycoprotein PO and antibodies to PO is difficult due to insolubility of PO in physiological solutions. We have overcome this problern by using the water-soluble recombinant form of the extracellular domain of PO (PO-ED) and describe newly developed assays which allow detection and quantitation of PO and antibodies to PO, in serum and cerebraspinal fluid (CSF). These sensitive and specific assays based on the ELISA technique were used to study humoral immune responses to PO during experimental autoimmune ("allergic") neuritis (EAN). In order to establish these tests, monoclonal antiborlies to different epitopes of rodent and human PO-ED were produced. A two-antibody sandwich-ELISA allowing quantitation of PO Oower detection Iimit of 0.5 ngjml or 30 fmoljml) and an antibody-capture ELISA (lower detection Iimit 1 ng specific antibody jml) to detect antiborlies to PO in serum and CSF were developed. EAN was induced in rats by active immunization with bovine myelin or the neuritogenic protein P2 or by adoptive transfer using P2 specific CD4 positive T cells. Serum and CSF were assayed for the presence of PO-ED and antibodies to PO-ED or P2. Antibodies to PO-ED were detected during active myelin-induced EAN, but not during P2-induced or adaptive transfer EAN. The anti-PO-ED antibodies in the CSF showed a correJation with disease activity. In contrast, in the same model antibodies to P2 persisted long after the disease ceased. No soluble PO-Iike fragments could be found in serum or CSF during any of the three types of EAN. We conclude that PO may be a B-eeil epitope in EAN. These findings warrant a screen for antibodies to PO-ED in human immune neuropathies.
Seven monoclonal antibodies were raised against the immunoglobulin-like extracellular domain of PO (POED), the major protein of peripheral nervous system myelin. Mice were immunized with purified recombinant rat PO-ED. After fusion, 7 clones (POI-P07) recognizing either recombinant, rat, mouse, or human PO-ED were selected by ELlS A and were characterized by Western blot, immunohistochemistry, and a competition assay. Antibodies belonged to the IgG or IgM class, and P04-P07, reacted with PO in fresh-frozen and paraffin-embedded sections of human or rat peripheral nerve, but not with myelin proteins of the central nervous system of either species. Epitope specificity of the antibodies was determined by a competition enzyme-linked immunosorbent assay (ELISA) and a direct ELlS A using short synthetic peptides spanning the entire extracellular domain of PO. These assays showed that POl and P02 exhibiting the same reaction pattern in Western blot and immunohistochemistry reacted with different distant epitopes of PO. Furthermore, the monoclonal antibodies P05 and P06 recognized 2 different epitopes in close proximity within the neuritogenic extracellular sequence of PO. This panel of monoclonal antibodies, each binding to a different epitope of the extracellular domain of PO, will be useful for in vitro and in vivo studies designed to explore the role of PO during myelination and in demyelinating diseases of the peripheral nervous system.
DNA synthesis and adenosine(S')tetraphosphate(S ')adenosine (Ap.A) levels decrease in cells treated with EDTA. The inhibitory effect of EDTA can be reversed with micro molar amounts of ZnCI2• ZnCh in micromolar concentrations also inhibits Ap.A hydrolase and stimulates amino acid-dependent Ap.A synthesis, suggesting that Zn2+ is modulating intracellular Ap.A pools. Serum addition to GI-arrested cells enhances uptake of Zn, whereas serum depletion leads to a fivefold decrease of the rates of zinc uptake. These results are discussed by regarding Zn2+ as a putative 'second messenger' of mitogenic induction and Ap.A as a possible 'third messenger' and trigger of DNA synthesis.
To investigate the influence of inflammatory cytokines on the potential of peripheral nerves to regenerate, we analyzed the effect of interferon-y (lFN-y) and tumor necrosis factor-a (TNF-a) on the ability of immortalized Schwann cells to mediate outgrowth of neurites from primary DRG neurons. We found that IFN-y and TNF-a synergistically inhibited the neurite outgrowth-promoting properties of the Schwann cells by spedfically dowllregulating myelin-associated glycoprotein (MAG) at the levels of mRNA and cell surface protein by approximately 60%. Antibodies to MAG inhibited the outgrowth of neurites on Schwann cells to the same extent as treatment with the two cytokines. Since MAG appears to be involved in both neurite outgrowth and myelination, our findings may provide evidence for a mechanism, by wh ich inflammatory cytokines interfere with Schwann cell-neuron interactions.