David W. Golde

The Sphingomyelin Pathway in Tumor Necrosis Factor and Interleukin-1 Signaling

Richard Kolesnick and David W. Golde The Laboratories of Signal Transduction and Molecular and Cellular Hematology Memorial Sloan-Kettering Cancer Center New York, New York 10021 E:vidence suggests that tumor necrosis factor a (TNFa) and interleukin-18 (IL-18) employ the sphingomyelin path- way to effect signal transduction by their receptors. This pathway is initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the action of a sphingomye- linase. Ceramide serves as asecond messenger, stimulat- ing a serine/threonine ceramide-activated protein kinase to transduce the cytokine signal, in part through mitogen- activated protein (MAP) kinase and transcription factors such as NF-KB (Figure 1). The extent to which this signal- ing system is used in inflammation, immune responses, and apoptosis is not known, but accumulating evidence suggests that it is a commonly employed pathway that could be exploited therapeutically. The Sphingomyelin Metabolic Pathway Sphingomyelin is preferentially concentrated in the outer leaflet of the plasma membrane of most mammalian cells; it is comprised of a long chain sphingoid base backbone (predominantly sphingosine), a fatty acid, and a phospho- choline head group (Figure 2). The fatty acid in amide linkage at the second position of the sphingoid base consti- tutes ceramide. Hydrolysis of the phosphodiester bond by a sphingomyelinase to yield ceramide and phosphocho- line is the only clearly defined mechanism for sphingomye- lin degradation in mammalian cells (Kolesnick, 1991). Sphingomyelin was considered only a structural ele- ment of the plasma membrane. However, 1,2-diacylglycerol (DG), a physiologic activator of protein kinase C, stimu- lated rapid sphingomyelin degradation toceramide in GH3 rat pituitary cells (Kolesnick, 1991). Little of the generated ceramide was deacylated to sphingoid bases, potential inhibitors of protein kinase C (Hannun and Bell, 1989), prompting a search for additional derivatives of ceramide. Several investigations established the existence of a spe- cific metabolic pathway from sphingomyelin to ceramide l-phosphate (see Bajjalieh et al., 1989; Kolesnick, 1991). The sphingomyelin metabolic pathway is similar to the phosphoinositide signal transduction pathway. The cen- tral lipids in these pathways, ceramide and DG, both serve as substrates for the same bacterial DG kinase, implying they possess structural similarity. Their phosphorylated forms, ceramide l-phosphate and phosphatidic acid, were, therefore, also structurally similar. Further, neutral sphingomyelinase, the enzyme that initiates the sphingo- myelin pathway, is a phospholipase C concentrated in the plasma membrane (Kolesnick, 1991) like the enzyme ini- tiating the phosphoinositide pathway. Ceramide also ap- pears to be an ideal candidate second messenger since it readily redistributes across a membrane bilayer (Lipsky and Pagano, 1985). Since DG utilized a specific kinase, protein kinase C, for signaling, it was considered that cera- mide might stimulate a kinase. Ceramide does not activate protein kinase C. identification of Ceramidektivated Protein Kinase and Phosphatase Because natural ceramide contains long and very chain saturated or monounsaturated fatty acids and is poorly soluble in aqueous solutions, cell-permeable ana- logs were synthesized. A ceramide was constructed con- taining octanoic acid, N-octanoyl sphingosine (CS-cer), which is analogous to 1,2dioctanoylglyceroI, the hydro-

Activation of human eosinophil and neutrophil functions by haematopoietic growth factors: comparisons of IL‐1, IL‐3, IL‐5 and GM‐CSF

Summary. We compared the effect of haematopoietic growth factors granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin (IL)‐1, IL‐3, and IL‐5 on the functional activation of human eosinophils and neutrophils from the same donor. All four colony‐stimulating factors (CSF) enhanced the phagocytosis of Candida albicans by eosinophils and increased staphylococcal, but not Candida, killing. GM‐CSF and IL‐5 had a profound stimulating effect on eosinophil staphylocidal activity. GM‐CSF and IL‐3 enhanced the generation of leukotriene C4 (LTC4) induced by calcium ionophore A23187 and the release of arylsulphatase and β‐glucuronidase from specific and small granules of eosinophils. In contrast, IL‐1 and IL‐5 had no effect on degranulation. GM‐CSF and IL‐1 enhanced phagocytosis of C. albicans by neutrophils, and GM‐CSF stimulated degranulation and the release of the enzymes β‐glucuronidase and arylsulphatase from neutrophils while IL‐1 stimulated the release of arylsulphatase only. This study indicates that the eosinophilactive colony‐stimulating factors can markedly enhance the host defence function of the eosinophil and even make it the equal of the neutrophil in staphylocidal activity. The CSF‐activated eosinophil, however, may cause inappropriate inflammation and normal tissue damage.

Effect of Recombinant Human Granulocyte–Macrophage Colony-Stimulating Factor on Myelopoiesis in the Acquired Immunodeficiency Syndrome

We administered recombinant (biosynthetic) human granulocyte-macrophage colony-stimulating factor (GM-CSF) to 16 patients with the acquired immunodeficiency syndrome (AIDS) and leukopenia (2225 +/- 614 cells per microliter [mean +/- SD]). Each patient first received a single intravenous dose; 48 hours later a 14-day continuous intravenous infusion of the agent was begun. The doses used were 1.3 X 10(3) (n = 4), 2.6 X 10(3) (n = 4), 5.2 X 10(3) (n = 4), 1.0 X 10(4) (n = 3), or 2.0 X 10(4) (n = 1) U per kilogram of body weight per day. Administration of recombinant GM-CSF resulted in dose-dependent increases in circulating leukocytes and in increases in circulating neutrophils, eosinophils, and monocytes. The peak leukocyte count ranged from 4575 +/- 2397 cells per microliter at the lowest dose, to 48,700 in the patient receiving the highest dose. Mild side effects--low-grade fever, myalgia, phlebitis, and flushing--were observed in some patients; there were no life-threatening toxic reactions. Our data demonstrate that recombinant human GM-CSF is well tolerated and biologically active in leukopenic patients with AIDS. Strategies to increase the number and function of circulating leukocytes may reduce the morbidity and mortality of infections in these and other patients with leukopenia.

Hairy Cell Leukemia: A Clinical Review Based on 71 Cases

We studied the clinical and laboratory characteristics of 71 patients with hairy cell leukemia to ascertain whether prognostic factors important for the therapy of the disease existed. Granulocytopenia of less than 500/mu 1 was associated with the occurrence of a serious infection in 46% of the patients, compared to 19% of patients whose absolute granulocyte count was greater than 500/mu 1 (P less than 0.05). In most patients with symptoms of thrombocytopenia or leukopenia, splenectomy led to improved blood counts. Patients treated with splenectomy only had a significantly longer survival than those treated with steroids only (P less than 0.05). A group of patients who were slightly older and had minimal splenomegaly and few hairy cells in the peripheral blood did well without therapeutic intervention. Twenty-four patients have died, 16 of an infectious process. The actuarial survival for the group is 51% at 4 years, and there has been no change subsequently.

Vitamin C crosses the blood-brain barrier in the oxidized form through the glucose transporters.

Vitamin C concentrations in the brain exceed those in blood by 10-fold. In both tissues, the vitamin is present primarily in the reduced form, ascorbic acid. We identified the chemical form of vitamin C that readily crosses the blood-brain barrier, and the mechanism of this process. Ascorbic acid was not able to cross the blood-brain barrier in our studies. In contrast, the oxidized form of vitamin C, dehydroascorbic acid (oxidized ascorbic acid), readily entered the brain and was retained in the brain tissue in the form of ascorbic acid. Transport of dehydroascorbic acid into the brain was inhibited by d-glucose, but not by l-glucose. The facilitative glucose transporter, GLUT1, is expressed on endothelial cells at the blood-brain barrier, and is responsible for glucose entry into the brain. This study provides evidence showing that GLUT1 also transports dehydroascorbic acid into the brain. The findings define the transport of dehydroascorbic acid by GLUT1 as a mechanism by which the brain acquires vitamin C, and point to the oxidation of ascorbic acid as a potentially important regulatory step in accumulation of the vitamin by the brain. These results have implications for increasing antioxidant potential in the central nervous system.

Molecular characterization and expression of the gene encoding human erythroid-potentiating activity.

Erythropoietin is the primary physiological regulator of erythropoiesis; however, in vitro studies have identified another class of mediators which appear to be important in stimulating erythroid progenitors. These factors have generally been referred to as burst-promoting activities (BPA), because they stimulate the growth of early erythroid progenitors referred to as burst-forming units-erythroid (BFU-E) which give rise to colonies of up to thousands of haemoglobinized cells1,2. We recently reported purification of a burst-promoting activity from medium conditioned by the Mo T-lymphoblast cell line infected with human T-cell lym-photropic virus type II (HTLV-II)3,4. This purified glycoprotein of relative molecular mass (Mr) 28,000 also stimulates colony formation by more mature erythroid precursors (CFU-E) and is therefore referred to as erythroid-potentiating activity (EPA)5. Purified EPA specifically stimulates human and murine cells of the erythroid lineage, unlike murine interleukin-3 (IL-3) which stimulates precursor cells from all haematopoietic lineages6. We report here the isolation of a complementary DNA molecular clone encoding EPA and its use in producing EPA in COS (monkey) cells and CHO (Chinese hamster ovary) cells. We also define the organization of the EPA gene in human DNA.

Identification of the colony-stimulating cell in human peripheral blood

Bone marrow colony formation in soft gel culture may be stimulated by substances elaborated by human peripheral blood leukocytes. In order to determine the cell type responsible for colony stimulation, peripheral leukocytes were separated by Ficoll-Hypaque gradients and differential glass adhesion. Morphologic, histochemical, and functional criteria were applied to determine the purity of the monocyte, lymphocyte, and neutrophil fractions. Using these cells as feeder layers and as a source of conditioned medium, evidence was obtained that the monocyte is the colony-stimulating cell of human peripheral blood. Activity greater than that of mixed white cells was obtained with monocyte underlayers, and only monocyte- and macrophage-conditioned media were shown to have significant colony-stimulating activity.

Chronic Myelogenous Leukemia — New Concepts

CHRONIC myelogenous leukemia (CML) is a clonal myeloproliferative disorder arising from neoplastic transformation at the level of the pluripotent stem cell and characterized clinically by a marked ...

A Second Isolate of HTLV-II Associated with Atypical Hairy-Cell Leukemia

THE human T-cell lymphotropic viruses Type I (HTLV-I) and Type II (HTLV-II) and the bovine leukemia virus, which are members of a family of leukemogenic mammalian retroviruses, share some of the sa...

Tissue inhibitor of metalloproteinase-2 (TIMP-2) has erythroid-potentiating activity

Tissue inhibitor of metalloproteinase (TIMP) was purified and molecularly cloned on the basis of its erythroid‐potentiating activity (EPA). TIMP/EPA appears to be a bifunctional molecule with both growth factor and anti‐enzymatic activity. Recently, a second TIMP‐related molecule was identified and we have investigated its possible erythroid‐potentiating activity. Native, purified human TIMP‐2 was assayed for erythroid‐potentiating activity using an in vitro erythroid burst formation assay and was compared with that of previously characterized recombinant EPA/TIMP‐1. The results demonstrate that both members of the tissue inhibitor or metalloproteinase family, TIMP‐1 and TIMP‐2, possessed erythroid potentiating activity which was inhibited by antibodies developed to neutralize EPA. These results suggest that TIMP‐2 shares a common structural domain with EPA/TIMP‐ I that is responsible for the erythroid‐potentiating activity of these inhibitors. Therefore, TIMP‐1 and TIMP‐2, with both anti‐protease activity and growth factor activity, join a family of bifunctional molecules such as Fibroblast growth factor and thrombin which have both enzymatic and growth factor activity.