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T M Aune

Publications and source records attributed to T M Aune.

51 records · Page 3Linked to original sources
PubMed1983

Purification and initial characterization of the lymphokine soluble immune response suppressor.

Two molecular forms of the lymphokine soluble immune response suppressor (SRIS), obtained from serum-free supernatant fluids of a T cell hybridoma producing SIRS, have been purified by a combination of gel filtration and high performance liquid chromatography (HPLC). Supernatant fluids (8 liters) from the hybridoma 393.D2.6 were concentrated and fractionated on Sephadex G-50 in 0.4 M pyridine/0.4 M acetic acid buffer. Active fractions containing approximately 1 X 10(7) units of SIRS activity and 300 mg protein were fractionated further by reverse phase HPLC on a Lichrosorb RP-18 column in a 1.0 M pyridine-0.5 M acetic acid buffer. A stepwise n-propanol gradient reproducibly eluted SIRS activity in the 20% n-propanol fraction. After three chromatography steps on the Lichrosorb RP-18 column, final purification was obtained with an Si-100 diphenyl column by employing the same buffer and elution system. SIRS eluted in the 30% n-propanol fraction as two discrete protein peaks. Purity of SIRS was assessed by SDS-polyacrylamide gel electrophoresis. The two protein peaks from the fractionation on the diphenyl column each exhibited only one band of protein with m.w. of 21,500 and 14,000, respectively. Additional studies of both molecular species of SIRS showed they possessed functional properties identical to those previously described for crude SIRS. Final purification of the 21,500 SIRS species yielded approximately 10 micrograms of protein and 6 X 10(8) units of SIRS activity or 6 X 10(10) units/mg protein; final purification of 14,000 SIRS species yielded approximately 30 micrograms of protein and a similar amount of SIRS activity or 2 X 10(10) units/mg protein.

Cell Line↗
PubMed1983

Properties of the SIRS suppressor pathway.

The SIRS suppressor pathway is initiated by activation of Ly 2+ T lymphocytes by either con A or IFN beta. SIRS is a protein which has been purified and exists as two species with mol. wts. of 14,000 and 21,500. The target of SIRS is the macrophage and macrophages appear to oxidize or activate SIRS in a peroxide dependent process. Catalase blocks SIRS or IFN beta action by consuming H2O2 and levamisole blocks SIRS or IFN beta by preventing activation or oxidation of SIRS by H2O2. Other agents which block SIRS or IFN beta action include electron donors which can inactivate SIRSox. SIRSox is a potent inhibitor of immune responses and proliferation of normal and neoplastic cells. The mechanism of SIRSox-mediated inhibition of proliferation appears to involve oxidation or modification of protein sulfhydryls. Although the applicability of this pathway to the regulation of immune responses and cellular proliferation remains to be determined, both IFN beta and levamisole have been found to affect a wide variety of cellular processes. The involvement of both IFN beta and levamisole in the SIRS pathway suggests that this pathway may be an important host mechanism for regulating both immune responses and cellular proliferation in general.

Animals↗
PubMed1982

Preparation of soluble immune response suppressor and macrophage-derived suppressor factor.

Concanavalin A-activated murine suppressor T cells act via the mediator, soluble immune response suppressor (SIRS) which non-specifically suppresses IgM and IgG antibody responses to a variety of antigens, cytotoxic T lymphocyte responses and proliferative responses to alloantigens and mitogens in vitro. SIRS is a protein with an apparent MW of 45,000-55,000; the target of SIRS is the macrophage (M phi). M phi following treatment with SIRS release a second factor, M phi-derived suppressor factor (M phi-SF), which is directly responsible for the observed suppression of responses. Moreover. M phi-SF appears to be modified SIRS by all criteria used to date; M phi-SF can be obtained by reacting SIRS with low concentrations of H2O2 in the absence of M phi. Thus, M phi appear to serve only as a source of H2O2 and the mechanism of M phi-SF action action appears to have an oxidative basis. M phi-SF activity is lost following treatment with sulfhydryl reagents such as 2-mercaptoethanol, dithiothreitol or cysteine, reducing agents such as NaBH4 and a variety of peroxidase substrates such as pyrogallol, phenylenediamine, and ascorbic acid. Additionally, M phi-SF-mediated inhibition can be reversed by high concentrations of 2 mercaptoethanol or dithiothreitol under appropriate conditions. Since M phi-SF appears to be modified SIRS, oxidized by peroxide, and not a distinct second mediator produced by M phi in response to SIRS, we propose eliminating the term M phi-SF and using SIRSox to denote the active form of SIRS produced either by the SIRS-H2O2 reaction or SIRS-treated M phi.

Animals↗
PubMed1982

Activation of a suppressor T-cell pathway by interferon.

In addition to antiviral activities, murine fibroblast (type I) interferon (IFN-beta) suppresses immune responses. The mechanism(s) by which IFN-beta suppresses antibody responses by murine spleen cells to sheep erythrocytes in vitro has been investigated. IFN-beta-mediated suppression is partially or completely prevented by catalase, 2-mercaptoethanol, and certain peroxidase substrates (ascorbic acid, potassium iodide, and tyrosine). These same reagents also block suppression by mediators from concanavalin A-activated murine suppressor T cells, soluble immune response suppressor (SIRS)/macrophage-derived suppressor factor (Mphi-SF), and act by inactivating Mphi-SF or preventing formation of Mphi-SF from SIRS. Therefore, these experiments suggested that IFN-beta may act by inducing production of a molecule that has properties of SIRS. Treatment of spleen cells with IFN-beta leads to generation of a population of Lyt2+ suppressor T cells that acts by elaborating a soluble factor. This IFN-beta-induced suppressor T-cell factor (IFN-TsF) has properties in common with SIRS. First, both SIRS and IFN-TsF suppress antibody responses with the same characteristic kinetic pattern; responses initiate normally but prematurely terminate after day 4 of culture. Second, IFN-TsF and SIRS are of comparable size (45,000-55,000 daltons) and are converted to Mphi-SF by low (1 microM) concentrations of H2O2 or by macrophages. Third, Mphi-SF obtained from IFN-TsF or SIRS is inactivated by similar concentrations of reagents such as ascorbic acid, potassium iodide, and 2-mercaptoethanol. These data show that the immunosuppressive properties of IFN-beta are due, at least in part, to its ability to activate suppressor T cells that produce mediators that appear to be analogous to those in the SIRS/Mphi-SF pathway of immunosuppression.

Animals↗
PubMed1981

Conversion of soluble immune response suppressor to macrophage-derived suppressor factor by peroxide.

After incubation with soluble immune response suppressor (SIRS), a product of concanavalin A-activated Ly2+ T cells, macrophages release a factor that suppresses in vitro antibody responses, DNA synthetic responses to T-cell and B-cell mitogens, and division of several tumor cell lines. This factor, macrophage-derived suppressor factor (M phi-SF), is a protein with an apparent Mr of 55,000 that is inactivated by sulfhydryl compounds, certain amines, and iodide but not by other halides. In experiments reported here, conventional SIRS and SIRS produced by a cloned T-cell hybridoma were used to analyze formation of M phi-SF by SIRS-treated macrophages. Formation of M phi-SF was insensitive to inhibitors of protein and prostaglandin synthesis but was sensitive to catalase and cyanide, indicating that M phi-SF was not a newly synthesized product and that peroxide was important to its formation. As M phi-SF and SIRS have similar molecular weights and other properties, it is possible that M phi-SF is SIRS modified by peroxide. To test this possibility, SIRS was treated with H2O2 and M phi-SF activity was determined. H2O2 at 0.1-1 pM was sufficient to convert SIRS to M phi-SF; the reaction required approximately 15-20 min and was sensitive to cyanide. Several conventional peroxidase substrates inactivated M phi-SF produced by the SIRS-H2O2 reaction or by SIRS-treated macrophages. In addition, catalase and several of the compounds that directly inactivate M phi-SF also partially interfere with SIRS-mediated suppression of antibody responses. Collectively, these data suggest that SIRS-treated macrophages produce H2O2, which converts SIRS to M phi-SF, which has properties of an oxidized peroxidase-like protein and acts by oxidizing cellular components essential for cell division.

Animals↗
PubMed1981

Mechanism of action of macrophage-derived suppressor factor produced by soluble immune response suppressor-treated macrophages.

After a 2-hr incubation with soluble immune response suppressor (SIRS), a product of concanavalin A-activated murine T cells, macrophages release a factor, M phi-derived suppressor factor (M phi-SF), which nonspecifically suppresses immune responses in vitro. The mechanism(s) of action of M phi-SF and range of cell types affected by M phi-SF have been investigated. M phi-SF suppressed antibody responses to background levels if added at culture initiation and by 80 to 90% if added as late as 2 hr before assay. Primary and secondary IgM and IgG antibody responses, proliferative responses to T cell and B cell mitogens, antibody and protein secretion, and the division of several tumor cell lines in culture were inhibited by M phi-SF. Division of synchronized tumor cells was inhibited when M phi-SF was added at any point prior to and during mitosis; this inhibition could be reversed with 2-mercaptoethanol. In the presence of M phi-SF, asynchronous tumor cells accumulated in the cell cycle just prior to cell division and could be released into mitosis by 2-mercaptoethanol. These data indicate that M phi-SF inhibits cell division by causing a block at or in mitosis and suggest that M phi-SF may be a general inhibitor of cellular proliferation and possibly of protein secretion.

Animals↗
PubMed1978

Susceptibility of Escherichia coli to bactericidal action of lactoperoxidase, peroxide, and iodide or thiocyanate.

The bactericidal action that results from lactoperoxidase-catalyzed oxidation of iodide or thiocyanate was studied, using Escherichia coli as the test organism. The susceptibility of intact cells to bactericidal action was compared with that of cells with altered cell envelopes. Exposure to ethylenediaminetetraacetic acid, to lysozyme and ethylenediaminetetraacetic acid, or to osmotic shock were used to alter the cell envelope. Bactericidal action was greatly increased when the cells were exposed to the lactoperoxidase-peroxide-iodide system at low temperatures, low cell density, or after alteration of the cell envelope. When thiocyanate was substituted for iodide, bactericidal activity was observed only at low cell density or after osmotic shock. Low temperature and low cell density lowered the rate of destruction of peroxide by the bacteria. Therefore, competition for peroxide between the bacteria and lactoperoxidase may influence the extent of bactericidal action. Alteration of the cell envelope had only a small effect on the rate of destruction of peroxide. Instead, the increased susceptibility of these altered cells suggested that bactericidal action required permeation of a reagent through the cell envelope. In addition to altering the cell envelope, these procedures partly depleted cells of oxidizable substrates and sulfhydryl components. Adding an oxidizable substrate did not decrease the susceptibility of the altered cells. On the other hand, mild reducing agents such as sulfhydryl compounds did partly reverse bactericidal action when added after exposure of cells to the peroxidase systems. These studies indicate that alteration of the metabolism, structure, or composition of bacterial cells can greatly increase their susceptibility to peroxidase bactericidal action.

Escherichia coli↗
PubMed1978

Cofactor role of iodide in peroxidase antimicrobial action against Escherichia coli.

The mechanism of antimicrobial activity of the peroxidase-hydrogen peroxide (H(2)O(2))-iodide (I(-)) system was investigated. Inhibition of respiration and loss of viability of Escherichia coli were used as measures of antimicrobial activity. Because the bacteria destroyed H(2)O(2), peroxidase antimicrobial action depended on the competition for H(2)O(2) between the bacteria and the peroxidase. Utilization of H(2)O(2) by the peroxidase was favored by (i) increasing either the peroxidase or the I(-) concentration, so as to increase the rate of oxidation of I(-), (ii) lowering the temperature to lower the rate of destruction of H(2)O(2) by the bacteria, and (iii) adding H(2)O(2) in small increments so as to avoid a large excess of H(2)O(2) relative to I(-). When utilization of H(2)O(2) by the peroxidase system was favored, the peroxidase system and iodine (I(2)) were equivalent. That is, antimicrobial action per mole of H(2)O(2) equaled that per mole of I(2). Also, identical antimicrobial action was obtained either by incubating the bacteria directly with the peroxidase system or by preincubating the peroxidase system so as to form I(2) and then adding the bacteria. On the other hand, peroxidase antimicrobial action could be obtained at low I(-) concentrations. These I(-) concentrations were lower than the concentration of I(2) that was required for antimicrobial action. It is proposed that peroxidase-catalyzed oxidation of I(-) yields I(2), which reacts with bacterial components to yield the oxidized components and I(-). The I(-) that is released can be reoxidized and participate again in the oxidation of bacterial components. In this way, I(-) acts as a cofactor in the peroxidase-catalyzed oxidation of bacterial components.

Coenzymes↗
PubMed1978

Oxidation of Escherichia coli sulfhydryl components by the peroxidase-hydrogen peroxide-iodide antimicrobial system.

The chemical modification of bacterial components was studied following incubation of Escherichia coli with the peroxidase-hydrogen peroxide (H(2)O(2))-iodide (I(-)) antimicrobial system or with iodine (I(2)). The oxidation of cell sulfhydryls and the iodination of cell components were measured. Both the peroxidase system and I(2) oxidized sulfhydryls. When the I(-) concentration in the peroxidase system was greater than 100 muM, the peroxidase system and I(2) were equivalent. That is, sulfhydryl oxidation or killing per mole of H(2)O(2) equaled that per mole of I(2). These results were consistent with peroxidase-catalyzed oxidation of I(-) to yield 1 mol of I(2) per mol of H(2)O(2). Sulfhydryls were oxidized to yield sulfenic acids and free I(-). With I(-) concentrations in the range of 10 to 100 muM, the amount of sulfhydryls oxidized by the peroxidase system could exceed the amount of I(-). Because the oxidation of sulfhydryls to sulfenic acids did not consume I(-), one I(-) ion could participate in the oxidation of many sulfhydryls. With I(-) concentrations lower than 10 muM, complete oxidation of sulfhydryls was not obtained. Incorporation of I(-) into iodinated derivatives of bacterial components partly depleted the system of I(-) and limited the formation of I(2). These results indicated that antimicrobial activity was due to peroxidase-catalyzed oxidation of I(-) to I(2), followed by I(2) oxidation of cell components. There was a direct relationship between sulfhydryl oxidation and antimicrobial action. Although iodination of bacterial components accompanied sulfhydryl oxidation, the amount of I(-) incorporation was not directly related to antimicrobial action. Also, incorporation of I(-) interfered with antimicrobial action at low I(-) concentrations.

Escherichia coli↗
PubMed1978

Lactoperoxidase, peroxide, thiocyanate antimicrobial system: correlation of sulfhydryl oxidation with antimicrobial action.

The antimicrobial activity of the lactoperoxidase, peroxide, thiocyanate system against Escherichia coli was directly related to the oxidation of bacterial sulfhydryls. Lactoperoxidase catalyzed the oxidation of thiocyanate, which resulted in the accumulation of hypothiocyanite ion, OSCN-. A portion of the bacterial sulfhydryls were oxidized by OSCN- to yield sulfenic acid and sulfenyl thiocyanate derivatives. The remaining sulfhydryls were not oxidized, although OSCN- was present in large excess. The oxidation of sulfhydryls to sulfenyl derivatives inhibited bacterial respiration. This inhibition could be reversed by adding sulfhydryl compounds to reduce the sulfenyl derivatives and the excess OSCN-. Also, this inhibition could be reversed by washing the cells so as to remove the excess unreacted OSCN-. After washing, the bacteria underwent a time-dependent recovery of their sulfhydryl content. This recovery resulted in recovery of the ability to respire. The inhibited cells were viable if diluted and plated shortly after the incubation with the lactoperoxidase, peroxide, thiocyanate system. On the other hand, long-term incubation in the presence of the excess OSCN- resulted in loss of viability. Also, the inhibition of respiration became irreversible. During this long-term incubation, the excess OSCN- was consumed and the sulfenyl derivatives disappeared.

Dithiothreitol↗