yo-pro-1 and batimastat

Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.

Topics: ADAM Proteins; ADAM10 Protein; Adenosine Triphosphate; Amyloid Precursor Protein Secretases; Animals; B-Lymphocytes; Benzoxazoles; Dipeptides; Fluorescent Dyes; Gene Expression Regulation; Humans; Hydroxamic Acids; Lymphocyte Activation; Membrane Proteins; Mice; Mice, Knockout; Microscopy, Fluorescence; Phenylalanine; Primary Cell Culture; Protease Inhibitors; Purinergic P2X Receptor Antagonists; Quinolinium Compounds; Receptors, IgE; Receptors, Purinergic P2X7; Signal Transduction; Spleen; Thiophenes