Activation of the leukocyte integrin LFA-1 on T lymphocytes

Leukocytes, by dynamically altering the adhesive state of the integrin LFA-1 (lymphocyte function-associated antigen-1) on their surfaces, can perform many necessary immunological activities such as phagocytosis, cytolysis, lymphocyte activation and leukocyte migration. LFA-1 is not constitutively adherent for its ligands ICAM (intercellular adhesion molecule)-1,-2 and-3 but requires prior activation before it can bind. Activation is achieved by triggering other cell surface receptors, such as the T cell receptor (TCR), or with phorbol esters through a process referred to as "inside-out" signalling. It is also possible to directly activate LFA-1 from the cell exterior by manipulations with divalent cations such as Mg2+. In this thesis, the intracellular signals responsible for induction of LFA-1 activation on T cells are investigated with the finding that tyrosine kinases and the serine/threonine kinase protein kinase C (PKC) are involved in LFA-1 activation. Through the use of intracellular Ca2+ [Ca2+]i mobilisers, such as ionomycin and thapsigargin, a role for [Ca2+]i in LFA-1 activation was discovered. Ca2+ does not mediate this induction through the common Ca2+- dependent signalling enzymes PKC or the serine/threonine phosphatase calcineurin but exhibits dependence on extracellular Ca2+ and cytoskeletal reorganisation. Through the analysis of two distinct protocols for inducing LFA-l-mediated adhesion it is shown that stimulation with divalent cations, such as Mg2+ induces adhesion through high affinity receptors which can bind soluble ICAM-1 and are recognised by the activation reporter antibody 24. This form of adhesion has little dependence on major intracellular signalling mechanisms. In contrast, stimulation with the phorbol ester PDBu does not induce high affinity receptors, instead adhesion is facilitated through cell spreading which is dependent on PKC and [Ca2+]i. The relevance of these findings to physiological integrin stimulation is discussed