New role for Agrin in T cells and its potential importance in immune system regulation
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Agrin plays a crucial role in the maintenance of the neuromuscular junction. However, it is expressed in other tissues as well, including T lymphocytes, where cell activation induces its expression. Agrin from activated T cells has the capacity to induce aggregation of key receptors and to regulate signalling. Interestingly, T cells isolated from patients with systemic lupus erythematosus over-express Agrin and its co-stimulation with the T cell receptor enhances production of pathogenic cytokines. These early studies point to an important function for Agrin in T cell biology and make the case for a more thorough and systematic investigation into its role in the immune system.
KeywordsHeparan Sulphate Glomerular Basement Membrane Lupus Patient Immunological Synapse Collapsin Response Mediator Protein
α3 subunit of the Na+/K+ ATPase
antigen presenting cell
collapsin response mediator protein 2
central supramolecular activation cluster
glomerular basement membrane
heparan sulphate proteoglycan
low density lipoprotein related protein 4
T cell receptor
T helper cell
T regulatory cell.
Immunity against pathogens and cancer requires cell-cell interactions, the type, strength, and duration of which determine to a large extent the final outcome of the immune response. In this process, T cells transiently interact with specialised antigen presenting cells (APCs) to sample the nature of the prevalent antigens in the body. Recognition of foreign antigen by the T cell receptor (TCR) results in the strengthening of the T cell- APC interaction, which is primarily mediated by an increase in the affinity of integrins for their corresponding ligand in a process known as 'inside-out signalling' . The resulting stable interaction between a T cell and its cognate APC is the formation of a synapse between the two cells, generally referred to as the immunological synapse (IS)  owing to its similarity to the neuronal synapse . The strength and duration of cell-cell interactions play a critical role in the activation of T cells; thus, a reduced capability to interact might result in failure to generate a good response when needed, whereas increased and/or sustained interaction might result in a breach of tolerance against self antigens, leading to the development of autoimmunity.
The formation of a mature IS has been characterized recently using advances in imaging techniques . Initial activation of the TCR leads to the rapid formation of microclusters that contain phosphorylated-active TCR associated with the proximal signalling proteins Lck, ZAP-70 and LAT [5, 6]. These microclusters are active signalling structures involving the actin cytoskeleton, since inhibition of actin polymerization prevents their formation . The TCR microclusters coalesce to form the central region of the IS - known as the central supramolecular activation cluster (cSMAC) - which also contains important coreceptors, such as CD4 and CD28, and key signalling proteins, such as PKCθ [7, 8, 9]. Receptors accommodated in the cSMAC are of small molecular mass, while large and heavily glycosylated proteins, such as LFA-1 (lymphocyte function-associated antigen 1), CD43, and the tyrosine phosphatase CD45, accumulate in a ring around the central region called the peripheral supramolecular activation cluster (pSMAC) [10, 11]. The mature IS with its defined areas is thought to control various cell-cell interaction-mediated processes by influencing signal transduction, leading to differential cell functions, and also signal termination and dissolution of cell conjugates [12, 13].
Interestingly, recent studies have shown that certain proteins with an established function in the neuronal synapse are also expressed by different T cell subsets. For example, regulatory T cells (Tregs), which are shown to be better poised to interact with APCs compared to naïve T helper (Th) cells, express neuropilin-1 . This molecule enhanced Treg interaction with APCs and its down-regulation by means of small interfering RNA resulted in a concomitant reduction in the ability of Tregs to form long-lasting synapses. Neuropilin-1 was not detected in naïve Th cells and this lack of expression correlated with their reduced capacity to form stable synapses. Interestingly, ectopic expression of neuropilin-1 resulted in a higher number of long-lasting interactions between Th cells and APCs . CRMP2 (Collapsin response mediator protein 2) is another neural protein found to be expressed in T cells and to have a role in their polarization and migration . Down-regulation of CRMP2 expression dampened the chemokine-induced transmigratory ability of human T cells. Significantly, CRMP2 was over-expressed in T cells from patients with neuroinflammatory disease, which were found to have elevated transmigratory activity . Relevant to this intense area of research is the recent association of the molecule Agrin with the formation of the T cell-APC IS.
The Agrin protein
Agrin is highly expressed in the brain, where its function has been linked to proper synaptic transmission of excitatory but not inhibitory synapses in the cerebral cortex. Mutant mice deficient in Agrin expression have non-functional NMJs and die before or shortly after birth due to asphyxiation . However, perinatal death can be rescued by the specific expression of Agrin in motor neurons . These mice exhibit a reduced number of presynaptic and postsynaptic specializations in the brain, indicating that Agrin has a role, at least in part, in maintaining synaptic structure in this tissue . In addition, Agrin is expressed at high levels in the brain microvasculature, where it could play a role in the maintenance of the blood-brain barrier . Reduction of Agrin expression could compromise the integrity of the blood-brain barrier, possibly resulting in uncontrolled immune cell infiltration.
Agrin in T cells and lupus autoimmunity
Genomic location of the Agrin gene in genomes available at the National Center for Biotechnology Information
Canis lupus familiaris
A working hypothesis on the function of Agrin in the immune system
Other reported functions of Agrin
Its widespread expression suggests that Agrin is important in other tissues. It has been reported that Agrin is required for efficient transcytosis of HIV-1 across epithelial cell monolayers by means of formation of the so-called 'virological synapse' . This structure, formed between HIV-infected cells and healthy mucosal epithelial surface, supports a more efficient viral transcytosis compared to cell-free virus. Agrin expressed on mucosal epithelial cells bound to the envelope glycoprotein gp41, and this interaction substantially enhanced HIV-1 trancytosis . Importantly, an anti- Agrin antibody reduced virus transcytosis in vitro, suggest ing that blocking Agrin on the surface of epithelial cells early on during infection could limit the initial viral load.
Agrin-null mice can be rendered viable by the restricted ectopic expression of Agrin specifically in motor neurons . A study of these mice revealed reduced growth and impaired skeletal development. Examination of long bones showed changes in the morphology and matrix composition of the growth plate, most notably in the thickness of the hypertrophic chondrocyte zone, which was reduced by up to 50% . Analysis of wild-type mice revealed high expression of Agrin in chondrocytes. These findings might point to an important role for Agrin in chondrocyte biology, the details of which, however, remain unexplored at present.
Agrin was found to be a major HSPG expressed in the glomerular basement membrane (GBM) of kidneys [36, 37, 38]. It wa s initially proposed that the high anionic content of HSPGs in GBM is a critical factor controlling glomerular permeability, and the observed reduction of heparan sulphate levels in various renal inflammatory diseases, including lupus nephritis, was associated with the increased GBM permeability seen in these diseases [39, 40]. However, recent studies have directly investigated the contribution of Agrin to GBM functionality by using mutant mice deficient for Agrin expression specifically in podocytes [41, 42, 43]. These mice, despite the strong reduction in the anionic content of the GBM, did not display any changes in glomerular architecture and had normal renal function, suggesting that Agrin is not required for establishment or maintenance of GBM architecture . Therefore, the role of Agrin in this tissue remains unresolved.
Potential Agrin receptors in the immune system
Agrin is a large HSPG containing many distinct structural domains (Figure 1), and to date several Agrin binding partners have been identified, including: fibroblast growth factor 2, which binds to Agrin heparan sulphate moieties ; α-dystroglycan and laminins [45, 46, 47], which are components of the extracellular matrix; adhesion molecules, such as neural cell adhesion molecule ; and integrins that contain the β1 subunit . Furthermore, the α3 subunit of the Na+/K+ ATPase (α3NKA) was found to serve as an Agrin receptor in neurons of the central nervous system . Agrin binding to cortical neurons inhibited α3NKA activity, resulting in membrane depolarization and increased action potential frequency . Also, α3NKA was found to mediate, at least in part, the effects of Agrin on cardiac myocyte contraction .
Recently, two papers have reported the long sought Agrin receptor expressed by the postsynaptic membrane at the NMJ. This was found to be Lrp4 (low-density lipoprotein related protein 4), a member of the lowdensity lipoprotein receptors [52, 53]. Lrp4, upon Agrin binding, forms a complex with MuSK, which initiates intracellular signalling leading to the aggregation of acetylcholine receptors. Lrp4 binds to the B/z+ form of Agrin with an affinity that is many fold higher compared to B/z- forms, confirming the selectivity of this receptor for the function of Agrin at the NMJ [52, 53]. It is conceivable that outside the NMJ, Agrin mediates its effects through additional receptors that have not been identified yet. One report, discussed above, implicates α-dystroglycan as an Agrin receptor in T cells . Nevertheless, a systematic analysis and identification of the types of Agrin receptors expressed will be an essential step in order to understand the biological function of Agrin in the immune system.
Although Agrin was initially identified as a factor critical for the function of the NMJ, its expression in other tissues, including T cells, implies a more widespread role. Results from studies in T cells suggest that Agrin function is linked to TCR signalling and cell activation. Despite these initial findings, there are big gaps in our knowledge regarding the function of Agrin in T cells and the immune system in general, at both the molecular level and at the level of the whole organism. At the molecular level important areas of investigation, although by no means the only ones, are: to understand the nature of Agrin modification induced early on during T cell activation, which increases its aggregating activity; to explore the role of Agrin in cell-cell adhesion during antigen presentation; and to identify the receptor(s) that mediate the effects of Agrin in immune cells. At the organism level, vital information will be generated by: studying how T cells and the immune system in general develop in the absence of Agrin expression; assessing changes in immune responses of viable Agrin-/- mice compared to wild type; and studying whether higher expression of Agrin in T cells in engineered mice predisposes to T cell hyperactivity and autoimmunity. Many tools are already available to study the biology of Agrin and we anticipate that these issues will be addressed in the near future by immunologists with different areas of expertise. These studies may well prove that Agrin has a critical role in the immune system as it has in the NMJ.
This work is supported by an arc Career Development (18106) and a University College London Hospital (CRDC) project grant (GCT/2008/EJ) award to ECJ and an arc project grant (16018) to PSK.
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