Lymphokine research最新文献

The positively charged rhodamine analog rhodamine 123 accumulates specifically in the mitochondria of living cells. Mitochondria-specific interaction of this molecules is apparently dependent on the high transmembrane potential maintained by functional mitochondria (1, 2, 3). The application of such potential-dependent probes in conjunction with flow cytometry allows the monitoring of mitochondrial membrane potential in living cells (4, 5, 6). In the present work, the uptake of rhodamine 123 by Molt 16 cells stimulated by IFN-gamma or IFN-beta was measured by flow cytometry. Marked elevations in mitochondria-associated probe fluorescence have been observed in cells as early as 30-60 min after exposure of IFN-gamma but not observed by IFN-beta exposure. This results suggest that the IFN-gamma and -beta induced biological functions are different in the control of energy metabolism and energy requirements at the cellular level.

General features of signal transduction by G proteins are described. Recent results on roles of beta gamma dimers, kinetic properties of Gi alpha subunits, structural diversity of Go alpha subunits and possible functions of the Go protein as a stimulator of phospholipase C activity are discussed.

The effect of Colony-Stimulating Factors (CSFs) on the growth of murine thymocytes was investigated. None among the following factors tested alone, i.e., Interleukin-3 (IL-3), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Granulocyte Colony-Stimulating Factor (G-CSF) or Macrophage Colony-Stimulating Factor (M-CSF) has been found to stimulate thymidine uptake by thymocytes. However, GM-CSF synergistically enhances thymocyte proliferation induced by Interleukin-1 (IL-1). Synergistic responses are obtained at a very pronounced level after 3 days of culture with very low factor concentrations (1.5 to 15 pM) and in the complete absence of mitogen. Similar effects are induced by IL-3, though to a lesser degree. In contrast, neither G-CSF nor M-CSF potentiate thymocyte proliferation promoted by IL-1. Kinetic studies show that the synergy between IL-1 and GM-CSF reaches its maximum after about 72 h of thymocyte culture and that it requires the simultaneous presence of both factors during the first 24 h. In addition, our data suggest that GM-CSF acts in synergy with IL-1 by an Interleukin-2 (IL-2)-independent pathway since: (i) incubation of thymocytes with GM-CSF in the presence of IL-1 does not significantly enhance the expression of the IL-2 receptors (IL-2R) as demonstrated by flow cytometry, and, (ii) specific monoclonal antibodies against murine IL-2 or IL-2R fail to reduce thymocyte proliferation in response to the synergistic combination. Similarly, the potentiating effect of GM-CSF on IL-1 thymocyte growth does not depend on Tumor Necrosis Factor alpha (TNF) since (i) the synergy for IL-1 and GM-CSF and that previously described for IL-1 and TNF cumulate and (ii) anti-TNF antibodies do not abolish the potentiating effect of GM-CSF.

Human serum ultrafiltrate (UFS) obtained from cancer patients contains material(s) which inhibit the cytolytic activity of tumor necrosis factor (TNF-alpha) and lymphotoxin (LT-TNF-beta) in vitro. These factors are found in UFS from patients with different types of cancers and are not detected in the sera of normal donors. Results from molecular sizing studies demonstrated that their molecular weights are over 30 kD. They appear to block by interfering with the early stages of cell lysis. It is not clear whether or not these TNF-LT blocking factor(s) are the same molecules. These factors may have adverse effects on the biological activities of TNF and LT in cancer patients.

An interferon-inducible synthetic promoter was constructed with oligonucleotides which correspond to two regions of the promoter of the human interferon-inducible gene 6-16 that interact in-vitro with nuclear proteins. Firstly, we cloned a direct repeat sequence that was necessary for interferon regulation and that interacted with a 55 kilodalton nuclear protein. 5' to the direct repeat we introduced a 45 bp long oligonucleotide located at -450 in the native 6-16 promoter that interacted in-vitro with an 80 kilodalton nuclear protein. Expression after transfection of these plasmids showed that the direct repeat is necessary for interferon-inducible control and the -450 oligonucleotide by itself has no effect on transcriptional activity while in conjunction with the direct repeat it decreased the basal and interferon-inducible transcriptional activity of the reporter promoter in human cells.

Using a novel flow cytometric assay (BrdU/Hoechst flow cytometry) for the visualization and quantitation of cell activation and cell progression through multiple cell cycles we show that (1) resting mononuclear cells from human venous blood respond to polyclonal activation in a highly asynchronous fashion, as some of these cells enter their first cell cycle as early as 30 and as late as 80 hrs after exposure to the mitogen; (2) reducing agents improve the mitogenic response of polyclonally activated human lymphocytes by increasing the recruitment of non-cycling G0/G1 cells; (3) of 8 reducing agents tested, alpha-thioglycerol proved most effective with respect to enhancing recruitment into the cell cycle; (4) the effective agents needs to be present permanently for maximum response; (5) the growth-promoting effect of alpha-thioglycerol is mediated, but not solely caused by increased IL2 production. The latter conclusion was arrived at by using the IL2-dependent CTLL cell line in conjunction with the BrdU/Hoechst flow technique as a sensitive bioassay system for the growth promoting activity of the cell cycle progression factor IL2; (6) exogenously added IL-2, however, did not improve cell activation towards proliferation. These data indicate that increased IL-2 levels are probably due to increased cell activation rates but are not the primary cause of improvement of human lymphocyte proliferation.

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2.

IL-4 has been shown to participate in interregulatory networks with other cytokines including IL-2, IFN-gamma, IL-1 beta, and TNF-alpha. The ability of recombinant IL-4 (rIL-4) to modulate the synthesis and release of IL-6 was investigated in vitro. LPS-stimulated adherent cells (AC) from human peripheral blood secreted IL-6 as determined by ELISA and bioassay with the B9 hybridoma cell line. The secretion of IL-6 peaked between 12-16 h after LPS stimulation. The addition of rIL-4 inhibited LPS-induced IL-6 production measured at 8 h. The inhibitory effect of rIL-4 on IL-6 secretion was dose-dependent and occurred at doses as low as 0.001 U/ml (0.01 pg/ml). Recombinant IL-4 was added to the cultures for various periods and removed by aspiration of the medium and washing the cells. The subsequent LPS-induction of IL-6 secretion was reduced in cultures exposed to rIL-4 for as little as 5 minutes indicating that the effect of rIL-4 on the monocytes was rapid and irreversible. Northern blot analysis revealed that the effect of rIL4 on LPS-induced production of IL-6 by AC occurred at least in part at the level of transcription. In contrast, PMA-induced IL-6 gene transcription was not affected by rIL-4. These findings indicate that rIL-4 can regulate the synthesis of IL-6 by adherent peripheral blood mononuclear cells.

Interferon-gamma (IFN-gamma) is a cytokine produced by T lymphocytes and Natural Killer cells which has a key function in resistance against infections. Baboon (Papio anubis) IFN-gamma was produced by stimulation of baboon splenocytes with a lysate of Staphylococcus aureus. This interferon was active on human cells and could be seroneutralized with a polyclonal antiserum against human IFN-gamma, but not with antisera against human interferon-alpha and interferon-beta. Poly(A)(+)-RNA was isolated from baboon splenocytes and fractionated according to its sedimentation coefficient by sucrose density centrifugation. BaIFN-gamma mRNA was present in the 15 S fraction as was shown by hybridization with a human IFN-gamma cDNA probe. A cDNA library was constructed and a clone containing the complete BaIFN-gamma cDNA was isolated. The cDNA codes for a polypeptide of 165 amino acids of which the 23 N-terminal may serve as signal peptide. BaIFN-gamma differs at 11 residues from human IFN-gamma. Southern analysis of chromosomal DNA confirmed some of the nucleotide sequence differences between baboon and human IFN-gamma. The baboon IFN-gamma cDNA was placed under control of a trc promoter and brought to expression in Escherichia coli cells. Recombinant baboon IFN-gamma could be seroneutralized with certain monoclonal anti-human IFN-gamma antibodies. The presented work leads to the availability of recombinant baboon IFN-gamma for animal experiments but also yields new insight in the structure-function relationship of IFN-gamma.

Supernatants of human mononuclear cells cultured in the presence of LPS (LPS-MNC-SN) directly induced production of superoxide by isolated polymorphonuclear leukocytes, measured as superoxide dismutase--inhibitable cytochrome c reduction or, more sensitively, Lucigenin-enhanced chemiluminescence. Of recombinant human cytokines, only TNF and to a lesser degree also LT, but not GM-CSF, showed this activity. Neutralising antibody to TNF reduced the activity of LPS-MNC-SN to activate an oxidative burst in polymorphonuclear leukocytes by 30-50%. In contrast, killing of TNF-sensitive mouse L 929 cells by LPS-MNC-SN was completely inhibited by anti-TNF, while the L929 killing assay was at least as sensitive for TNF as the granulocyte chemiluminescence assay. Material with granulocyte chemiluminescence inducing (GCI) activity but devoid of TNF was obtained by sequential chromatography of LPS-MNC-SN on Mono Q anion exchange and phenyl sepharose hydrophobic interaction chromatography. The time course of GCI-induced superoxide generation was prolonged with a tmax of approximately 60 minutes. Subsequent separation of this GCI-active material on Superose gel filtration yielded two overlapping activity peaks with apparent molecular weights of approximately 40-50 and 200 kDa. Further, two distinct GCI activity peaks were found when Mono Q--and phenyl-sepharose--separated material was subjected to reversed phase chromatography. Thus, in addition to TNF, LPS-triggered mononuclear cells produce also other granulocyte chemiluminescence inducing mediators which appear distinct from presently known cytokines.