Identification and quantitation of human T-cell antigen by antisera purified from antibodies crossreacting with hemopoietic progenitors and other blood cells

Anti-T cell globulin (ATCG) prepared from antihuman thymocyte serum by absorption with kidney, cells from patients with chronic lymphatic leukemias, and several lymphoblastoid cell lines was shown to react specifically with human thymus-derived lymphocytes. While high activity against thymocytes and a T-lymphoblastoid cell line could be demonstrated, ATCG remained negative against several chronic lymphatic leukemias and B-lymphoblastoid cell lines. The ATCG was used in the cytotoxic test, electronmicroscopy, and immunoautoradiography for identification of T cells in thymus, tonsils, spleen, blood, bone marrow, lymphatic leukemias, and lymphoblastoid cell lines. A comparison of these results with the ability to form spontaneous SRBC-rosettes revealed remarkable deviations between both markers in leukemias. Absorption with human brain failed to remove specific activity of ATCG. Labeling experiments by immunoautoradiography and investigations by complement fixation permitted quantitation of relative T-cell antigen concentration on different cell populations. As further evidence for specificity it could be shown that ATCG was no longer toxic for hemopoietic progenitors, whereas unabsorbed globulin reduced the number of colonyforming cells considerably.Abbreviations ALL acute lymphatic leukemia - ATCG anti-human T cell globulin (absorbed) - ATG anti-human thymocyte globulin (not absorbed) - Bm-C bone marrow cells - CFA complete Freund's adjuvant - CLL chronic lymphatic leukemia - EBV Epstein-Barr virus - GPC' guinea pig complement - HBSS Hanks' balanced salt solution - Ig immunoglobulin - PBS phosphate buffered saline - Per-Ly peripheral blood lymphocytes (normal) - Spl-Ly spleen lymphocytes - SRBC sheep red blood cells - VBS veronal buffered saline - Thy-Ly thymus lymphocytes - Ton-Ly tonsil lymphocytes     

Models for recognition of virally modified cells by immune thymus-derived lymphocytes

Virus-immune effector T cells generated in vivo are specific for both the virus used and theH-2 type of the infected mouse. Genes mapping at H-2K or H-2D apparently serve as self markers. The mechanism underlying this function is, however, not understood. This paper summarizes models that seem, at present, to be feasible explanations for this phenomenon, and considers possible implications of the hypothesis that self-recognition may be mediated by way of the same V gene subset that codes for alloreactivity.     

Tumor necrosis factor signal transduction. Cell-type-specific activation and translocation of protein kinase C

We have investigated the changes in protein kinase C (PKC) activity after treatment of several cell lines with TNF. Binding studies with [3H]phorbol dibutyrate (PBt2) on whole cells revealed rapid and transient activation of PKC in Jurkat, K562, and U937 cells with a maximum of phorbol ester binding at 6 min after TNF treatment. As shown by Scatchard analysis, the TNF-induced increase of [3H]PBt2 binding reflected increments of phorbol ester binding site numbers rather than greater binding affinities. Upon subfractionation of TNF-treated U937 cells a transient increase of PBt2 binding in the membrane fraction was accompanied by a long term loss of PBt2-binding in the cytosol, indicating a TNF-induced translocation of PKC from the cytosol to the cell membrane. With histone III-S as a substrate, the determination of specific PKC activity revealed similar kinetics of PKC translocation in U937 cells. TNF also induced PKC translocation in K562 and Jurkat cells. However, although TNF caused long term down-regulation of cytosolic PKC activity in U937 cells, the cytosolic PKC activity only transiently decreased in both Jurkat and K562 cells and then recovered to near basal levels. In the human nonmalignant fibroblast cell line CCD18, PKC was not activated by TNF. Our data suggest that PKC activation may play a major role in TNF signal transduction in some, but not all target cells.     

Complement factor I is upregulated in rat hepatocytes by interleukin-6 but not by interferon-gamma, interleukin-1beta, or tumor necrosis factor-alpha.

Complement factor I (FI) is a regulatory serine protease of the complement system which cleaves three peptide bonds in the alpha-chain of C3b and two bonds in the alpha-chain of C4b and thus prevents the assembly of the C3 and C5 convertases. We have investigated the proinflammatory cytokines IL-6, IL-1beta, TNF-alpha and IFN-gamma for their potential role in the regulation of FI expression. Of the investigated cytokines, only IL-6 increased the FI-specific RT-PCR signal in isolated hepatocytes, in the two rat hepatoma-derived cell lines FAO and H4IIE or in HUVECs. Quantitative competitive RT-PCR showed an IL-6 induced upregulation of FI-specific mRNA by about ten-fold. These data are in accord with Northern blot analyses in which the FI-mRNA was upregulated by IL-6 between five- and seven-fold. IL-6, but not IL-1beta, TNF-alpha or IFN-gamma also increased FI-protein levels in cell culture supernatants by about five-fold as determined by a semiquantitative immunoblot using a novel monoclonal antibody specific for rat FI.     

Local estradiol metabolism in osteoblast- and osteoclast-like cells

Bone is an estradiol-responsive tissue. Estrogen withdrawal during the menopause causes loss of bone mass and clinically relevant osteoporosis in a third of all women. Sufficient or impaired local production, as well as degradation of estradiol in cells present in the bone microenvironment might be an important mechanism of rescue or might contribute to the development of osteoporosis, respectively. We therefore investigated aromatase and 17β-hydroxysteroid dehydrogenase type IV (17β-HSD IV) expression in osteoblast- and osteoclast-like cells. Aromatase mRNA was increasingly expressed in myeloid THP 1 cells differentiated along the monocyte/phagocyte pathway exploiting vitamin D and either granulocyte-macrophage-stimulating factor (GMCSF) or macrophage-stimulating factor (MCSF). In long-term cultures, when sequentially exposed to vitamin D (days 0–21) and GMCSF (days 5–10) and plated on collagen, the amount of expression of aromatase mRNA steadily increased along with the increasing expression of osteopontin mRNA, ν integrin mRNA, c-fms (MCSF-receptor) mRNA and multinucleated cells developing. The conversion of estradiol from testosterone (10⁻⁷ M/1) in the supernatants of dishes mirrored changes in aromatase mRNA expression and by day 21 rose to 30,000 ng/10⁷ cells/24 h. 17β-HSD IV mRNA expression was abundant in undifferentiated THP 1 cells and was decreased to approximately 50% by day 21. Unstimulated SV-40 immortalized fetal osteoblasts did not express aromatase mRNA, but the expression was stimulated by the addition of the phorbol ester phorbol myristate acetate (PMA). Unstimulated osteoblasts from primary cultures did not express aromatase mRNA. Osteoblast-like osteosarcoma cells MG 63 expressed faint levels of aromatase mRNA in contrast to the osteosarcoma cell line HOS 58. 17β-HSD IV mRNA was expressed in fetal osteoblasts as well as in osteoblasts from primary culture, MG 63 and HOS 58 cells. In summary, we can show the expression of estradiol metabolizing enzymes in cells which are present in the bone microenvironment. Impaired aromatase expression and/or enhanced expression of 17β-HSD IV may contribute to the pathogenesis of osteoporosis.     

Is interleukin-6 the necessary pyrogenic cytokine?

(1) This paper analyses arguments suggesting a critical role for interleukin-6 (IL-6) in the manifestation of fever.

(2) Evidence supporting such a role for IL-6 derives from the excellent correlation between IL-6 in plasma and the accompanying fever, from neutralization experiments with anti-IL-6-antibodies, and from studies in IL-6-deficient mice.

(3) Peripherally administered IL-6 has rather poor pyrogenic properties and needs co-factors to elicit pronounced fever.

(4) Circumventricular organs, which lack a tight blood–brain barrier, have been identified as brain sites where circulating IL-6 causes a direct genomic activation of cellular elements in astrocytes and endothelial cells.

(5) Due to the fact that the onset of fever frequently preceeds the formation of sufficient amounts of IL-6 and the activation of brain cells by this cytokine, it is suggested that IL-6 contributes to the maintenance rather than to the induction of the initial phase of fever.

(6) During the time course of fever IL-6 seems to be involved in the induction of endogenous anti-inflammatory/antipyretic mediators and thus in the manifestation of defervescence.     

Pyrexia, anorexia, adipsia, and depressed motor activity in rats during systemic inflammation induced by the Toll-like receptors-2 and -6 agonists MALP-2 and FSL-1

Macrophage-activating lipopeptide-2 (MALP-2) from Mycoplasma fermentans has been identified as a pathogen-associated molecular pattern of Mycoplasmas that causes activation of the innate immune system through the activation of the heterodimeric Toll-like receptors (TLRs)-2 and -6. The aim of this study was to characterize the ability of MALP-2 and a synthetic analog fibroblast-stimulating lipopeptide-1 (FSL-1; represents the NH2-terminal sequence of a lipoprotein from M. salivarium) to act as exogenous pyrogens, to induce formation of cytokines (endogenous pyrogens), and to cause sickness behavior, such as depressed motor activity, anorexia, and adipsia. For this purpose, body temperature, activity, food intake, and water intake were recorded for 3 days by use of telemetry devices in several groups of rats treated with MALP-2/FSL-1 or the respective control solutions. Intraperitoneal injections of FSL-1 caused fever at doses of 10 or 100 microg/kg, which was preceded by a pronounced phase of hypothermia in response to a dose of 1,000 microg/kg. The maximal fever (a peak of 1.5 degrees C above baseline) was caused by the 100 microg/kg dose with almost identical responses to both MALP-2 and FSL-1. Fever was accompanied by pronounced rises of the proinflammatory cytokines TNF and IL-6 in plasma. Treatment with the TLR-2 and -6 agonists further induced a dose-dependent manifestation of anorexia and adipsia, as well as a reduction of motor activity. We could thus demonstrate that activation of TLR-2 and -6 can induce systemic inflammation in rats accompanied by the classical signs of brain-controlled illness responses.     

Transfer of resistance against the beet cyst nematode from radish (<Emphasis Type="Italic">Raphanus sativus</Emphasis>) to rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) by monosomic chromosome addition

In rape (Brassica napus), no resistance to the beet cyst nematode (BCN) Heterodera schachtii is available. This study was carried out to determine the specific chromosome(s) of resistant radish (Raphanus sativus) carrying the gene(s) for nematode resistance as a prequisite to convert rape from a host into a trap crop for this pest. A Raphanobrassica progeny of 25 plants was analyzed which segregated for all nine chromosomes of the Raphanus genome in a genetic background of synthetic rape. The number of radish chromosomes was determined by fluorescence in situ hybridization, using the Raphanus-specific DNA probe pURsN; and their type was identified by chromosome-specific randomly amplified polymorphic DNA markers. Five different multiple rape–radish chromosome additions (comprising the whole set of nine radish chromosomes, a–i) were selected and crossed to rape. For each cross-progeny, the number of cysts on plant roots was counted 42 days after inoculation with a L2 larvae suspension. Simultaneously, the plants were characterized for the presence or absence of individual radish chromosomes, using sets of chromosome-specific markers. Thus, the effect of each radish chromosome on cyst number was tested. Chromosome d had a major resistance effect, whereas the presence/absence of the other radish chromosomes had nearly no influence on cyst number. Plants with added chromosome d showed a resistance level comparable with that of the radish donor parent. The analysis in the cross to rape of a plant monosomic only for chromosome d confirmed the strong effect of this chromosome on nematode resistance. A further experiment comprising seven crosses using winter rape breeding lines and monosomic addition line d as pollen parent provided the same results on a broader genetic basis. In each case, the added chromosome d in a single dosage caused nearly the full resistance of the radish donor. Resistance was independent of the glucosinolate content in the roots. The possibilities for stabilizing BCN resistance in rape and its use for other crops and nematodes are discussed.Communicated by C. Möllers