T-helper lymphocytes specific for myelin basic protein: activation-induced refractoriness of IL-2 production pathways augments an anti-CD4-mediated proliferative deficit.

Cloned and uncloned lines of encephalitogenic rat T cells produce IL-2 when activated with myelin basic protein (MBP) in the presence of irradiated splenocytes (SPL). Although these T cells use IL-2 as a primary mediator of autocrine growth, regulatory mechanisms controlling production of IL-2 have yet to be fully defined. This study shows that T cells reactivated within approximately 7 days of a prior activation were refractory to the reinduction of MBP-stimulated IL-2 production. In contrast, T cells rested for > 7 days regained the ability to produce optimal levels of IL-2 during activation with MBP. Cultures containing both activated and resting T cells responded to MBP by producing levels of IL-2 that were similar to those obtained from control cultures of resting T cells. The lack of IL-2 production during this refractory phase was associated with lowered responsiveness to MBP in proliferative assays as evidenced by right-shifted dose-response curves. However, this refractory phase did not affect the magnitude of responses elicited by optimal concentrations of MBP. The dissociation of proliferation from IL-2 production suggested parallel pathways of autocrine growth. Indeed, anti-MBP-proliferative responses were mediated by two distinct mechanisms distinguished by differential susceptibility to the anti-CD4 mAb W3/25. The W3/25-sensitive proliferation was desensitized in chronically activated T cells as well as in T cells activated once in the presence of the anti-CD4 mAb W3/25. Conversely, MBP responsiveness of W3/25-insensitive proliferation was unchanged by both chronic activation and by a prior activation in the presence of W3/25. In cultures of T cells recently activated by MBP in the presence of W3/25, the use of nonirradiated SPL rather than irradiated SPL reversed W3/25-mediated tolerance but did not restore MBP-stimulated IL-2 production. In summary, this study reveals mechanisms whereby the engagement of TcR and CD4 negatively regulates subsequent responsiveness of IL-2 production pathways and thereby impairs restimulation of IL-2-dependent proliferation by MBP-specific T-helper cells.

Parallel costimulatory pathways promote myelin basic protein-stimulated proliferation of encephalitogenic rat T cells.

Activation pathways responsible for myelin basic protein (MBP)-stimulated proliferation of encephalitogenic T cells were studied by derivation of new monoclonal antibodies against rat T cell surface proteins. These monoclonal antibodies were derived by immunization of Balb/c mice with THYB-2 T cell hybrids or with the GP2.E5 line of encephalitogenic T-helper cells. The LRTC1 mAb inhibited MBP-stimulated IL-2 production by THYB-2 hybrids but not by THYB-1 hybrids and did not inhibit the alternative response of MBP-induced growth inhibition by either hybrid subset. Although LRTC1 labeled virtually all rat leukocytes, it selectively inhibited proliferative responses to T cell mitogens but not to B cell mitogens. LRTC1 inhibited MBP-stimulated IL-2 production by GP2.E5 T cells but did not effectively block MBP-stimulated proliferation. Rather, LRTC1 acted in synergy with a second mAb (LRTC2) to effectively inhibit MBP-stimulated proliferation by GP2.E5 T cells. The observation that LRTC1 did not exhibit synergy with a third biologically active mAb (LRTC3) supported the hypothesis that LRTC1 and LRTC2 represented a specific combination of synergistic mAb. In contrast to the inhibitory activity on GP2.E5 T cells, LRTC1 and LRTC2 synergistically stimulated antigen-independent IL-2 production by the THYB-1 hybrid LSS-A1. These results support the hypothesis that GP2.E5 T cells respond to parallel costimulatory pathways that are respectively inhibited by LRTC1 and LRTC2 mAb. Furthermore, these synergistic mAb exhibited inhibitory or stimulatory activities that may be diagnostic of distinct T-helper cell subsets. These novel mAb may thereby facilitate studies of costimulatory pathways and T-helper cell subsets in the pathogenesis of autoimmune disease.

Differentiation of encephalitogenic T cells confers resistance to an inhibitory anti-CD4 monoclonal antibody.

The anti-CD4 mAb W3/25 inhibits experimental autoimmune encephalomyelitis (EAE) in Lewis rats by blocking Th cell responses to encephalitogenic determinants of myelin basic protein (MBP). However, it has yet to be resolved how W3/25 modulates CD4 to inhibit EAE-associated T cell responses. This study revealed that W3/25 profoundly inhibited MBP-stimulated proliferation by sensitized lymph node cells but only partially inhibited the respective response of uncloned and cloned lines of MBP-specific T cells. That is, low concentrations of W3/25 blocked 30 to 60% of MBP-stimulated proliferation, but 100-fold higher concentrations did not result in additional inhibition. W3/25 also inhibited MBP-induced acquisition of EAE transfer activity, but only in cultures of freshly isolated lymph node cells and not in cultures of continuously propagated T cells. Studies focusing on the GP2.E5 T cell line revealed that the lack of sensitivity to W3/25 in encephalitogenic and proliferative assays was nevertheless associated with an effective blockage of MBP-stimulated IL-2 production. Importantly, W3/25 specifically inhibited antigenic but not mitogenic stimulation of IL-2 production. Reverse transcriptase/polymerase chain reaction analyses revealed that MBP-activated GP2.E5 T cells produced mRNA for both IL-2 and IL-4, and that W3/25 selectively inhibited accumulation of IL-2 as compared to IL-4 mRNA. Thus, GP2.E5 T cells apparently express a IL-4-dependent pathway that confers resistance to the inhibitory activity of W3/25. Studies focusing on two CD4+ T cell hybridomas revealed that W3/25 profoundly inhibited MBP-stimulated IL-2 production but did not affect the alternative response of MBP-induced growth inhibition. Several other hybrids also mediated MBP-stimulated IL-2 production but did not express CD4 and were not affected by W3/25. These results indicate that: 1) interactions of W3/25 with CD4 do not necessarily block class II MHC-restricted recognition of MBP; and 2) expression of CD4 is not necessary for Ag recognition by several clonotypes of MBP-reactive T cells. Rather, the results of this study are consistent with the concept that W3/25 inhibits transduction of costimulatory signals that are required specifically for initiation of IL-2 production. These findings may have important implications for understanding the therapeutic potential of anti-CD4 mAb in autoimmune disease.

Purification and quantitation of a rat plasma selenoprotein distinct from glutathione peroxidase using monoclonal antibodies.

Studies with 75Se have shown the existence of a rat plasma selenoprotein in addition to glutathione peroxidase. Because the function of the protein is not known, it has been referred to as selenoprotein P. A partially purified preparation was used to produce a monoclonal antibody to selenoprotein P. The antibody did not bind glutathione peroxidase as evidenced by its failure to remove glutathione peroxidase activity from rat plasma by immunoprecipitation. An immunoaffinity column was prepared with the monoclonal antibody, and selenoprotein P was purified 1270-fold from rat plasma in a two-step procedure. The purified selenoprotein P migrated in a single band with an Mr of 57,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography demonstrated that this band contained 75Se when the protein was purified from rats which had received 75SeO2-(3). A competitive radioimmunoassay for selenoprotein P was developed. The selenoprotein P concentration in plasma of selenium-replete rats was determined with this assay to be 51 +/- 3.7 micrograms/ml. It was less than 5 micrograms/ml in plasma from selenium-deficient rats. Injection of 50 micrograms of selenium into selenium-deficient rats caused an increase in selenoprotein P from less than 10% of control to 52% of control in 6 h. Plasma glutathione peroxidase activity increased only from 2.2 to 3.1% of control. These experiments demonstrate that rat plasma contains a selenoprotein distinct from glutathione peroxidase. The concentration of this selenoprotein is depressed in selenium deficiency, as is glutathione peroxidase activity, but selenoprotein P increases more rapidly when selenium is supplied than does glutathione peroxidase activity.

Molecular characterization of Mycoplasma genitalium species-specific and cross-reactive determinants: identification of an immunodominant protein of M. genitalium.

Species-specific proteins of Mycoplasma genitalium as well as proteins cross-reactive with M. pneumoniae have been identified using monoclonal antibodies generated against these mycoplasmas in enzyme-linked immunosorbent assays (ELISA) and Western blot analyses. Specificity of the antibodies was examined using M. hominis, M. orale M. salivarium and Acholeplasma laidlawii. A 140-kDa (kilodalton) protein of M. genitalium that appeared to be immunodominant in mice was also shown by radioimmunoprecipitation to be immunodominant in experimentally infected chimpanzees.

Identification of a 32-kilodalton protein of Mycoplasma pneumoniae associated with hemadsorption.

Monoclonal antibodies directed against a 32-kilodalton (kDa) protein of Mycoplasma pneumoniae have been used to characterize a hemadsorption-negative (HA-) mutant class whose protein profile was previously indistinguishable from the wild-type, hemadsorbing (HA+) strain. Electron microscopy and colloidal gold labeling techniques were applied for ultrastructural analysis of the 32-kDa protein. Results indicate that this protein clusters in the tip structure of M. pneumoniae (HA+) wild-type organisms. Additionally, the protein is precipitated by infected hamster sera.

Shared epitopes between Mycoplasma pneumoniae major adhesin protein P1 and a 140-kilodalton protein of Mycoplasma genitalium.

Previous serological data have demonstrated cross-reactive antigens between two pathogenic species of mycoplasmas, M. pneumoniae and M. genitalium. Preliminary analysis of sera and monoclonal antibodies (MAbs) to protein antigens of these species showed an immunodominance of adhesin P1 (165 kilodaltons [kDa]) of M. pneumoniae in mice and hamsters and a 140-kDa protein of M. genitalium in mice and experimentally infected chimpanzees. To further characterize these two proteins, we assayed multiple anti-P1 and anti-140-kDa protein MAbs by enzyme-linked immunosorbent assay, immunoblot, and radioimmunoprecipitation techniques. The 140-kDa M. genitalium protein was shown to be surface accessible and insensitive to levels of trypsin which readily degrade protein P1. Peptide mapping was used to identify a unique class of MAbs which bound a cross-reactive molecule common to both the major adhesin protein P1 of M. pneumoniae and the 140-kDa protein of M. genitalium. MAbs generated against both M. pneumoniae and M. genitalium which were reactive with this determinant blocked M. pneumoniae attachment to chicken erythrocytes.

Biological effects of anti-lipid and anti-protein monoclonal antibodies on Mycoplasma pneumoniae.

Monoclonal antibodies directed against Mycoplasma pneumoniae surface components were examined for their ability to block mycoplasma attachment to chicken erythrocytes. Purified preparations of antibodies which recognize the major mycoplasma ligand mediating cytadherence (protein P1, 165 kilodaltons) inhibited attachment by more than 85% of the control values. Monoclonal antibodies reactive with two other surface proteins of 110 and 32 kilodaltons also blocked attachment. Surprisingly, monoclonal antibodies specific for M. pneumoniae lipids (J. Morrison-Plummer, D. H. Jones, and J. B. Baseman, J. Immunol. Methods 64:165-178, 1983) enhanced mycoplasma-erythrocyte binding. All antibodies examined had no effect on thymidine incorporation by M. pneumoniae.

Analysis of proteins in rabbit pulmonary surfactant using monoclonal antibodies.

We have used monoclonal antibodies developed against the apolipoproteins associated with pulmonary surfactant purified from rabbit lavage fluid to study the expression of epitopes common to these proteins. The pulmonary surfactant contained nearly 20 proteins, of which at least 10 were not derived from serum. Electrophoresis, with sulfhydryl reduction of these proteins indicated apparent molecular weights of approximately 155, 135, 125, and 115 X 10(3) (high-molecular-weight group); 80, 70, and 60 X 10(3) (intermediate group); and 18 through 10 X 10(3) (low-molecular-weight group). Two-dimensional polyacrylamide gel electrophoresis, in which the proteins were electrophoresed without reduction in the first dimension, but with sulfhydryl reduction in the second dimension, revealed that the 80, 70, and 60 X 10(3) proteins dissociated into proteins of nominal molecular weights of 40, 35, and 30 X 10(3), respectively. In contrast, the 125 and 115 X 10(3) proteins of the high-molecular-weight group contained a protein which could only be reduced to a minimum molecular weight of 55 to 60 X 10(3). Monoclonal antibodies generally were of three types: those that reacted strongly with the high-molecular-weight group and weakly with the intermediate group; those that reacted conversely; and those that reacted only with the low-molecular-weight group. Our results indicate that at least two different surfactant apolipoproteins, with differing minimum molecular weights in SDS-polyacrylamide gel electrophoresis, have common epitopes. Although these results cannot certify a physiological relationship between these proteins, they suggest that the intracellular synthesis or extracellular processing of surfactant apolipoproteins may be more complicated than predicted by the findings of previous experiments, perhaps involving the posttranslational assembly of one surfactant protein into oligomers which resist dissociation under the conditions used for the analyses.

Detection of the major adhesin P1 in triton shells of virulent Mycoplasma pneumoniae.

Filamentous structures designated Triton shells were obtained from virulent Mycoplasma pneumoniae by treatment with Triton X-100. Monoclonal antibodies directed against M. pneumoniae were used in conjunction with radioimmunoprecipitation and Western blotting to detect immunologically reactive polypeptides in Triton shells. The major adhesin, protein P1, was associated with these structures.

Monoclonal antibodies to Treponema pallidum: recognition of a major polypeptide antigen.

Hybridomas secreting monoclonal antibodies that reacted with a 45 000 dalton surface polypeptide and major immunogen of T pallidum were produced. This polypeptide was also found in T pertenue but not in T hyodysenteriae or T phagedenis biotype Reiter.

Synthesis of sigma 29, an RNA polymerase specificity determinant, is a developmentally regulated event in Bacillus subtilis.

Using an immunological probe, we have determined that the synthesis of the Bacillus subtilis RNA polymerase promoter specificity determinant sigma 29 is a developmentally regulated event. sigma 29 is absent from vegetatively growing cells but is abundant in sporulating cells for a restricted (2-h) period during differentiation (hour 2 to hour 4 into the sporeforming process). The narrowness of this period suggests that sigma 29 is a regulatory factor that directs the transcription of a subpopulation of genes at a precise, intermediate stage of spore formation. This view predicts that sigma 29 should be dispensable for early sporulation events. We verified this prediction by an analysis of sigma 29 accumulation in mutants that are blocked at different stages of sporulation in which we show that cells can advance to at least an intermediate point in development (stage III) in the absence of detectable sigma 29. Lastly, our anti-sigma 29 antibody probe detected a second, previously unrecognized protein in Bacillus cell extracts that may be a precursor to sigma 29. This protein, P31 (molecular weight, 31,000) is synthesized earlier in sporulation than is sigma 29. It has a peptide profile that is similar to sigma 29 and is present in all Bacillus subtilis Spo- mutants that were tested and found to still be able to accumulate sigma 29.

An ELISA to detect monoclonal antibodies specific for lipid determinants of Mycoplasma pneumoniae.

An enzyme-linked immunosorbent assay (ELISA) was developed for analysis of monoclonal antibodies directed towards lipid determinants of Mycoplasma pneumoniae. Chloroform-methanol lipid extracts as well as chromatographed lipid fractions of M. pneumoniae were bound to polyvinylchloride microtiter wells. Of 293 clones positive to M. pneumoniae as detected by a whole cell ELISA, a total of 78 clones produced antibodies which selectively bound to lipid extracts of M. pneumoniae. The simplicity and reproducibility of the assay permit rapid screening for detection of antibodies directed against non-protein cellular antigens.

Enzyme-linked immunosorbent assay for the detection of serum antibody to outer membrane proteins of Treponema pallidum.

A highly sensitive enzyme-linked immunosorbent assay was used for the analysis of serum IgG reactivity against specific immunogenic Treponema pallidum proteins. Outer membrane treponemal proteins purified by preparative SDS-polyacrylamide gel electrophoresis were used as antigenic probes at concentrations as low as 100 ng per ml (5 ng per well in microtitre plates). Detection of anti-treponemal antibody was possible using rabbit syphilitic sera diluted to 1/10 000. The sensitivity of the assay was equal to or greater than that detected by radioimmuno-precipitation combined with gel electrophoresis and fluorography techniques and was capable of monitoring host IgG responses throughout the progress of the disease.