Induction of T-Cell Differentiation In Vitro by Thymin, a Purified Polypeptide Hormone of the Thymus

165

Interferon produced by human diploid fibroblast cells in culture has been purified approximately 5000-fold. The purified interferon, when analyzed by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate, contains only one polypeptide component of 20,000 molecular weight. The interferon activity comigrates with this polypeptide, indicating identity of the activity and the polypeptide. Oxidation of this polypeptide with periodic acid and subsequent staining with Fuchsin base indicates that it contains carbohydrate and suggests that the human fibroblast interferon is a glycoprotein.Interferon, a broad-spectrum antiviral protein, has been the subject of numerous biological studies, yet its chemical characterization from any source has remained elusive (see refs. 1 and 2 for reviews). Human leukocyte (3) and human diploid fibroblast (3-5) interferons have been purified by various techniques. This paper reports (i) a procedure for purifying human diploid fibroblast interferon and (ii) some chemical and physical properties of the purified interferon. MATERIALS AND METHODSProduction of Crude Interferon. The human diploid fibroblast cells (neonatal foreskin fibroblasts, FS-4) were a gift from Drs. E. Havell and J. Vilcek. The procedures for growing the cells and producing the interferon have been described (6). For production of interferon, cells were grown at 370 in glass roller bottles 20 cm long and 9.5 cm internal diameter. Induction of interferon with poly(I)-poly(C) was performed exactly as previously described (6). Briefly, cell cultures 10-12 days old, 4 X 107 cells per bottle, were induced, then allowed to produce interferon at 340 for 20 hr in serum-free medium, 50 ml per bottle. Crude interferon was collected and stored at 40 until a sufficient quantity was accumulated for purification. Titers of crude interferon varied from 4000 to 20,000 units/ml. The crude interferon was concentrated 10-to 20-fold prior to purification, as described for mouse L cell interferon (7). Interferon was assayed on FS-4 cells with vesicular stomatitis virus as the challenge virus (6). All units are expressed as reference units.Polyacrylamide Gel Electrophoresis. Preparative electrophoresis was performed in cylindrical separating gels, 1 cm X 20 cm, using the discontinuous buffer system of Laemmli (8) with sodium dodecyl sulfate (NaDodSO4). Stacking gels 2 cm in length of 5% acrylamide were cast on top of the separating gel. The separating gel was an exponential gradient gel (7, 9) of 9-18% acrylamide. The gradient was prepared by mixing 20 ml of 9% acrylamide with 3 ml of 18% acrylamide kept at constant volume, and the ratio of acrylamide to bis-acrylamide was 38:1. All gels and buffers contained 0.1% NaDodSO4. Analytical slab gels, 9-18% acrylamide, were prepared as described (7, 9). Protein in the gels was fixed and stained for 15 min in a solution of methanol:acetic acid:water 50:10:40 (vol/vol/vol) containing 1.25 mg/ml of Coomassie blue R 250. Gels were destained in 7% acetic-5% methanol and dried un...

Section: Methodsmentioning

confidence: 99%

“…Titers of crude interferon varied from 4000 to 20,000 units/ml. The crude interferon was concentrated 10-to 20-fold prior to purification, as described for mouse L cell interferon (7). Interferon was assayed on FS-4 cells with vesicular stomatitis virus as the challenge virus (6).…”

Section: Methodsmentioning

confidence: 99%

Interferon: purification and initial characterization from human diploid cells.

Knight¹

1976

165

“…Thymopoietin, a 49 amino acid polypeptide hormone of the thymus discovered by its effect on neuromuscular transmission (1), was later shown to induce T-cell differentiation (2,3) and to affect immunoregulatory balance (4)(5)(6)(7).…”

mentioning

confidence: 99%

Contrasting biological activities of thymopoietin and splenin, two closely related polypeptide products of thymus and spleen.

Audhya¹,

Scheid²,

Goldstein³

1984

Thymopoietin, a 49 amino acid polypeptide hormone of the thymus discovered by its effect on neuromuscular transmission, was later shown to induce T-cell differentiation and to affect immunoregulatory balance. A radioimmunoassay for thymopoietin revealed a crossreaction with a product found in spleen and lymph node but not other tissues. This product, named splenin, differs from thymopoietin only in position 34, aspartic acid for bovine thymopoietin and glutamic acid for bovine splenin. Synthetic pentapeptides corresponding to residues 32-36, called thymopentin and splenopentin, reproduce biological activities of thymopoietin and splenin, respectively. Thus thymopoietin and thymopentin affect neuromuscular transmission and induce the phenotypic differentiation of T precursor cells in vitro while inhibiting phenotypic differentiation of B cells. Splenin and splenopentin, in contrast, do not affect neuromuscular transmission, and they induce both T-and B-cell precursors.Thymopoietin, a 49 amino acid polypeptide hormone of the thymus discovered by its effect on neuromuscular transmission (1), was later shown to induce T-cell differentiation (2, 3) and to affect immunoregulatory balance (4-7).Initial studies leading to the discovery of thymopoietin concerned myasthenia gravis, a human disorder in which neuromuscular impairment accompanies disease of the thymus (8). Pathological and immunological evidence suggested that the thymic lesion might be due to autoimmune thymitis, and an animal model of experimental autoimmune thymitis was shown to be associated with neuromuscular impairment by electromyographic and other neurophysiological techniques (9,10). Results with this model implied that a product of the thymus can impair neuromuscular transmission. Neurophysiological evidence from thymectomized and thymusgrafted rats indicated that this product, now termed thymopoietin, was secreted by the normal thymus (11,12).Neuromuscular transmission was impaired 1-5 days after injection of preparations of thymus but not of spleen or other tissues (1). The delay in appearance of this effect implies a regulatory rather than direct influence on neuromuscular transmission. This assay led to the isolation of two closely related polypeptides of linear 49 amino acid sequence, thymopoietin I and thymopoietin II (1). These shared a common pentapeptide at residues 32-36, Arg-Lys-Asp-Val-Tyr (13), named thymopentin or TP-5, which reproduced the biological activity of thymopoietin (3, 14, 15) and thus was considered to represent the active site. Since thymopoietins I and II were isolated from pooled bovine thymus and are not functionally distinguished, they could be products of a single polymorphic gene.A radioimmunoassay (RIA) for thymopoietin gave identical displacements for thymopoietins I and II (16). The fact that serum levels of thymopoietin, measured by RIA in rats, failed to decline after thymectomy led to the finding that spleen and lymph nodes, but not other tissues, yield a product that reacts in the RIA for thymopoietin...

“…Lymphocytes that differentiate under the influence of thymopoietin subsequently give rise to distinct subclasses of T cells with potential to develop discrete effector, helper, or suppressor functions after encounter with antigen (10). The relationship of thymopoietin to the later stages of the functional differentiation of T cells remains to be elucidated.Thymopoietin can be measured in vitro by its ability to differentiate precursor cells from bone marrow or spleen to cells that express characteristic surface antigens of T cells (7,11,12). Induction of T cell differentiation by thymopoietin is accomplished by the generation of cyclic AMP (13), which accounts for the ability of other agents that elevate intracellular cyclic AMP to induce T cell differentiation in a relatively nonspecific fashion (14 (17); this may be due to null lymphocyte membranes having distinct receptor sites for thymopoietin and ubiquitin.…”

mentioning

confidence: 99%

Bioassay determinations of thymopoietin and thymic hormone levels in human plasma.

Twomey¹,

Goldstein²,

Lewis³

et al. 1977

Thymopoietin is a thymic hormone that induces differentiation of thymocytes from precursor cells which arise in hemo oietic tissues. This paper describes a sensitive in vitro assay Ior the induction of Thy 1.2 antigen on null lymphocytes from germ-free athymic (nu/nu) mice. The sensitivity and specificity of the bioassay were increased by adding hig concentrations of ubiquitin (a nonspecific inducer) to the induction incubations. The bioassay was sufficiently sensitive to detect thymopoietin at <0.25 ng/ml. A dose-response relationship was shown between thymopoietin concentration and the percentage of cells induced to eress Thy 1.2 antigen. When normal human plasma was assayed, induction was registered with activity corresponding to thymopoietin at >1 ng/ml in plasma from infants or young adults. Activities in the thymopoietin range of 0.25 ng/ml were registered with plasma from healthy subjects over 50 years of age. Thymectomy was followed by loss of this inductive activity from the plasma. This bioassay permits clinical studies on T (thymus-derived) cell inducers released by the human thymus into the circulation. The function of the thymus is to promote differentiation of lymphocytes (T cells) that participate in effecting and regulating immune responses. Abnormalities of the thymus have been detected with certain human diseases such as myasthenia gravis (1), systemic lupus erythematosus (2), and immune deficiency states (3-5). Thymic dysfunction may be implicated in a broader range of human disorders and also with aging (1). The lack of sensitive and precise tests for thymic function has hampered attempts to clarify the clinical significance of this immunologic organ.Thymopoietin (molecular weight 5562) is a polypeptide hormone of defined amino acid sequence (6) that induces differentiation of precursor lymphoid cells to thymocytes (7) and also has secondary effects upon neuromuscular transmission (8). Thymopoietin is secreted by epithelial cells of the thymus (9). Lymphocytes that differentiate under the influence of thymopoietin subsequently give rise to distinct subclasses of T cells with potential to develop discrete effector, helper, or suppressor functions after encounter with antigen (10). The relationship of thymopoietin to the later stages of the functional differentiation of T cells remains to be elucidated.Thymopoietin can be measured in vitro by its ability to differentiate precursor cells from bone marrow or spleen to cells that express characteristic surface antigens of T cells (7,11,12). Induction of T cell differentiation by thymopoietin is accomplished by the generation of cyclic AMP (13), which accounts for the ability of other agents that elevate intracellular cyclic AMP to induce T cell differentiation in a relatively nonspecific fashion (14 (17); this may be due to null lymphocyte membranes having distinct receptor sites for thymopoietin and ubiquitin.We have utilized these differences in induction with thymopoietin and ubiquitin to develop a sensitive bioassay for thymopoietin. ...