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Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the β1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin–containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin
Lβ2. Although C3 treatment of PB T cells did not prevent adhesion to the β1 integrin substrate Fn, it did inhibit β1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited β1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.
Key Words: integrins • cytoskeleton • interleukin 2 • cell adhesion • extracellular matrix
Dr. Woodside's present address is Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037.
Abbreviations used: 4,5-PIP2, phosphatidylinositol 4,5-bisphosphate; ADP, adenosine diphosphate; Fn, fibronectin; GTP, guanosine triphosphatase; PB, peripheral blood; PIP 5-kinase, phosphatidylinositol 4-phosphate 5-kinase; PLC, phospholipase C.
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