Our group investigates the function of the adhesion receptors known as integrins that are used by immune cells to migrate into tissues and to interact with each other. We are particularly interested in how the β2 integrin, LFA-1, expressed by all immune cells, controls this migration. We have focussed on LFA-1 signalling and the cooperation of LFA-1 with other partners, that is so essential for cell spreading and migration. The absence of active integrin on immune cells in Leukocyte Adhesion Deficiency-III patients leads to fatal infections and this year we have identified the mutated adhesion protein responsible for this disease. Another topic concerns early events of the response to microbial stimuli that require cooperation between macrophages and mast cells. This combined effort leads specifically to the recruitment of neutrophils into tissues, one of the first events of an immune response.
The behaviour of LFA-1 on migrating T lymphocytes: signaling leading to migration
The integrin LFA-1 is a promigratory adhesion receptor that immune cells utilise at many stages of an immune response. For example, when cells are recruited from the circulation, they use LFA-1 to migrate across the blood vessels into lymph nodes and infected tissues. How the integrin directs this migration has been a focus of interest for the Leukocyte Adhesion Laboratory. We have discovered that different conformations of LFA-1 are organised into distinct activity zones on the T cell membrane and that these zones operate together enabling the cell to migrate (Smith et al. J. Cell Biol 2005;170:141-151; Stanley et al. EMBO J 2008; 27: 62-75). Our model is that high affinity LFA-1 in the mid-cell 'focal zone' provides support for the more dynamic intermediate affinity LFA-1 attachments at the leading edge. These findings highlight how cooperation between two active conformations of LFA-1 are needed for key lymphocyte activities such as scanning the surfaces of blood vessels and other immune cells.
When LFA-1 binds its ligand ICAM-1, signals are transmitted into the T cell causing cells to adhere and then migrate. Our aim has been to dissect this 'outside in' signalling pathway that is immediately downstream of LFA-1. We find that the Src and Syk tyrosine kinases, Lck and ZAP-70 are both physically associated with LFA-1. This LFA-1/Lck/ZAP-70 signalling complex is absolutely essential for the behaviour of T cells in a shear flow assay that mimics the condition of lymphocytes leaving the circulation and attaching to the vasculature. The assay has revealed that ZAP-70 is required for conversion of short-lived intermediate affinity LFA-1 contacts with substrate to the stable contacts formed by high affinity LFA-1. Therefore the presence of LFA-1 already in complex with Lck/ZAP-70 and primed for action, offers an explanation as to how arrest of T cells under shear flow conditions occurs with sub-second timing. Such fast action would have particular relevance during an on-going immune response when it is essential for T cells to respond rapidly to stimulation at sites of tissue injury.
LFA-1 adhesion and de-adhesion
The detail of how T cells use LFA-1 for adhesion events on ICAM-1-expressing substrates is becoming clearer. However migration requires de-adhesion and much less is understood about what drives this essential part of the process. Surprisingly we find that the turnover of LFA-1 adhesions depends on the cytosolic enzyme, Ca2+-activated calpain 2. Inhibition of calpain activity results in the inefficient disassembly of LFA-1 adhesions, causing cell elongation and trails of LFA-1 clusters left behind as the cell struggles to migrate. Calpain 2 is distributed throughout the T cell, but the most active enzyme is found at the trailing edge and its activity is constantly maintained as the T cells migrate on ICAM-1. The major Ca2+ channel in lymphocytes is the ORAI1- expressing CRAC channel mediating Ca2+ influx and release from the Ca2+ stores. However it is not this ion channel, but the 'stretch-sensitive' TRPM7 channel that delivers the extracellular Ca2+ essential for calpain activity. Our data support a model whereby constitutive Ca2+ influx maintained by the TRPM7 channel is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this calpain is essential for the turnover of LFA-1 adhesions.
Figure 1. Migrating human T lymphocytes need to detach integrin LFA-1 to effectively translocate. The cytosolic enzyme calpain 2 is essential for deadhesion and is activated through the action of the TRPM7 ion channel. When calpain 2 activity is blocked (see image), T lymphocytes migrate inefficiently leaving behind punctate trails of LFA-1.
Leukocyte Adhesion Deficiency-III
About 10 years ago a group of patients was identified that expressed normal levels of the β1, β2 and β3 integrins on their haematopoietic cells, but with a failure of integrin function due to defective integrin signalling (McDowall et al. J. Clin Invest 2003; 111: 51-60). These Leukocyte Adhesion Deficiency-III (LAD-III) patients suffer from life-threatening bleeding and infections unless they are bone marrow transplanted. Now we have identified mutations in the FERMT3 gene, that specifies the kindlin-3 adhesion protein, as the cause of LAD-III disorder (Svensson et al Nat. Med 2009;. 15: 306). Two independent mutations in the FERMT3 gene in Maltese and Turkish patients result in decreased kindlin-3 mRNA and loss of protein expression. Importantly, the lack of kindlin-3 is responsible for LAD-III as transfection of the patients' lymphocytes with wild type kindlin-3 cDNA reverses the LAD-lll defect restoring integrin-mediated adhesion and migration. There is evidence to suggest that kindlin-3 enables the cytoskeletal adaptor protein talin to bind to integrin, but much remains to be discovered about how this new adhesion player promotes the activity of integrins on immune cells and platelets.
Neutrophil migration during the early stages of an immune response
Neutrophils are very frequently the first immune cells to migrate into an infected tissue and thus it is essential to understand how they are recruited. In an in vivo model of inflammation, we found that the de novo production of neutrophil chemoattractants, KC and MIP-2 by resident tissue macrophages scattered throughout tissue is a very early event in the innate immune response to microbial challenge (De Filippo et al., J Immunol 2008; 180: 4308-4315). What has not been appreciated is that mast cells also have a role in this early recruitment. We find that they are closely associated with the blood vessels and release pre-formed granules containing KC and MIP-2. Our evidence indicates that these two cell types with their distinct tissue locations cooperate to create an extended chemokine gradient that is initiated at the blood vessel and spreads throughout the infected tissue. This extended gradient is essential for recruiting neutrophils and directing them toward the centre of an infection.
For a list of refereed research papers, see Publications (in navigation on left).
