Regulation of proteasome complexes by gamma-interferon and phosphorylation

Proteasomes play a major role in non-lysosomal proteolysis and also in the processing of proteins for presentation by the MHC class I pathway. In animal cells they exist in several distinct molecular forms which contribute to the different functions. 26S proteasomes contain the core 20S proteasome together with two 19S regulatory complexes. Alternatively, PA28 complexes can bind to the ends of the 20S proteasome to form PA28-proteasome complexes and PA28-proteasome-19S hybrid complexes have also been described. Immunoproteasome subunits occur in 26S proteasomes as well as in PA28-proteasome complexes. We have found differences in the subcellular distribution of the different forms of proteasomes. The gamma-interferon inducible PA28 alpha and beta subunits are predominantly located in the cytoplasm, while 19S regulatory complexes (present at significant levels only in 26S complexes) are present in the nucleus as well as in the cytoplasm. Immunoproteasomes are greatly enriched at the endoplasmic reticulum (ER) where they may facilitate the generation of peptides for transport into the lumen of the ER. We have also investigated the effects of gamma-interferon on the levels and subcellular distribution of inducible subunits and regulator subunits. In each case gamma-interferon was found to increase the level but not to alter the distribution. Several subunits of proteasomes are phosphorylated including alpha subunits C8 (alpha7) and C9 (alpha3), and ATPase subunit S4 (rpt2). Our studies have shown that gamma-interferon treatment decreases the level of phosphorylation of proteasomes. We have investigated the role of phosphorylation of C8 by casein kinase II by site directed mutagenesis. The results demonstrate that phosphorylation at either one of the two sites is essential for the association of 19S regulatory complexes and that the ability to undergo phosphorylation at both sites gives the most efficient incorporation of C8 into the 26S proteasome.     

Regulation of sophorose lipid production by Candida (Torulopsis) apicola

Summary When growing on glucose, fructose or sucrose, Candida apicola produces large amounts of sophorose lipid during the stationary growth phase. In contrast, no sophorose lipid formation is observed with galactose or maltose independently whether hydrocarbons are present or not. The biosynthesis of the biosurfactant is therefore not simply a prerequisite for the degradation of extracellular hydrocarbon.     

The regulation of gamma-interferon production by interleukins 1 and 2

Purified interleukins 1 and 2 (IL-1 and IL-2) were used to investigate their role in the production of gamma-interferon (gamma-IFN). Macrophage depletion from human peripheral blood mononuclear leukocytes (PBML) inhibited gamma-IFN production. Addition of purified IL-1 partially restored IFN production of macrophage-depleted PBML induced by three T cell mitogens (phytohemagglutinin, PHA; concanavalin A, con A; and staphylococcal enterotoxin A, SEA), but had no effect on induction of IFN production by undepleted PBML. Therefore endogenous IL-1 production by macrophages is probably one of the mechanisms by which they act as accessory cells for IFN production by lymphocytes. A monoclonal antibody 9.6 which binds to the sheep erythrocyte (E) receptor found on human T cells inhibited IFN production. Addition of IL-2, but not IL-1, was found to reverse this inhibition. Prostaglandin E2, a macrophage product, inhibited gamma-IFN production induced by PHA, Con A, and OKT3 but usually not SEA. This inhibitory effect was reversible by the addition of IL-2 but not IL-1. In the absence of mitogen IL-1 alone rarely induced any IFN production, although some IFN was produced by PBML from a small minority of donors. Without mitogen IL-2 induced IFN production only at very high concentrations and the added presence of IL-1 did not enhance this induction.     

Regulation of lymphokine response during reinfection by influenza virus. Production of a factor that inhibits lymphokine activity

Mononuclear cells, obtained from the spleens and lungs of influenza virus-seropositive C57BL/6 mice at 2 to 4 days after re-infection with homologous virus (strain A/Bangkok/1/79), produced a low m.w. factor in vitro that prevents the biologic expression, but not production, of the lymphokine, leukocyte migration inhibition factor (LIF). The low m.w. factor inhibited LIF activity without destroying the LIF molecule inasmuch as simple dialysis restored lymphokine activity to culture supernatants. Production of the low m.w. factor was observed from 2 to 4 days after re-infection, at which time the delayed-type hypersensitivity response to viral Ag was suppressed. In contrast, LIF was produced by splenocytes and lung mononuclear cells obtained at all times tested after re-infection (from 2 to 30 days). Production of the low m.w. factor required re-infection of influenza A virus-seropositive mice with type A virus; re-infection with influenza B virus failed to induce production. Ag specificity was also required in vitro for splenocytes to produce the factor; cells from type A virus-re-infected mice required type A Ag stimulation. Cell depletion studies with mAb plus C revealed that macrophages and T cells along with Ag stimulation were required for factor production by spleen cells. However, mononuclear cells obtained within 4 days from the lungs of re-infected mice did not require in vitro Ag stimulation for production of the low m.w. factor, and factor production was dependent upon the presence of CD4+ (L3T4) cells in the culture. Fractionation of culture supernatants over a Sephadex G-50 column indicated that the factor had a molecular mass of 2 to 3 kDa, and by FPLC chromatofocusing over a Mono P column, the factor eluted at a pH of approximately 8.2. Thus, re-exposure of influenza virus-seropositive mice to homologous virus resulted in the production of a low m.w. factor that prevented the biologic expression of LIF, but not its production. Lymphokines are an important component of the delayed-type hypersensitivity response; the presence of mononuclear cells secreting a low m.w. factor and LIF concomitantly at the site of virus replication (lungs) and the capacity of the factor to block the biologic expression of LIF in vitro suggest that the factor may have a role in the regulation of a delayed-type hypersensitivity response in vivo during re-infection.     

Interleukin 1 (IL 1)-dependent lymphokine production by human leukemic T cell line HSB.2 subclones

Cloning of a human T cell leukemic cell line, HSB.2, was performed by a limiting dilution method to obtain clones with high levels of IL 2 production. None of the subclones that were obtained produced IL 2 constitutively, and only a low level of IL 2 was produced by the stimulation of these subclones with phytohemagglutinin (PHA) alone. High levels of IL 2 production (greater than 300 U/ml) were observed in several clones when stimulated with a cocktail of PHA and IL 1. Among them, HSB.2-A7-D2, A7-D9, or C5-B2 subclones, which were selected after cloning twice, were most effective in IL 1-dependent IL 2 production. HSB.2 subclones exhibited IL 1-dependent production of a variety of lymphokines other than IL 2, e.g., interferon-gamma (IFN-gamma), B cell growth factor (BCGF), and colony-stimulating factor (CSF). We observed that subclones with high IL 2-producing capacity tended to produce high levels of IFN-gamma or BCGF as well, while the capacity of CSF production was not parallel to these properties. Although several subclones were found to produce IFN-gamma and BCGF simultaneously with minimal IL 2 activity, no subclones with an exclusive BCGF production were obtained. Furthermore, when supernatants from the stimulated A7-D9 subclone were applied to an Ultro-gel AcA54 gel chromatography, it was revealed that IL 2 activity (m.w. 17K to 18K) and IFN-gamma (40K to 45K) were clearly separated, whereas two peaks of BCGF activity coincided with each peak of IL 2 and IFN-gamma, respectively. On the other hand, CSF activity was eluted at a different peak (30K to 35K). These data indicate that IL 2, IFN-gamma, and CSF activities are based on distinct molecules, whereas BCGF activities are indistinguishable from IL 2 and IFN-gamma. The HSB.2 subclones thus selected will provide a useful model for delineating the mechanism of IL 1-dependent lymphokine(s) production, and are a promising candidate for better lymphokine(s) producers.     

A quantitative analysis of antigen-triggered lymphokine production by activated T cells

We have developed a theoretical model to describe the triggering of lymphokine release from antigen-specific, activated T (T') cells, and we have used this model to define parameters that regulate this interaction. Under assay conditions of T' cell excess, the efficiency of triggering is a function of the target cell type. When various H-2k-bearing target cells were used to trigger B10.AQR T' cells activated against B10.A cells (anti-Kk), a hierarchy of triggering efficiency was observed with B10.A Con A blasts greater than R1.1 tumor cells greater than B10.A spleen cells greater than B10.A lymph node cells. There was a 10-fold difference in triggering efficiency between Con A blasts and lymph node cells. A similar pattern of reactivity was observed for various H-2d-bearing target cells used to trigger CBA T' cells specific for BALB/c antigens (anti-H-2d). Under assay conditions of T' cell excess, the order of reaction, i.e., the number of target cells per T' cell required to trigger lymphokine release, remained constant for the different target cell types. However, the order of reaction can vary with conditions of T cell activation. CBA T' cells activated against BALB/c spleen cells exhibited pseudo-first-order reactivity when triggered for lymphokine release with P815 target cells, whereas CBA T' cells activated against UV-irradiated P815 cells exhibited pseudo-second-order reactivity when triggered by the same tumor cells. Thus, T cells with the same apparent specificity can be qualitatively different in their reactivity with antigen. Under assay conditions of target cell excess, our analysis indicated that no T-T interaction was involved in the triggering reaction. Finally, our analysis was applied to study the nature of cross-reactivity. The results indicated that the triggering of lymphokine elicited by a cross-reactive antigen was due to the reactivity of a discrete subset of T' cells.     

Stimulation of lymphokine production by teleocidin, aplysiatoxin, and debromoaplysiatoxin

Previous studies showed that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and several structurally related tumor-promoting compounds stimulate lymphocytes to produce immune interferon (IFN-gamma) and interleukin 2 (IL-2). This study shows that three compounds structurally unrelated to TPA, previously shown to mimic TPA in some other biological activities, are similar to TPA in stimulating IFN-gamma and Il-2 production in cultures of human peripheral blood lymphocytes. The production of another lymphokine, termed lymphotoxin (LT), was also enhanced by TPA and the other three compounds examined. Maximal enhancement of lymphokine production was observed in cultures costimulated with TPA or one of the other tested compounds and phytohemagglutinin (PHA). TPA was separated from IFN-gamma during a multistep purification procedure.     

An alternative pathway of induction of lymphokine production by T lymphocyte clones

We have previously described a variant murine CTL clone that in contrast to all other clones tested, exhibited a novel capacity to produce IFN-gamma in response to IL-2. This alternative pathway of IFN-gamma induction differed from the conventional TCR complex-mediated pathway in that it was independent of elevated intracellular Ca2+ and insensitive to cyclosporine A. We report here the presence of an analogous pathway in the majority of T lymphocyte clones tested, when these clones are stimulated with IL-2 in the presence of syngeneic or third-party splenocytes. The accessory function of splenocytes in this alternative pathway is mediated by the MAC-1+ subpopulation and apparently involves cell-cell contact. However, the structure with which the MAC-1 antibody reacts probably is not involved directly. No involvement of Ag or the TCR for Ag could be demonstrated in this alternative pathway of lymphokine induction. The array of lymphokines induced by this alternative pathway is only a subset of those induced by antigenic stimulation. Finally, as with the previously described variant clone, IL-2-mediated induction of IFN-gamma production by the normal T lymphocyte clones is independent of normal extracellular Ca2+ levels and insensitive to cyclosporine A. Thus, this alternative pathway of lymphokine induction apparently constitutes a distinct signaling pathway in cloned T lymphocytes.     

Regulation of IGF-I receptors by corticotropin and angiotensin-II in cultured bovine adrenocortical cells

The effects of angiotensin II (A-II) and corticotropin (ACTH) on insulin-like growth factor-I (IGF-I) receptors of bovine adrenocortical cells were investigated. Pretreatment of cells for 48 h with ACTH or A-II induced in a dose-dependent manner an increase in [125I]IGF-I binding (ED50 congruent to 10(-11)M, Vmax = 10(-10) M with ACTH; ED50 congruent to 3.10(-9) M, Vmax = 10(-7) M with A-II). This resulted from an increase in the number of binding sites without modification of the binding affinity. Pretreatment with 8-Bromo-cAMP (10(-3) M), a phorbol ester (PMA 10(-7) M) + ionophore A23187 (10(-7) M) produced a positive regulation of IGF-I receptors. Glucocorticoids did not mediate the effect of A-II and ACTH on IGF-I receptors. Since previous studies have shown that IGF-I increased ACTH and A-II receptors the present data indicate the existence of a reciprocal positive trophic effect between IGF-I and the two hormones on the regulation of their specific membrane-bound receptors.     

Regulation of pheromone production by female pink bollworm moths Pectinophora gossypiella (Sanders) (Lepidoptera: Gelechiidae)

Abstract. We present in this study data which indicate that there is a diel periodicity in the pheromone production of the pink bollworm moth Pectinophora gossypiella (Sanders) (Lepidoptera: Gelechiidae) but that it is not well defined. Moreover the control mechanism of pheromone production differs somewhat from that reported for other moths. No pheromonotropic response was obtained when photophase females were injected with synthetic Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN). After decapitation for 24 h, Hez-PBAN did not induce pheromonotropic activity above control levels, which themselves remained relatively high. No effect on pheromone production was observed after treatment with the non-steroidal ecdysone agonist (RH5999). Decapitation for 72 h resulted in a significant drop in the control levels of pheromone titres. After decapitation for 72 h, stimulation by injections of Hez-PBAN and pink bollworm head extracts was observed. In addition, an enhancement of the PBAN stimulation was observed when combined with severance of the ventral nerve cord before injection. On the other hand, pink bollworm head extracts did not cross-react with Hez-PBAN antiserum in a radioimmunoassay, indicating that the pheromonotropic factor present is sufficiently different from Hez-PBAN and does not recognize the antigenic binding sites. In studies using isolated abdomen and pheromone gland cultures in vitro , no stimulation of de novo pheromone biosynthesis was observed but a 3-fold increase in the de novo fatty acid biosynthesis was detected in pheromone gland cultures.     

Regulation of ATP production by mitochondrial Ca(2+)

Stimulation of mitochondrial oxidative metabolism by Ca(2+) is now generally recognised as important for the control of cellular ATP homeostasis. Here, we review the mechanisms through which Ca(2+) regulates mitochondrial ATP synthesis. We focus on cardiac myocytes and pancreatic β-cells, where tight control of this process is likely to play an important role in the response to rapid changes in workload and to nutrient stimulation, respectively. We also describe a novel approach for imaging the Ca(2+)-dependent regulation of ATP levels dynamically in single cells.     

A lymphokine resembling transfer factor that stimulates MIF production by nonsensitive lymphocytes

High potency preparations of a new heat-labile, low m.w. (less than 5000) lymphokine (LMWL) were obtained by culturing tuberculin-sensitive guinea pig peritoneal exudate cells (PEC) with PPD in geometric conditions that promote amplified lymphokine production. This LMWL has the ability, in the presence of PPD, to stimulate nonsensitive PEC to produce a heat-stable molecule(s) resembling MIF with a m.w. in the range 50,000 to 100,000. The effects of the LMWL (less than 5000 daltons) and the MIF-like molecule(s) (50,000 to 100,000 daltons) were defined by the indirect macrophage migration assay and a macrophage deoxyglucose uptake assay. It is possible that LMWL represents a form of transfer factor with the ability to recruit unsensitized lymphocytes to produce MIF.     

Regulation of corticoliberin and corticotropin incretion in old age

Experiments on adult and old rats have shown age-related decrease of the CRF incretion in the hypothalamus, weakening of its response to dexamethasone, as well as a decrease of receptor corticosterone binding. Against this background in the hypophysis of old rats sensitivity ox CRF and serotonin increased, in stress the same content of ACTH as in adult rats secreted, it more markedly reacted to dexamethasone, and receptor of corticosterone binding increased in hypophysis. In old age, the role of hypophyseal link in control of the adrenocortical function is assumed to increase in stress.     

Leukotrienes: positive signals for regulation of gamma-interferon production

Leukotrienes B4, C4, and D4 were capable of replacing the helper cell or interleukin 2 requirement for gamma-interferon (IFN gamma) production by Lyt-1-,2+ cells from C57BL/6 mouse spleen cells at leukotriene concentrations as low as 0.002 microM. An antioxidant inhibitor (butylated hydroxyanisole) of lipoxygenase metabolism of arachidonic acid suppressed IFN gamma production. The suppression was significantly reversed by leukotriene C4, which further suggests that leukotrienes and possibly other substances produced by the lipoxygenase pathway of arachidonic acid metabolism play an important role in the regulation of IFN gamma production. All of these events may be related to activation of guanylate cyclase activity, since cyclic GMP also significantly reversed the suppressor effects of butylated hydroxyanisole in IFN gamma production. The leukotriene help for IFN gamma production was independent of DNA synthesis or cellular proliferation. The data are consistent with the hypothesis that lipxoygenase products of arachidonic acid metabolism may play a role in the mediation of interleukin 2 help in IFN gamma production. Cells that are rich sources of leukotrienes, then, should play important roles in positive regulation of lymphokine production.     

Regulation of T-cell activation and T-cell growth factor (TCGF) production by hydrogen peroxide

Activated macrophages are known to release a variety of immunoregulatory substances including the low-molecular-weight substances hydrogen peroxide and lactate. We report here that lactate but not hydrogen peroxide is capable of supporting a substantial production of T-cell growth factor (TCGF) in cultures of accessory cell-depleted splenic T-cell populations after stimulation with concanavalin A. Hydrogen peroxide and its biosynthetic precursor superoxide anion (O2-) mediate, however, a strong augmentation of the TCGF production by accessory cell-depleted T-cell populations in the presence of lactate. Lactate inhibits the incorporation of [3H]thymidine in short-term cultures (18-26 hr) of accessory cell-depleted T cells. This confirms the rule that (optimal) production of T-cell growth factor requires a growth inhibitory signal. Concentrations of hydrogen peroxide which augment TCGF production most effectively (i.e., 1 X 10(-5) M) do not inhibit the incorporation of [3H]thymidine; and higher concentrations (3 X 10(-5)-1 X 10(-4) M) of hydrogen peroxide inhibit both the production of TCGF and the incorporation of [3H]thymidine. In agreement with the augmenting effect of hydrogen peroxide on TCGF production, it was observed that the proliferative response in mixed lymphocyte cultures is suppressed by catalase and augmented by 1 X 10(-5) M H2O2. Proliferative and cytotoxic responses in mixed lymphocyte cultures with an external source of interleukin 2 (IL-2) in contrast, are not augmented by 1 X 10(-5) M H2O2. The relatively high concentration of 1 X 10(-4) M hydrogen peroxide was found to inhibit the proliferative responses in mixed lymphocyte cultures with or without external IL-2 but not the cytotoxic response in the presence of IL-2. This indicates that CTL precursor cells may be relatively resistant against H2O2.     

Augmentation of murine lymphokine (rIL-2)-activated killer cell activity by indomethacin

The effect of indomethacin on murine lymphokine-activated killer (LAK) cell activity was investigated using a natural killer-resistant, spontaneously developed, weakly immunogenic, and highly tumorigenic syngeneic murine mammary adenocarcinoma, mimicking that of human disease, as the target. When used in combination with human recombinant interleukin-2 (rIL-2), indomethacin was found to augment LAK cell activity, which was generated from culture of the normal mouse splenocytes with rIL-2, as compared to that with rIL-2 alone. This increase in LAK cell activity was shown to be indomethacin dose-dependent, and was demonstrated only when indomethacin was added to the rIL-2-containing medium at the beginning of culture. The enhancement of LAK cell activity by indomethacin was abrogated when the nylon-wool nonadherent "macrophage-poor" splenocytes were incubated with rIL-2 plus indomethacin. These results indicated that the rIL-2-induced LAK cell activity generated from murine splenocytes could be augmented by indomethacin, and the macrophages may be involved as the mediator.     

Amplification of plasmid (L factor) DNA and increased production of a plasmid gene product (gamma-interferon) in mouse L cells.

It has previously been reported that composite DNAs derived from L factor, a polyoma-related mammalian plasmid, can be established in several mouse cell lines after transfection. Here, we report that the copy number of a plasmid composite DNA consisting of L factor, pBR DNA, dihydrofolate reductase (dhfr) gene, and gamma-interferon (gamma-IFN) gene was increased more than 10-fold after two successive adaptations of the plasmid-bearing mouse L cells to increasing concentrations of methotrexate (MTX), an inhibitor of dhfr. The structure of the amplified L factor plasmid remained intact during prolonged cell culture, but the copy number remained to be amplified only when the selective pressure (presence of MTX in the medium) has been exerted during the culture. Cells bearing the amplified plasmid produced a higher level of gamma-IFN compared with the original clone, which was likely to be derived from the plasmid gamma-IFN gene amplified along with L factor and the dhfr gene.