Kenneth E. Dombrowski

Secretion of IL-2 and IFN-γ, But Not IL-4, by Antigen-Specific T Cells Requires Extracellular ATP

Extracellular ATP and other nucleotides transmit signals to cells via surface-associated molecules whose binding sites face the extracellular milieu. Ecto-nucleoside triphosphate diphosphohydrolase is such an ATP-binding enzyme that is expressed by activated lymphocytes. We have previously shown that nonhydrolyzable ATP analogs block the lytic activity of NK cells and CD8+ T cells as well as their E-NTPDase activity. These results suggest that the hydrolysis of ATP may play a role in lymphocyte function. Here we report that E-NTPDase activity is up-regulated within 15 min of T cell stimulation and that reversible and irreversible enzyme inhibitors profoundly reduce secretion of IL-2 and IFN-γ, but not IL-4. TNF-α, IL-10, and IL-5 production showed intermediate sensitivity to these ATP analogs. Depletion of extracellular ATP also inhibited secretion of IFN-γ, but not IL-4, supporting the interpretation that extracellular ATP is required for secretion of some, but not all, cytokines. E-NTPDase antagonists reduced transcription of IL-2 mRNA and inhibited TCR-mediated intracellular calcium flux. These results suggest that extracellular ATP plays an essential role in the TCR-mediated signal transduction cascade for expression of certain cytokine genes.

Role of Ecto-ATPase in Lymphocyte Function

Extracellular nucleotides are now recognized as mediators of immune and non-immune cell function. The effects of extracellular nucleotides also vary with tissue. For example, extracellular ATP in micromolar concentrations can form pores in cell membranes, resulting in osmotic changes that are detrimental to the cell1. In bone marrow and thymocytes, extracellular ATP stimulates DNA synthesis, but it inhibits DNA synthesis in spleen, lymph nodes and peripheral blood lymphocytes2. The nucleotide also has cytostatic and cytotoxic effects on some tumor cells3, but the mechanism is unknown. In addition, ATP triggers histamine secretion from mast cells4–6 and the secretion of granules from neutrophils and monocytes7, 8, but inhibits macrophage-9, 10, NK cell-11–13 mediated cytotoxicities. Furthermore, extracellular ATP may serve as a substrate for ectoprotein kinases14 which have been identified on neutrophils13, erythrocytes16, neuronal cells17 and fibroblasts18.

MHC-Restricted Presentation of a Single Repeat of MUC1 Mucin

Major histocompatibility complex (MHC) restriction of antigen presentation of a single mucin1 (MUC1) variable number of tandem repeats peptide (VNTR1) was examined by generating cytotoxic T lymphocytes (CTL) derived from peripheral blood mononuclear cells (PBMC) stimulated with a single repeat MUC1 peptide presented by allogeneic (MHC-independent) or autologous (MHC-dependent) Epstein-Barr Virus (EBV) immortalized B lymphocytes. The ability to generate greater CTL activity against MUC1-expressing tumor cells by stimulation with autologous versus allogeneic EBV-B supports the hypothesis that presentation of a single repeat of MUC1 peptide is MHC-restricted (MHC-dependent).

X-Ray Photoelectron Spectroscopy of Amino Acids, Polypeptides and Simple Carbohydrates

X-ray photoelectron spectroscopy (XPS) is a surface sensitive analytical technique which measures the binding energy of electrons in atoms and molecules. The binding energy can be related to the molecular bonding or oxidation state of an element in the outermost layer of a material, that is 100 A. Thus, XPS is able to identify chemical species present on the surface of a molecule. In this paper XPS is briefly described. Spectra demonstrating its potential use for probing the surface properties of amino acids, polypeptides, proteins, carbohydrates and glycoproteins are discussed.