Biochemical characterization of lymphocyte regulatory molecules. II. Purification of a class of rat and human lymphokines

Rat spleen cells activated in vitro by concanavalin A produce lymphokine molecules that possess biologic activity in a number of murine lymphocyte response assays. A single class of lymphokine most adequately described as T cell growth factor (TCGF, Interleukin-2) with a m.w. of 15,000 as estimated from gel filtration studies and with an isoelectric range of 5.4 to 5.6 stimulates i) the growth of established T cell lines in culture, ii) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is non-mitogenic, iii) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) mouse spleen cell cultures, and iv) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures and in nude mouse spleen cell cultures. We suggest that in each of the assay systems tested, this class of rat lymphokine acts directly on activated T cells. Nonactivated T cells must be stimulated by either mitogen or antigen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued lymphokine-dependent proliferation. Similarly, human peripheral blood mononuclear cells activated by phytohemagglutinin (PHA) produce a class of lymphokines of identical size with an isoelectric point of 6.0 to 6.5 that possess the same biologic properties as measured in murine lymphocyte response systems.     

Properties of T-cell growth factor obtained from diucifon-stimulated human lymphocytes

The properties of T-cell growth factor (TCGF), obtained by diucifon (Dc) stimulation of human mononuclear cells (MNC) (TCGF-Dc) have been studied. Taking into account the fact that Dc alone does not, like other TCGF inductors, cause proliferation, differences between TCGF-Dc and TCGF were suggested. Partial purification of supernatant from cells, activated by Dc was performed on Sephadex G-100 column. TCGF-Dc biological activity in these fractions was determined in the system of mitogen activated human MNC and mice thymocytes, as well as in the system of concanavalin A transformed cells. 2 peaks of TCGF-Dc activity have been revealed that are indicative of TCGF-Dc molecular mass heterogeneity. In contrast to TCGF, low molecular mass TCGF-Dc (8000-12000) and TCGF-Dc from the whole supernatant were capable of absorbing on intact human MNC. TCGF-Dc may be constantly present on MNC membrane, but TCGF-Dc fixation is not sufficient for proliferation induction, the receptor activation is necessary as well. Receptors to TCGF-Dc were suggested to consist of fixing and triggering sites.     

The production of T-cell growth factor (TCGF) in vivo in sheep

Lymph and supernatants derived from efferent lymphocytes leaving the popliteal lymph nodes of sheep responding to human red cells or dinitrophenylated bovine serum albumin were examined for the presence of T-cell growth factor (TCGF). Efferent cells from normal sheep, but not from antigen-stimulated sheep, were found to release low levels of TCGF when incubated in medium for 12 hr in the absence of any exogenous stimulus. High levels of TCGF were found in normal lymph and also in immune lymph collected from sheep during the first 6 hr of immune responses. There were no detectable levels of TCGF in lymph collected later in the response. The lymphokine appeared to be a single molecular species of 10,000–20,000 molecular weight as assessed by exclusion chromatography. Efferent cells expressing receptors for TCGF were found in efferent lymph during the first 12 hr of the response. The results demonstrate for the first time that TCGF is produced in vivo and that asynchrony exists between TCGF production and expression of receptors for TCGF on efferent cells released by the stimulated node. Based on the known kinetics of previously reported synergistic factors, mitogenic factors, and T-cell-replacing factors in sheep efferent lymph and their physical characteristics it was concluded that the TCGF detected in lymph is distinct from these factors.     

Ligand-activated T cell growth factor-induced proliferation: absorption of T cell growth factor by activated T cells.

The fate in culture of the T cell growth factor (TCGF), which is required for continued growth of human cultured T cells (CTC) in vitro, was studied. TCGF activity was stable for 7 days at 37 degrees C. However, it was no longer detectable after incubation with actively growing CTC at 37 degrees C for 3 days. This loss of TCGF activity also occurred quite rapidly and was detectable within 1 hr of incubation of 0.3 ml supernatant with 2 to 5 x 10(7) CTC at 23 degrees C. 2 x 10(8) mononuclear peripheral blood leukocytes were not effective in removing TCGF activity, and incubation with similar numbers of cells from B and T cell lines had no effect. Three-day-old concanavalin A and phytohemagglutinin blasts were very reactive with TCGF, so that 10(7) or 2 x 10(7) cells consistently removed TCGF activity. These experiments suggested specific absorption of TCGF by activated T cells, and led us to develop a model of ligand-activated TCGF-induced proliferation of T cells: Ligands induce production of TCGF by T-producer cells and deliver a first signal to the T-responder cells. This causes a receptor for TCGF to appear on T-responder cells. Only then does TCGF deliver the obligatory second signal that is needed to drive the T-responder cells into proliferation.     

Tissue distribution of mammalian aldose reductase and related enzymes

Activities of aldose reductase (AR) and related NADPH-dependent enzymes were examined in extracts of human, cat, dog, guinea pig, mouse, monkey, pig, rabbit, rat and sheep lenses and a variety of other tissues. The activity of the tissues against DL-glyceraldehyde, D-glucuronic acid, and 3-pyridinecarboxaldehyde (PCA) was determined. High glyceraldehyde:glucuronic acid activity ratios, a characteristic of aldose reductase, were found in all lenses, except from mouse. An analytical thin-layer isoelectric focusing system which separates the mammalian NADPH-dependent enzymes was developed. AR appears to be present as two or more isozymes in all mammalian lenses studied with the exception of mouse. Other tissues contain one or more isozymes which have the same isoelectric point and substrate specificity as the AR present in the lens of that species. This AR activity, however, may represent only a small proportion of the total NADPH reducing activity present. AR and HDH isozymes reduce the aromatic substrate, PCA, and thus have the general characteristics of an aldehyde reductase.     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Tumour inhibitory effects of TCGF/IL-2/-containing preparations

Summary Supernatants from ConA-stimulated rat spleen cell cultures and from cultures of PMA-stimulated murine lymphoma subline EL-4TF were found to contain TCGF and to inhibit growth of a transplantable, MC-induced sarcoma MC11 in syngeneic mice. Tumour-inhibitory effects of the supernatants were dependent on local and repeated administration. Prior to use of the supernatants obtained from PMA-stimulated EL-4TF cell cultures, the dialysable PMA had to be removed; contamination with PMA was found to abolish the tumour-inhibitory effect of the supernatants and to produce enhancement of tumour growth. A significant tumour-inhibitory effect has also been obtained with partially purified TCGF prepared from culture supernatants of cloned EL-4TF cells by ammonium sulphate precipitation, ion-exchange (FPLC) chromatography, and AcA 44 Ultrogel filtration.Abbreviations TCGF T cell growth factor - IL-2 interleukin 2 - Con A concanavalin A - ³H-TDR tritiated thymidine - PMA 4--phorbol-12--myristate-13--acetate - NK natural killer cells - B10 C57BL10/ScSnPH mice - FPLC fast protein liquid chromatography - BSA bovine serum albumin - OVA ovalbumin - SBI trypsin inhibitor from soybean - Cyt C cytochrome C - OD optical density - PBS phosphate-buffered saline - MC 3-methylcholanthrene     

Purification and partial sequence analysis of the soluble catechol-O-methyltransferase from human placenta: comparison to the rat liver enzyme

Catechol-o-methyltransferase from human placenta was purified 1400-fold by hydroxyapatite adsorption, ammonium sulfate precipitation, gel filtration, high performance anion- exchange and reversed-phase chromatography. The purified enzyme has an apparent molecular weight of 26.000, an isoelectric point of 5,3 and is activated ten-fold in the presence of 20mM cysteine. The enzyme shows primary structure homology to the corresponding rat liver soluble enzyme, based on the sequenced tryptic peptides.     

Preparing T cell growth factor from rat splenocytes

Maintenance of antigen-specific T cell lines or clones in culture requires rounds of antigen-induced activation separated by phases of cell expansion. Addition of interleukin 2 to the culture media during the expansion phase is necessary to prevent cell death and sufficient to maintain short-term T cell lines but has been shown to increase Th1 polarization (3). Replacement of interleukin 2 by T cell growth factor (TCGF) which contains a mix of cytokines is more effective than interleukin 2 in maintaining long-term T cell lines in vitro (3). Moreover, TCGF can easily be prepared in large amounts in the laboratory and is much cheaper than recombinant interleukin 2. Here, we show how to prepare TCGF from rat splenocyte culture supernatants. For this procedure, we harvest spleens from naive Lewis rats euthanized for thymus and blood collection. We prepare single-cell suspensions from the spleens, lyze the red blood cells by osmotic shock, and seed the splenocytes in culture medium. The cells are stimulated with concanavalin A, a mitogen that non-selectively activates all rat T lymphocytes, inducing the production of cytokines. The culture supernantant is collected 48 hours later and excess concanavalin A is bound to alpha methyl mannoside to prevent it from activating T cell lines to which TCGF will be added. The TCGF is then sterile-filtered, aliquoted, and stored at -20 degrees C.     

Biological and biochemical characterization of bovine interleukin 2. Studies with cloned bovine T cells

Bovine peripheral blood lymphocytes stimulated with the T cell mitogen concanavalin A (Con A) secrete a lymphokine with biological properties similar to T cell growth factor (TCGF) or interleukin 2 (IL 2) from other species. The material supports proliferation of Con A-derived T cell blasts, limiting dilution cloning of T cell blasts, and continuous growth of T cell clones for over 6 mo in vitro. A quantitative microassay with the use of TCGF-dependent, Con A-unresponsive cloned T cells was used to determine the biological activity during purification of IL 2. A single peak of activity with an apparent m.w. of 25,000 to 28,000 was recovered after gel filtration. This material eluted from DEAE-Sephacryl between 135 and 165 mM NaCl. After isoelectric focusing, high pressure liquid chromatography, and gel electrophoresis under reducing conditions, peak IL 2 activity was associated with proteins having m.w. of 20,000 and 23,000.     

Capacity of thymic cells to effect target cell lysis following treatment with concanavalin A

The capacity of lymph node, thymus and bone marrow cells to mediate a cytolytic effect on target monolayers in the presence of concanavalin A was investigated. It was found that freshly prepared rat lymph node cells, but not thymus or bone marrow cells, lyse mouse fibroblasts in cultures treated with concanavalin A. A population of thymic cells did, however, manifest cytolytic capacity in the presence of concanavalin A after having been first sensitized and selected on mouse fibroblasts. Thus, sensitized thymus cells possess a lytic potency which can be activated under proper conditions.     

Mechanism of T cell activation. II. Antigen- and lectin-dependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells

Small resting T cells, which do not respond to T cell growth factor (TCGF), acquire responsiveness upon a short (4-hr) pulse of specific ligands by presenting growth receptors for TCGF. The results demonstrate that the same mechanisms operate in the specific induction of primary MLR in that a 5-hr MLR is sufficient to render the responder cells reactive to TCGF. Furthermore, the results demonstrate that an active "response" by the resting T cells is required for expression of functional growth receptors, as demonstrated by the fact that: 1) a 4-hr pulse of concanavalin A (Con A) at 4 degrees C did not result in gain of reactivity to TCGF, whereas a 4-hr pulse at 37 degrees C did; 2) this metabolic requirement for acquisition of responsiveness to TCGF was not due to a secondary requirement for cap-formation of Con A-binding membrane structures, as normal responses were observed in the presence of cytochalasin B (cyt B); 3) the process of Con A-induced acquisition of susceptibility to TCGF was puromycin sensitive.     

Human angiotensinogen. Purification partial characterization, and a comparison with animal prohormones.

The renin-angiotensin system appears to play a major role in the regulation of sodium excretion and fluid intake in a wide variety of animal species from mammals to teleosts. In mammals the system has evolved further importance in terms of blood pressure homeostasis. This hormonal system in all species appears to involve a serum protein prohormone, angiotensinogen, a proteolytic enzyme, renin, and angiotensin I, the decapeptide product of the reaction between renin and angiotensinogen. The importance of this system to the organism appears to correlate directly with the necessity to conserve sodium while an abnormality of this process may underlie the development of hypertension in man. As the starting point of the system, angiotensinogen assumes special importance as a possible index of evolutionary development. In addition, it has been known for many years that human (viz. primate) angiotensinogen differs from that found in other mammals in its inability to be a substrate for animal renins while animal angiotensinogens readily react with human renin. Thus, the enzymatic specificity appears to reside with the prohormone. The biochemical basis for this difference is unresolved due primarily to the lack of purified human angiotensinogen. In this paper we describe methods for the purification of human angiotensinogen which have direct applicability to animal angiotensinogens. Our approach utilizes ammonium sulfate precipitation, Sephadex G-150 chromatography, multiple isoelectric focusing, and concanavalin A-Sepharose affinity chromatography. With the availability of highly purified human angiotensinogen we compared the molecular weights, heterogeneity, isoelectric points, and thermal lability of hog, rabbit, and human angiotensinogen in order to define the biochemical basis of the species variation in renin reactivity...     

T cell growth factor from human splenic cell cultures: conditions for its production, and its utilization for maintenance of cytotoxic T cell lines

We prepared the T cell growth factor (TCGF) from human spleen cell cultures stimulated with phytohemagglutinin (PHA). Various cell culture conditions and agents supporting the active TCGF production of the spleen cells were examined. The highest TCGF activity was obtained in the supernatants under the conditions that 2 x 10(6)/ml spleen cells were stimulated with PHA for 48 hr. Production of TCGF from spleen cells depended markedly on their individual sources. Addition of indomethacin to the culture or irradiation of the responding spleen cells increased TCGF activity in the supernatant of the culture. Further, addition of irradiated cells of an Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) to spleen cell cultures stimulated with PHA greatly enhanced TCGF production. Human splenic TCGF facilitated the establishment of human cytotoxic T cell (Tc) lines specific for EBV-transformed LCL cells when those Tc line cells were stimulated periodically with irradiated autologous LCL cells but not with the other two types (K-562 or Molt-4) of cells. Allogeneic LCL stimulators allowed the Tc line cells to proliferate. However, Tc line cells cocultured once with allogeneic LCL stimulators no longer exhibited EBV-specificity in their cytotoxicities.     

Crystallization and comparative characterization of reduced nicotinamide adenine dinucleotide phosphate-ferredoxin reductase from sheep adrenocortical mitochondria

1. Sheep NADPH-ferredoxin reductase (E.C. 1.18.1.2) was purified from the adrenocortical mitochondria. The reductase was typical flavoenzyme and crystallized in ammonium sulfate solution. 2. The properties of the reductase were investigated physicochemically and immunochemically. The minimum molecular weight of the reductase was 52,000 and the reductase has one FAD per mole as a coenzyme. 3. The sheep NADPH-ferredoxin reductase showed a precipitate line against antibody to bovine NADPH-ferredoxin reductase. 4. The compositions and sequences of amino acid residues of this reductase and porcine, bovine, and human enzymes were compared. In spite of differences of mammalian species, the sequence of amino acid residues in the amino-terminal regions were highly homologous. 5. It is suggested that the amino-terminal region may be essential for the function of the NADPH-ferredoxin reductase.     

Mouse monoclonal antibodies can nonspecifically inhibit the proliferation of human T-cell growth factor-dependent T cells

A large series of mouse monoclonal antibodies was found to inhibit the proliferation of T-cell growth factor (TCGF)-dependent human T-cell blasts as measured by the incorporation of tritiated thymidine. The specificity of the antibody appeared to be irrelevant for inhibition and two T-cell-specific antibodies did not prevent the absorption of TCGF by treated T cells. It is suggested that the antibodies function by the indirect release of suppressor factors by Fc receptor-bearing TCGF-dependent cells.     

Platelet derived growth factor(s) for a hormone-responsive rat mammary tumor cell line.

Mammary tumor cell growth factor(s) has been identified in extracts of platelets from both male and female rats, as well as in extracts prepared from pooled outdated human platelets. When assayed by the growth promotion of MTW9/PL rat mammary tumor cells in culture, platelet extracts alone were able to support growth 50--75% as well as whole serum. The mitogenic activity from crude human platelet lysates was shown to be trypsin sensitive, relatively stable to extremes of pH, labile to heat treatment at 70 degrees, non-dialysable, ammonium sulfate precipitable, not removed by 56 degrees charcoal treatment, and of apparent molecular weight of 30,000 to 50,000 daltons as estimated by G-100 Sephadex chromatography. The platelet derived mammary growth factor activity was not replaced or potentiated by thrombin or known hormones and growth factors such as prolactin, insulin, 17-beta-estradiol, progesterone, hydrocortisone, L-thyroxine, and mouse epidermal growth factor. The experimental report demonstrates that platelets are a rich source growth factor activity for rat epithelial mammary tumor cells, and that the activity appears to be a polypeptide(s) different from other mitogenic activities known to influence growth of mammary tissue.     

Renin substrate in plasma of various mammalian species: electrophoresis on polyacrylamide gel.

The plasma of eight different species was subjected to electrophoresis on polyacrylamide gel, and the position of renin substrate was determined. There are considerable differences in the electrophoretic mobility of the renin substrates tested. Sheep substrate shows the slowest migration and mouse substrate the most rapid. The species tested appear to fall into two groups: slow-moving substrates occuring in the plasmas of sheep, cow, pig, and rabbit and fast moving substrates in man, dog, rat, and mouse. In most species only a single peak of renin substrate appeared, but in man and dog a minor peak was often observed in addition to the prominent major one. The classification of human renin substrate as an alpha-2-globulin is questioned.     

The allogeneic effect: the mechanism of allosuppression by Lyt-1, Ia- T cells

The kinetics of allohelp mediated by diffusable factors revealed that help by nonirradiated T cells (TOR) peaked at 48 to 72 hr, followed by a sharp decline if the T cells remained in the cultures. The temporal decrease in help after 72 hr was not mediated by suppressor lymphokines because mixtures of early (24 to 48 hr) and late (120-hr) allogeneic supernatants enhanced help synergistically. Lyt-1, Ia- T cells mediated the temporal decline in help and suppressed allogeneic B cell activation in co-cultures, and this "down-regulatory" activity (allosuppression) was radiosensitive. Help by irradiated T cells (T1000R) increased gradually until it plateaued between 96 and 120 hr. The helper activities of the allogeneic supernatants were directly proportional to their T cell growth factor (TCGF) activities. In addition, their kinetics were identical, and the removal of TCGF from 48-hr allogeneic supernatants by adsorption with TCGF-dependent HT-2 cells depleted both helper and TCGF activities. Help was restored to depleted 48-hr and 120-hr allogeneic supernatants by preparations of TCGF obtained from concanavalin A (Con A)-stimulated FS6-14.13 hybridoma cells that were adsorbed with lipopolysaccharide (LPS)-activated B cells or normal spleen cells (NS), but not with HT-2 cells. These results indicate that allohelp is dependent on TCGF. Moreover, help was dependent on at least one factor in addition to TCGF, because a high level of synergy occurred between TCGF and the "help-deficient" 120-hr allogeneic supernatant. In conclusion, the mechanism whereby Lyt-1, Ia- T cells regulated B cell activation with positive and negative allogeneic effects was through the production and subsequent exhaustion of TCGF, respectively. The production of TCGF and help was radioresistant, but exhaustion of TCGF and suppression was radiosensitive.