Interleukin 1-dependent induction of both interleukin 2 secretion and interleukin 2 receptor expression by thymoma cells

The macrophage-derived product, interleukin 1 (IL 1) is thought to play an important regulatory role in the proliferation of T lymphocytes; however, its mechanism of action is unknown. We describe in this report a variant subline of EL4 thymoma cells (EL4-6.1) that displays a high degree of responsiveness to IL 1. We show that recombinant IL 1 can induce both the secretion of interleukin 2 (IL 2) and the expression of IL 2 receptors (IL 2-R) by these cells. EL4-6.1 cells do not constitutively secrete IL 2, nor do they express IL 2-R; but when cultured in the presence of recombinant IL 1, they secrete detectable amounts of IL 2 (5 to 15 U/ml). In the presence of either suboptimal levels of phorbol ester (PMA) or Ionomycin, the addition of IL 1 resulted in up to an 80-fold enhancement in the amount of IL 2 secreted. Stimulation with IL 1 alone or in combination with Ionomycin was unable to induce detectable IL 2-R expression by EL4-6.1 cells. However, in the presence of suboptimal concentrations of PMA, IL 1 induced expression of about 3000 high affinity (dissociation constant, Kd of 31 pM) and 50,000 low affinity (Kd of 2800 pM) IL 2-R. These IL 2-R were functional, based on their ability to rapidly internalize IL 2. This model system will allow a detailed analysis of the mechanisms involved in the regulation of the immune response by IL 1 and IL 2.     

Thymic epithelium in vitro. III. Cytokine production by a thymic epithelial subset

We have previously shown that two populations of thymic epithelium can be separated in culture on the basis of their differential growth rates and their adherence to the culture substratum, and maintained as long-term, morphologically distinct cell cultures, TECs and TECL. We have also described the effects of supernatants from the small epithelial cell (TESs) on the proliferative responses of thymocytes cocultured with mitogen and TESs over a 72-hr period. We now describe the effects in thymic epithelial supernatants (TESL) of soluble factors produced by TECL (the large epithelial cell) on thymocytes costimulated with mitogen and compare their effects to those derived from TECs. Both TESL and TESs suppress optimally stimulated thymocytes and enhance the proliferative responses of suboptimally stimulated thymocytes over a 72-hr period. The suppressive activities produced by TECL and TECs appear distinct, based upon markedly different molecular weights, but have similar sensitivities to heat treatment. The enhancing activities are of similar molecular weight, but have different sensitivities to heat treatment. In addition, TECL synthesize four- to fivefold less PGE2 than TECs. These results provide additional distinctions between the two cell types, and taken in conjunction with data on the anatomic distribution of similar cells, suggest that although they have similar functional effects in vitro, they may prove to have separable roles in vivo.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Growth of AKR T cell leukemia lymphoblasts in medium containing interleukin 2 (IL-2)

Long-term cloned mouse leukemic T cell lines were established in vitro using interleukin-2 (IL-2) conditioned media. The cell lines were tested for retention of both antigenic expression and tumorigenicity, as well as for growth characteristics. Several important findings resulted from these studies. A reliable method was developed for consistent success in the culturing and cloning of leukemic T cell lines. Cultured cells from IL-2-dependent lines were found to retain their original histocompatibility and differentiation antigen characteristics for up to 2 yr. Mortality patterns, comparing long-term cloned leukemic T cell lines with fresh AKR leukemia cells, showed that the cloned cells had greater tumorigenicity. An especially interesting finding was that cell lines established both from different mice and from a single organ of an individual mouse were heterogeneous with respect to antigenic makeup, cell growth kinetics, and tumorigenicity. Finally, because of their dependence on IL-2, the cloned tumor cell lines provided excellent index cells to quantify IL-2 activity.     

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical and functional characterization of a molecule expressed by a subset of thymic medullary epithelial cells.

A monoclonal antibody (mAb), named TE-4F 10, was produced by fusing P3X-Ag8 myeloma cells with splenocytes of BALB/c mice immunized with a rat medullary thymic epithelial cell (TEC) line, (TE-R 2.5), previously established in our Institute. Flow cytometry showed that 85-95% TE-R 2.5 cells expressed the TE-4F10 antigen. The mAb immunoprecipitated a 29 kDa molecule from the TE-R2.5 cell lysate. Immunohistochemical analysis using single and double staining of the thymus with anti-cytokeratin (CK) mAb, showed that TE- 4F10 mAb selectively stains a subpopulation of medullary TEC. Hematopoietic and lymphoid cells were negative. The expression of the TE-4F10 antigen on TE-R 2.5 cells in vitro was significantly upregulated by interleukin 1 (IL-1) and tumor necrosis factor (TNFalpha). Other cytokines IL-4, IL-6, IL-10 and granulocyte - macrophage colony stimulating factor (GM-CSF) showed lesser stimulation on its expression, whereas interferon gamma (IFN) and dexamethasone were without significant effect. The TE-R 2.5 cell line strongly bound and induced apoptosis of a rat / mouse thymocyte heterohybridoma (BWRT8), phenotypically alphabetaTCRhiCD4hiCD8lo. TE-4F10 mAb significantly inhibited binding (40-50%) of both BWRT8 cells and the BWRT8 - MDP.1 subclone to TE-R 2.5 cells. The inhibition was enhanced when TEC were stimulated with IL-1 + TNFalpha. The mAb also significantly blocked apoptosis of BWRT8 but did not modulate cell death of the BWRT8 - MDP.1 subclone, which was resistant to TEC-induced apoptosis. These findings indicate that the TE-4F10 antigen might be selectively involved in adhesion and selection processes in the medullary thymic microenvironment. The mAb of the same characteristics has not been described so far.     

High-level secretion of dipetarudin, a chimeric thrombin inhibitor, by Pichia pastoris

Dipetarudin is a potent direct thrombin inhibitor that was genetically engineered as a chimera between dipetalogastin II and hirudin. Dipetarudin was initially cloned and purified from Escherichia coli, but with a very low yield of about 0.3 mg/l of culture medium. In this study, we report the production of dipetarudin in the methylotrophic yeast Pichia pastoris using pPIC9 vector. The His+ transformants were screened for the best expression performances by prolongation of the ecarin clotting time. An optimal dipetarudin's expression was reached by addition of methanol in culture medium to a final concentration of 0.5%, every 8h during 4 days. Secreted dipetarudin was purified essentially using a two-step purification scheme: anion exchange chromatography in a Resource Q column, followed by C18-reversed phase HPLC. About 150 mg purified dipetarudin was obtained from 1l culture supernatant. This yield is 500-fold higher than the yield obtained with the E. coli system. The molecular mass of dipetarudin calculated by MALDI-TOF (7450 Da) was in agreement with the mass calculated by the amino acid composition (7454 Da), indicating correct processing of the signal sequence. The Ki value of dipetarudin was 399+/-83 fM, which is in agreement with that calculated for the inhibitor isolated from E. coli. This efficient and cost-effective expression system facilitates large-scale production and purification of dipetarudin for further structural, functional and pharmacological investigations.     

Qa-2 expression in the adult murine thymus. A unique marker for a mature thymic subset

The MHC Ag, Qa-2, is expressed on all peripheral T cells, a subset of bone marrow cells, and to a lesser extent on B cells. The Qa-2 Ag is also expressed on 5 to 6% of normal adult murine thymocytes. Through the use of flow cytometry, counterflow centrifugal elutriation and acridine orange staining, we have analyzed the cell surface phenotype, cell size, and cell cycle status of this thymic population. Our studies indicate that Qa-2+ thymocytes are large, non-mitotic, G1 cells which have the cell surface phenotype of CD5+, CD3+, J11dLO and lack receptors for peanut agglutinin. This population can be further subdivided into three categories; CD4+/CD8-, CD4-/CD8+, and CD4-/CD8-. These data indicate that Qa-2 surface expression can only be detected on thymocytes in the final stages of differentiation. The Qa-2 Ag can be used as a cell surface marker to identify a unique subset of mature thymocytes.     

Immunohistochemical studies on a human thymic epithelial cell subset defined by the anti-cytokeratin 18 monoclonal antibody

Summary Two monoclonal antibodies respectively recognizing cytokeratins (CK) 18 and 19 were applied to the human thymic epithelium (in vivo and in vitro) in normal and pathological conditions, including 12 thymomas. We observed that in both normal and hyperplastic thymuses (from patients with myasthenia gravis) virtually the entire epithelial network was CK19-positive as were the majority of cells growing in culture. In four thymomas, however, the expression of cytokeratin 19 was not detected by immunofluorescence. On the other hand, CK18 was expressed by a discrete subset of medullary thymic epithelial cells in normal and in hyperplastic thymuses. Among the thymomas a large majority was either negative or contained few isolated CK18-positive cells scattered within the tumour. Conversely, in the two undifferentiated epithelial thymomas, virtually all the tumoral network was strongly labeled with the anti-CK18 monoclonal antibody. The present investigation thus not only defines the human thymic epithelial cell subset on the basis of differential cytokeratin expression but also indicates that anti-CK antibodies with single cytokeratin specificities can be regarded as useful tools to study the heterogeneity of thymomas.     

Interleukin 1-driven secretion of interleukin 2 is highly temperature-dependent

To investigate the temperature dependence of T lymphocyte activation, we have examined the IL 1-induced secretion of IL 2 by the murine T lymphoma cell line LBRM-33-1A5. During 24-hr incubations, there are modest increases in IL 2 secretion as culture temperatures are increased from 33 degrees to 37 degrees C, but IL 2 secretion declines at higher temperatures. However, the kinetics of IL 2 release during the first 8 hr of culture are highly temperature-dependent. The rate of IL 2 release increases linearly with temperature over the range from 33 degrees to 41 degrees C, demonstrating temperature coefficients (Q10) greater than 70. In contrast, IL 2-promoted proliferation of a continuous T cell line is much less temperature-dependent with Q10 values of less than 4.0.     

High-level secretion of growth hormone by retrovirally transduced primary human keratinocytes

A gene therapy clinical trial for treatment of growth hormone (GH) deficiency has not been reached yet, but several strategies using different gene transfer methodologies and animal models have been developed and showed successful results. We have set up an ex vivo gene therapy protocol using primary human keratinocytes transduced with an efficient retroviral vector (LXSN) encoding the human (hGH) or mouse GH (mGH) genes. These stably modified cells presented high in vitro expression levels of hGH (7 μg/10⁶ cells/d) and mGH (11 μg/10⁶ cells/d) after selection with geneticin. When the hGH-secreting keratinocytes were grafted onto immunodeficient dwarf mice (lit/scid), hGH levels in the circulation were about 0.2–0.3 ng/mL during a 12-d assay and these animals presented a significant body weight increase (p<0.01) compared to the control. Substitution of conventional grafting methodologies with organotypic raft cultures revealed a peak value of up to 20 ng mGH/mL in the circulation of grafted lit/scid mice at 1 h postimplantation, followed by a rapid decline to baseline (≈2 ng/mL) within 24 h. One week after grafting, however, the cultured excised implants still presented approx 45% of their original in vitro secretion efficiency. Further studies are being carrier out to identify the main factor(s) that still constitute one of the major impediments to the success of this promising model of cutaneous gene therapy.     

High-level expression and secretion of recombinant mouse endostatin by Escherichia coli

The expression of murine endostatin was achieved by placing its gene downstream of an alkaline phosphatase gene (phoA) promoter. To ensure proper folding and secretion of the recombinant protein, the mouse endostatin was fused with alkaline phosphatase signal peptide. SDS/polyacrylamide gel electrophoresis analysis of the culture medium of recombinant Escherichia coli cells revealed that endostatin was efficiently secreted. The signal peptide was efficiently cleaved during secretion as demonstrated by N-terminal amino acid sequencing. The maximum yield of secreted endostatin during fermentation was 40 mg/liter. Up to 28 mg of endostatin was purified from 1 liter of cell culture broth. The biological activity of recombinant protein was tested in a bovine aortic endothelial (BAE) cell proliferation assay. The recombinant endostatin inhibited the growth of BAE cells stimulated by basic fibroblast growth factor, and its ED50 was comparable to that from a previous report. Flow cytometric measurements of BAE cells cultivated in medium with endostatin demonstrated a cell cycle arrest mainly in the G0/G1 phase and a decrease in the S phase.     

Shigella enterotoxin-2 is a type III effector that participates in Shigella-induced interleukin 8 secretion by epithelial cells

We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.     

Differences in turnover between thymic medullary dendritic cells and a subset of cortical macrophages

To investigate the turnover of thymic accessory cells, we performed vascular thymus transplantation in RT7 congenic rats. mAb specific for one of the two allelic variants of the RT7 molecule, as well as mAb specific for either medullary interdigitating cells or a subset of cortical macrophages (M phi), were used on cryostat sections and cell suspensions prepared from grafted thymuses to monitor the turnover of these two cell types. In contrast to the complete turnover of interdigitating cells within 3 wk after transplantation, ED2-labeled cortical M phi showed a very slow turnover. Seventy-six days after transplantation, more than 30% of these M phi were found to be still of donor origin. The different turnover rates of these thymic accessory cells could reflect their function in T cell development.     

Induction of interleukin 2 responsiveness in thymocytes by synergistic action of interleukin 1 and interleukin 2

Thymocyte cultures from C3H/HeJ mice were stimulated for proliferative responses with purified preparations of interleukin 1 (IL 1) and interleukin 2 (IL 2). Synergistic responses were obtained in the absence of mitogen. In the presence of excess IL 2, the thymocyte proliferation response was strictly dependent on the amount of IL 1 in the cultures. Antibodies to IL 1 inhibited the response in a dose-dependent manner. The combination of IL 1 plus IL 2 induced the appearance of IL 2 receptors on murine thymocytes as detected with a monoclonal antibody directed against the IL 2 receptor. Neither IL 1 nor IL 2 alone had this effect. The thymic subpopulation found to become IL 2 responsive upon IL 1 stimulus was the peanut agglutinin-negative (PNA-) medullary fraction.     

High-level secretion of a virally encoded anti-fungal toxin in transgenic tobacco plants

Ustilago maydis killer toxins are small polypeptides (7–14 kDa) whichkill susceptible cells of closely related fungal species. The KP4 toxin is a single polypeptide subunit with a molecular weight of 11.1 kDa. In this work, a transgenic tobacco plant was constructed which secretes the KP4 toxin at a high level. The KP4 toxin expressed in this transgenic plant was of the same size and specificity as the authentic Ustilago KP4 toxin. The expression level was at least 500 times higher than that of the KP6 toxin expressed in plants. Transgenic crop plants producing the KP4 toxin could be rendered resistant to KP4-susceptible fungal pathogens.     

Acute lowering of circulating fatty acids improves insulin secretion in a subset of type 2 diabetes subjects

We tested the effects of acute perturbations of elevated fatty acids (FA) on insulin secretion in type 2 diabetes. Twenty-one type 2 diabetes subjects with hypertriglyceridemia (triacylglycerol >2.2 mmol/l) and 10 age-matched nondiabetic subjects participated. Glucose-stimulated insulin secretion was monitored during hyperglycemic clamps for 120 min. An infusion of Intralipid and heparin was added during minutes 60-120. In one of two tests, the subjects ingested 250 mg of Acipimox 60 min before the hyperglycemic clamp. A third test (also with Acipimox) was performed in 17 of the diabetic subjects after 3 days of a low-fat diet. Acipimox lowered FA levels and enhanced insulin sensitivity in nondiabetic and diabetic subjects alike. Acipimox administration failed to affect insulin secretion rates in nondiabetic subjects and in the group of diabetic subjects as a whole. However, in the diabetic subjects, Acipimox increased integrated insulin secretion rates during minutes 60-120 in the 50% having the lowest levels of hemoglobin A(1c) (379 +/- 34 vs. 326 +/- 30 pmol x kg(-1) x min(-1) without Acipimox, P < 0.05). A 3-day dietary intervention diminished energy from fat from 39 to 23% without affecting FA levels and without improving the insulin response during clamps. Elevated FA levels may tonically inhibit stimulated insulin secretion in a subset of type 2 diabetic subjects.     

Separation of non-proliferating from proliferating thymic lymphocytes based on light scatter analysis

Summary The migration of the ameloblasts in the continuously erupting incisors of the rat is accompanied by cell loss. Ameloblasts degenerate near the mesial and lateral cemento-enamel junctions in the secretory zone and in the middle two thirds of the region of postsecretory transition, degeneration being most marked where these areas merge. These findings support the hypothesis that the prism decussation in the enamel results from alternating transverse rows of secretory ameloblasts sliding past each other whilst elaborating their rods. The distribution of the degenerating cells suggests, however, that the sliding cell rows are not exactly transverse but arcuate, with the opening facing incisally. The progress of structural alterations of the nuclei in the degenerating ameloblasts appears to follow the pattern earlier described in vinblastine-damaged ameloblasts.