Idiotypic determinants on human T cells and modulation of human T cell responses by anti-idiotypic antibodies

The role of idiotypic anti-idiotypic interactions in the regulation of the human T cell response to tetanus toxoid (TT) antigen was examined in three subjects. Rabbit anti-idiotypic (anti-Id) antisera were raised against IgG (Fab')2 anti-TT obtained 7 to 10 days after booster immunization with TT. F(ab')2 fragments of rabbit-anti-Id IgG were used in conjunction with fluorescein-conjugated goat anti-rabbit Ig in an indirect immunofluorescence assay to determine the frequency of Id-positive cells in T cell-enriched preparations. This frequency was 24, 29, and 38 per 10,000, respectively, in the three subjects studied. Significant contribution of contaminating B cell to fluorescence-staining was ruled out by capping experiments using goat anti-human Ig (GAHIG) and by double staining experiments using rhodamine-conjugated GAHIG. Absorption of anti-Id antisera with Epstein Barr virus (EBV)-transformed B cell lines from the IgG (Fab')2 anti-TT donor, but not with EBV-B cell lines from unrelated donors, removed their reactivity with the T cells. Rabbit anti-Id IgG caused minimal proliferation (two-threefold) of T cells and had no effect on T cell proliferation in response to TT antigen when added to the cultures. Preincubation of T cells for 48 hr with rabbit anti-Id IgG (Fab')2, but not with preimmune rabbit IgG (Fab')2, resulted in the generation of antigen-specific suppressor cells that inhibited T cell proliferation in response to TT, but not in response to diphtheria toxoid (DT). These cells also inhibited the synthesis of IgG anti-TT in response to in vitro stimulation with TT antigen, but not the synthesis of IgG anti-DT in response to DT antigen. Adsorption of T cells over plates coated with rabbit anti-Id IgG (Fab')2 enhanced the proliferative response of the T cells to TT, but not to DT antigen, and enhanced the helper activity of the T cells for the in vitro synthesis of IgG anti-TT but not of IgG anti-DT antibodies. These results suggest that idiotypic-anti-idiotypic interactions play a role in the human T cell response to antigen.     

Toxic shock syndrome toxin-1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells

Toxic shock syndrome toxin-1 (TSST-1) is a 22-kDa exotoxin produced by most Staphylococcus aureus strains responsible for toxic shock syndrome. TSST-1 is a mitogen for human T cells. The mechanism of T cell activation by TSST-1 was investigated. TSST-1 induced IL-2R expression, IL-2 synthesis, and proliferation in T cells in a monocyte-dependent fashion. Neither IL-1 nor IL-2, alone or in combination, substituted for monocytes in supporting TSST-1-induced mitogenesis. We investigated the mechanism by which TSST-1 induces initogenesis. TSST-1 failed to induce ADP-ribosylation of T cell membrane proteins. However, the toxin induced transient translocation of protein kinase C from cytosol to plasma membranes and also induced the mobilization of cellular Ca2+ stores in both PBMC and the Jurkat human tumor T cell line, suggesting that TSST-1 triggered inositol phospholipid turnover. This was directly demonstrated to be the case in both cellular preparations studied. TSST-1 induced the increased synthesis of the inositol phospholipid phosphatidyl inositol, phosphatidyl inositol-4 phosphate, and phosphoinositol inositol-4,5-bisphosphate, and induced the breakdown of inositol phospholipid as evidence by the accumulation of phosphatidic acid and inositol phosphates. We conclude that the action of TSST-1 involves the induction of inositol phospholipid turnover, protein kinase C activation, and mobilization of cellular Ca2+ stores. This effect is similar to that of mitogenic lectins and of anti-CD3 antibodies.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Developmental regulation of transmembrane signaling via the T cell antigen receptor/CD3 complex in human T lymphocytes.

We have examined transmembrane signaling events via the TCR/CD3 complex (TCR/CD3) at various stages of T cell development for evidence of developmental regulation. Engagement of TCR/CD3 induced defective activation of phospholipase C (PLC) in thymocytes relative to peripheral blood T lymphocytes. The defect in PLC activation via TCR/CD3 was restricted to immature thymocytes (CD3low, CD4+CD8+). Mature thymocytes (CD3high, CD4+CD8-/CD8+CD4-) were similar to PBL in signaling via TCR/CD3. Both immature and mature thymocytes expressed a similar profile of PLC isoenzyme mRNA species, indicating that the defect in signaling in immature thymocytes was not due to altered expression of PLC isoenzymes. Activation of tyrosine phosphorylation pathways implicated in the coupling of TCR/CD3 to PLC was impaired in immature thymocytes, as evidenced by depressed phosphorylation of CD3 zeta subunit after stimulation with anti TCR/CD3 mAb. This was associated with lower levels of p59fyn tyrosine kinase and minimal or undetectable stimulus-induced kinase activation in immature thymocytes relative to mature thymocytes. We conclude that the capacity to signal via TCR/CD3 is regulated during T cell development by mechanisms acting at the level of TCR/CD3-associated tyrosine phosphorylation pathways.     

Characteristics and mechanisms of action of the concanavalin A-activated suppressor cell in man.

Human peripheral blood lymphocytes treated for 24 to 48 hr with optimally mitogenic doses of concanavalin A suppressed the proliferative response of autologous T cells to mitogens and antigens. Con A-treated cells also suppressed the proliferative response and the immunoglobulin synthetic response of autologous B cells stimulated in vitro by T cell helper factor. The human Con A suppressor cell was sensitive to treatment with mitomycin C and to exposure to radiation doses exceeding 1000 rads. The Con A suppressor cell was shown to reside in the nylon wool-nonadherent, sheep red cell rosette-forming, histamine receptor-bearing population of lymphocytes and to lack surface DRW antigens. One mechanism of action of Con A suppressor cells was shown to be the inactivation of nonspecific T cell helper factor.     

Substrate and inhibitor specificities for human monoamine oxidase A and B are influenced by a single amino acid

Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines. It exists as two isoforms, A and B, which have a 70% amino acid identity and different substrate and inhibitor specificities. This study reports the identification of residues responsible for conferring this specificity in human MAO A and B. Using site-directed mutagenesis we reciprocally interchanged three pairs of corresponding nonconserved amino acids within the central portion of human MAO. Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for beta-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor). The reciprocal mutant MAO B-Y326I exhibited an increased preference for 5-hydroxytryptamine, a decreased preference for beta-phenylethylamine, and, similar to MAO A, was more sensitive to clorgyline than to deprenyl. These mutants also showed a distinct shift in sensitivity for the MAO A- and B-selective inhibitors Ro 41-1049 and Ro 16-6491. Mutant pair MAO A-T245I and MAO B-I236T and mutant pair MAO A-D328G and MAO B-G319D reduced catalytic activity but did not alter specificity. Our results indicate that Ile-335 in MAO A and Tyr-326 in MAO B play a critical role in determining substrate and inhibitor specificities in human MAO A and B.     

Analysis of conserved active site residues in monoamine oxidase A and B and their three-dimensional molecular modeling

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.     

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.