Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases.

When cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals.

Detection of ERK activation by a novel monoclonal antibody.

The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.

Ca2+-dependent interaction of S100A2 with muscle and nonmuscle tropomyosins.

Zero-length chemical crosslinking with 1-ethyl-3-[3-(dimethyl amino)propyl]carbodiimide (EDC) indicated an association of the Ca2+-binding protein S100A2 with tropomyosin (TM) in vitro. The mobility of the crosslinked product on SDS-PAGE gels indicated the formation of a 1:1 complex between S100A2 and TM and the interaction was Ca2+ dependent. Monoclonal antibodies were raised against S100A2 and used to determine its cellular localization in the porcine epithelial cell line LLC PK1. It was found that the localization of S100A2 depended on the differentiation state of the cells, being absent from actin stress fibers in sparsely seeded cultures, but present in the actin-containing microvilli characteristic of differentiated cells. Immunoprecipitations of [35S]methionine-labeled extracts using S100A2 as well as TM-specific antibodies failed to co-precipitate TM and S100A2, indicating a transient association between these two molecules in solution. Affinity chromatography of cell extracts on immobilized recombinant TMs, however, confirmed the Ca2+-dependent interaction between S100A2 and both muscle TMs as well as with high and low molecular mass nonmuscle TMs, suggesting that the binding site resides in one of the conserved regions of TM. Our data demonstrate the possible interaction of S100A2 with TM that is not bound to the microfilaments and indicate a differentiation-related function for S100A2 in LLC PK1 cells. The possible functional implications of this interaction are discussed.

Actin isoform compartments in chicken gizzard smooth muscle cells.

Differentiated smooth muscle cells typically contain a mixture of muscle (alpha and gamma) and cytoplasmic (beta and gamma) actin isoforms. Of the cytoplasmic actins the beta-isoform is the more dominant, making up from 10% to 30% of the total actin complement. Employing an antibody raised against the N-terminal peptide specific to beta-actin, which labels only the beta-isoform on two-dimensional gel immunoblots, we have shown that this isoform has a restricted localisation in smooth muscle. Using double-label immunofluorescence and immunoelectron microscopy of ultrathin sections of chicken gizzard, beta-actin was localised in the dense bodies and in longitudinal channels linking consecutive dense bodies that were also occupied by desmin. It was additionally found in the membrane-associated dense plaques, but was excluded from the actomyosin-containing regions of the contractile apparatus. Taken together with earlier results these findings identify a cytoskeletal compartment containing intermediate filaments, cytoplasmic actin and the actin cross-linking protein filamin. Using an antibody specific only for muscle actin, labelling was found generally around the myosin filaments of the contractile apparatus, but was absent from the core of the dense bodies that contained beta-actin. Thus, if dense bodies act as dual-purpose anchorage sites, for the cytoskeletal actin and the contractile actin, the thin filaments of the contractile apparatus must be anchored at the periphery of the dense bodies. A model of the structural organisation of the cell is presented and the possible roles of the cytoskeleton are discussed.

Beta-actin specific monoclonal antibody.

Using a synthetic peptide mimicking the NH2-terminus of beta-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the beta isoform, by its exclusive recognition of the synthetic beta-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the beta-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function.

A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T lymphocyte-mediated autoimmune disease, which serves as a model for several human ocular inflammations of an apparently autoimmune nature. EAU pathology in some rodents and in monkeys can readily be induced by immunization with several different retinal proteins; however, advancing research into the cellular mechanisms of this disease has raised the need for an EAU model in an immunologically and genetically well defined species. We report here the induction of EAU in the mouse, which has hitherto been considered a species refractory to EAU, with two retinal Ag, the retinal soluble Ag and the interphotoreceptor retinoid-binding protein. Although all the mouse strains tested exhibited lymphocyte responses and antibody titers to both retinal Ag, EAU was inducible in only some of the strains, and the uveitogenic responses to retinal soluble Ag and interphotoreceptor retinoid-binding protein appeared to be mutually exclusive. The EAU model in mice was found to differ in several respects from the EAU model in other rodent species. Induction of the disease was achieved with a relatively high dose of Ag and an intensified immunization protocol, and the onset of disease was later, the duration was longer, and the course was less acute. Anterior segment involvement was slight or nonexistent, and damage to the retina and uvea was of a focal rather than of a diffuse nature. Murine EAU appeared to approximate some types of human uveitis more closely than the EAU models described in other rodent species with respect to its pathologic manifestations as well as its more chronic course. The relatively longer duration of the active stage of disease in murine EAU should facilitate therapeutic intervention in established disease, which was not feasible in the more acute models of EAU. The extensive knowledge of the immunologic parameters of the mouse and the availability of genetically defined strains should be of great value in the study of cellular mechanisms and immunogenetics of ocular autoimmune disease.

Induction of experimental autoimmune uveoretinitis by T-cell lines.

Experimental autoimmune uveoretinitis was induced in genetically susceptible Lewis rats by passive transfer of T-lymphocyte cell lines from long-term cultures primed against soluble retinal antigen (S-Ag). A continuous T-cell line was established from non-adherent lymph node cells of S-Ag-immunized Lewis rats. The lymphoid cells were propagated in vitro by serially restimulating them with S-Ag in the presence of irradiated syngeneic spleen cells and expanding them in IL-2-containing media. The cell lines exhibited markers specific for T lymphocytes and the majority had the helper phenotype. When naïve rats were inoculated intravenously with anti S-Ag T-cell lines re-exposed to the antigen prior to injection, they developed uveoretinitis with both clinical and histological characteristics in half the time required by S-Ag to induce the disease by active immunization. The rats exhibited a delayed hypersensitivity skin reaction towards S-Ag.

Monoclonal antibodies to Müller's cells of the retina.

The immunohistologic properties of two monoclonal antibodies produced by hybridomas generated from bovine retinal S-antigen (S-Ag) immunized mice were investigated. These monoclonal antibodies demonstrated a low antibody titer to the original S-Ag preparation by the ELISA method. Immunohistologic studies using avidin-biotin-peroxidase complex (ABC) showed strong specific binding to the retinal Müller cells of all species tested (human, bovine, guinea pig and rat), a weaker binding to cell bodies and proximal component of the outer segments of the photoreceptor, but no apparent binding to the distal component of the outer segments of the photoreceptor cells.

Long-term antigen specific and non-specific T-cell lines and clones in uveitis.

T-cell lines, initially expanded with either mitogens or the uveitogenic S-antigen obtained from the eyes of three uveitis patients, were maintained in vitro for 4-8 weeks. S-antigen specific T-cell clones were derived from the peripheral blood of a fourth patient. The surface marker characteristics clearly demonstrate these cells to be T-cells. Both OKT4+ and OKT8+ clones and cell lines were found using immunofluorescence microscopy, an ELISA technique, or the laser cytofluorograph, but some could be only definitely identified as T-cells with the antibody to the IL-2 receptor, and not the OKT series of monoclonal anti-sera. These studies underscore how dynamic the expression of T-cell membrane markers can be, and that commercially available monoclonal anti-sera to T-cell membrane markers may not identify all T-cells in long term culture. This is the first report of the isolation of an antigen specific T-cell clone relevant to the eye, and the technique described will allow clonal expansion of ocular cells in order to study their functional characteristics.

Experimental autoimmune encephalomyelitis mediated by T-cell line. II. Specific requirements and the role of pertussis vaccine for the in vitro activation of the cells and induction of disease.

Murine T-cell lines derived from (SJL/J X BALB/c)F1 mice were established which are specifically proliferating in response to myelin basic protein (BP) and are also functional in mediating experimental autoimmune encephalomyelitis (EAE) in normal recipients. Partial characterization of the cells, the requirements of their selection and in vitro activation, and the role of pertussis vaccine for mediation of EAE were studied. The EAE-effector line cells were characterized as Lyt 1+2- cells, suggesting delayed-type hypersensitivity mechanism as a major EAE-effector mechanism in mice. Activation in vitro of EAE-effector line cells by stimulation with BP or concanavalin A in the presence of irradiated syngeneic accessory cells was required to facilitate their capacity to mediate EAE in normal recipients. (SJL/J X BALB/c)F1 EAE-effector line cells recognize BP presented by F1-specific accessory cells to facilitate adequate specific proliferation of the cells. Pertussis vaccine was found nonessential for mediation of EAE by BP-specific effector line cells, but was found essential for uncovering T cells responding to BP. Thus, the pertussis vaccine may play a more crucial role at the sensitization phase, by enhancing a T-cell response to BP, rather than by altering the blood-brain barrier at the effector phase of EAE.

Association of human T-cell leukaemia/lymphoma virus with the Tac antigen marker for the human T-cell growth factor receptor.

Certain adult T-cell lymphoproliferative disorders are associated with human T-cell leukaemia virus (HTLV), a unique human type C retrovirus. (The strains of HTLV used in these studies belong to the subgroup HTLV-I.) HTLV is not an endogenous agent in man, but rather is an acquired virus with T-cell tropism. Neoplastic cells from patients infected with HTLV generally express receptors for T-cell growth factor (TCGF) (interleukin-2), and do not require prior activation with antigens or lectins to undergo TCGF-induced proliferation. Furthermore, neoplastic T-cell lines originating from such patients may constitutively produce TCGF, TCGF receptors and HTLV virions. HTLV is transmissible from cell to cell, and the infection of human T cells in vitro is associated with the expression of TCGF receptors, which can be identified by the monoclonal antibody termed anti-Tac. In our experience to date, T-cell populations that produce HTLV without exception also express epitopes found on TCGF receptors. Recognition of an association between HTLV virions and the Tac antigen would have clinical and theoretical implications. We now present evidence that during the replication or release of HTLV, the virion becomes preferentially associated with the Tac antigen.

Analysis of cross-reactive antigen-specific T cell clones. Specific recognition of two major histocompatibility complex (MHC) and two non-MHC antigens by a single clone.

Two T cell clones, one specific for I-Es/d plus myelin basic protein (BP) and another specific for I-Ak plus influenza virus have been demonstrated to cross-react with DBA/2 cells. Genetic and serological analyses have shown that each clone recognizes its respective priming antigen in association with self-major histocompatibility complex (MHC) determinants and each recognizes DBA/2 minor H antigens in association with allo I-Ad MHC antigens. Further analysis of these clones suggests (a) that the allo I-Ad MHC epitopes recognized by these clones are not shared with self-I-A epitopes, (b) that the virus or BP antigens do not cross-react with DBA/2 minor H antigens, (c) that these clones recognize different determinants on the DBA/2 minor H antigens, and (d) that there is a requirement for a specific association between the different MHC antigens and the non-MHC antigens to stimulate these clones. This specific associative recognition argues strongly for the "altered self" hypothesis.

Detection of autoimmune cells proliferating to myelin basic protein and selection of T cell lines that mediate experimental autoimmune encephalomyelitis (EAE) in mice.

A procedure is described for the detection of an in vitro proliferative response to the autologous mouse myelin basic protein in mice injected with mouse spinal cord homogenate (MSCH) or with myelin basic proteins (BP) of mouse (MBP) or rat (RBP) origin. The administration of MSCH, but not of MBP or RBP, in a suitable adjuvant could produce a reproducible clinical disease. Nevertheless, a proliferative response to the autologous MBP could not be detected after either inoculation. Only the removal of the adherent cell fraction from the immunized cell population and its replacement with fresh naive accessory cells could reveal a proliferative response to the autologous MBP and to the heterologous RBP. A heteroclitic cross-reactivity between MBP and RBP is demonstrated. The possibility of detecting an in vitro proliferative response to BP allowed the selection and propagation in vitro of cells specific to BP. T cell lines were established that specifically proliferated in response to BP and mediated EAE in normal mice. Intravenous inoculation of as few as 10(6) line cells was capable of producing clinical signs of EAE in normal recipients within 5 to 6 days. Thus, although an inoculation of MSCH was required for active induction of the disease, T cells specifically reactive against BP are sufficient for mediation of EAE in mice.

Lack of H-2 restriction of suppressor factor specific to myelin basic encephalitogen.

A cell-free extract has been prepared from spleen cells of (SJL/J x BALB/c)F1 mice which were rendered non-susceptible to EAE by treatment with mouse spinal cord homogenate in incomplete Freund's adjuvant. Such extract has been previously shown to have suppressive activity on the induction of EAE, as well as on the immune response in syngeneic mice towards the mouse basic protein. We have now demonstrated that this factor is as effective in several other strains of mice, of different H-2 haplotype. The activity of this factor was manifested both in vivo by inhibiting the DTH response to MBE in the various mouse strains, and in vitro, by blocking the MIF activity of the allogeneic lymphocytes. A suppressor factor was prepared by a similar procedure from the EAE-resistant BALB/c mice. This factor was as active as the factor from the (SJL/J x BALB/c)F1 hybrid in blocking the anti-MBE antibody binding to MBE. It is also suppressive, and it inhibited the DTH response to MBE in both syngeneic and semi-allogeneic (SJL/J x BALB/c)F1 mice. It is thus demonstrated that the MBE-specific soluble suppressor factor involved in the EAE system in mice shows no indication of H-2 restriction.

Unresponsiveness to experimental allergic encephalomyelitis in mice: replacement of suppressor cells by a soluble factor.

A soluble suppressor factor has been prepared from cells of mice rendered nonsusceptible to experimental allergic encephalomyelitis (EAE) by treatment with mouse spinal cord homogenate in incomplete Freund's adjuvant. The specific activity of this factor can be augmented by using a cell population enriched on plates coated with anti-mouse Fab and the specific antigen, mouse basic encephalitogen (MBE). The resultant suppressor factor had the same biologic activities as the cells from which it originated. Thus, it suppressed specifically the delayed-type hypersensitivity (DTH) response to MBE in vivo, and blocked in vitro the effector lymphocytes that adoptively transfer the DTH response. The suppressor factor reactivity was manifested also by the capacity to suppress the activity of macrophage migration inhibition factor produced by sensitized lymphocytes in the presence of the specific antigen MBE. The suppressor factor is antigen-specific and can bind the MBE in vitro and thus compete with its antibody binding. The most significant activity of the soluble suppressor factor is its ability to interfere with the induction of clinical EAE.

The immunologic response in mice unresponsive to experimental allergic encephalomyelitis.

The suppressor cells that are involved in antigen-induced protection against EAE in mice were investigated with respect to their effect on the immune response. The cellular immune response to the basic encephalitogenic protein (BE) and to PPD were studied in mice with either actively induced or adoptively transferred unresponsiveness to EAE. The results demonstrate that the DTH response to BE, as assayed in the radiometric ear skin test, was suppressed in mice protected against EAE. Moreover, the passive transfer of DTH response to BE by effector lymphocytes was also inhibited by the preinjection of suppressor cells. On the other hand, the suppressor cells did not affect the response to PPD in all these experiments. The results indicate that suppressor cells that mediate unresponsiveness to EAE regulate also the cellular immune response to BE in a specific manner. These suppressor cells are probably active both at the induction and the effector phase of the immune response.

Effect of cyclophosphamide on suppressor cell activity in mice unresponsive to EAE.

Protection against experimental allergic encephalomyelitis (EAE) was induced in susceptible mice of (SJL/J X BALB/c)F1 hybrid, by injection of either mouse spinal cord homogenate, the small mouse basic protein, or Cop 1 in incomplete Freund's adjuvant, before EAE induction. It was demonstrated that the unresponsiveness induced by the three antigens is mediated by suppressor T cells residing in the spleen cell population and can be adoptively transferred to normal syngeneic recipients. Low dose of cyclophosphamide (20 mg/kg) administered 2 days before the encephalitogenic challenge abrogated the unresponsiveness to EAE and reverted the protected mice sensitive to disease induction. Cyclophosphamide was also active on adoptively transferred unresponsiveness, thus donors that had been treated with cyclophosphamide were unable to further transfer unresponsiveness to EAE. These results indicate the elimination by cyclophosphamide of suppressor cells that interfere with the effector mechanisms leading to EAE.