Innate and adaptive immunity

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What is the evidence supporting a role for synaptotagmin in regulating immunomediator secretion from mast cells? Could it be a novel drug target for the treatment of chronic inflammatory diseases?

Mast cells have long been established as generators of inflammation as well as playing a role in innate and adaptive immunity. Mast cells play a role in inflammatory disorders such as asthma, inflammatory bowel disease, rheumatoid arthritis and anaphylaxis among many others. (Metcalfe 2008) As indicated by the wide ranging disorders mast cells are implicated in, they can be found in numerous tissues around the body and are particularly easy to identify in microscopic slides due to the large granules seen in the cytoplasm. These granules contain many of the mediators of immune and inflammatory responses. The mediators can be classed into five separate groups; preformed mediators (Histamine, Serotonin etc), Lipid mediators (LTC4, PGD2 etc), cytokines (TGF-B, IL-2 etc), chemokines and growth factors. (Metcalfe) The granules in mast cells are able to undergo a process of degranulation. This is the fusion of the granule with the plasma membrane causing the exocytosis of granule contents, also known as immunomediator secretion. However, what sets degranulation apart from normal exocytosis is the movement of nearly all the intracellular granules to the plasma membrane upon stimulation. Degranulation causes the release of the preformed mediators and occurs via activation which causes cross-linking of the high affinity IgE receptor (FceRI). This process is highly regulated and like exocytosis at the synapse, is thought to be controlled at the level of membrane fusion. Due to this it can be suggested that degranulation is a form of regulated exocytosis rather that constitutive exocytosis, where vesicles fuse with the membrane as soon as they are formed. Regulated exocytosis also only occurs when an appropriate signal is received to the cell along with an increase in intracellular calcium. Regulation of exocytosis including degranulation occurs via the SNARE proteins. These consist of vesicular V-SNARE's such as VAMP's (vesicle-associated proteins, e.g. synaptobrevin) and target membrane T-SNARE's such as Syntaxin and SNAP-25. The SNARE's form a highly stable complex in which the core consists of a four-helix bundle. The SNARE proteins possibly mediate bilayer mixing and allow the contents of the vesicle to be released. Regulation is also controlled by other membrane bound proteins; these include the Munc18 proteins, Rab3D and possibly most importantly Synaptotagmin. Synaptotagmin (Syt) has been shown to be a calcium sensor within the regulation of the degranulation process. Sudhof (1993) showed this by equilibrium dialysis in presence of phospholipids showed binding of Ca2+ greatly increased, indicating that this was the calcium sensor of the vesicles*****(nb - not what it showed, need to read paper to say how he did this). It has also been well established that within neurons that Syt has a role in regulated exocytosis. This was first shown by injecting modified Syt fragments that then inhibited dopamine B-hydrolyase staining, a measure of regulated staining in cells being studied.

There are at least 15 Syt isoforms found within mammals although not all are involved in regulated exocytosis. Within mammals the isoforms of Syt 1, 2, 7 and 9 are the main calcium sensors for vesicular exocytosis (sudhof 2009 paper). Through experiments that cause point mutations in Syt1 without altering structure it can be confirmed that Syt1 is a calcium sensor. This is because the mutations that cause a change in the affinity of Syt1 for SNARE's or phospholipids these then cause a corresponding change in Ca2+ affinity for neurotransmitter release. The structure amongst the Syt isoforms is highly conserved with a single transmembrane domain, intravesicular n-terminus, variable linker domain and two C-domains (C2A and C2B) See fig.

Syt2 has been identified as being expressed as well as being the most abundant form of Synaptotagmin within the mast cell line RBL-2H3. This was shown in Baram (1999) by using RNase protection assay of the RBL cell transcripts followed by autoradiography of the products. (picture) Syt1 was shown not to be found within this cell line supporting the criticism of findings of studies based on Syt1 as being unrepresentative of mast cell studies. Possible note - theory that syt1 and 2 highly similar, variation in point, possible alteration of affinity for ca2+ in C2A domain? Baram eludes possible post translational modification is the difference, However, other studies show syt1 and 7 in the mast cells. This is a problem with research in this area, not much on mast cells, need to apply other findings.

Syt2 : Positive or negative regulator of exocytosis?

Baram 1999 suggested that Syt2 acted as a negative regulator in the release of Β-hexosaminidase, whether stimulated by Ca2+ or by FceRI trigger. The action for Syt 2 was shown by creating a down-regulated form of Syt (Syt2-) that evoked a higher level of Β-hexosaminidase release than a wild-type or up-regulated form of Syt (Syt2+). However, contrary to this there is evidence put forward by Melicoff (2009) that suggests the opposite function for Syt2, as a positive regulator of b-hxo release. This was shown by creating Syt2 knock-out mice (Syt-/-) and triggering the mast cell via FceRI. This caused release of b-hxo to be decreased relative to the wild-type. The reason for the difference in these two experiments seems to lie within the experimental procedures. The work by Baram shows that when placed on a continous sucrose gradient, the Syt2- tranfected cells comigrated with 60% of b-HXO activity and not the histamine containing granules. The authors then go on to show a increase in release of cathepsin-d in the syt2- cells similar to that of b-hxo. This suggests that there are different sub-populations of granules within the mast cells and b-hxo is found within both these subpopulations. Thus the reliability of using B-hxo to measure the effectiveness of Syt2 has to be questioned as it is difficult to determine which subpopulation of granules is being studied. * Another possible reason for the difference's noted could arise from the use of BMMC's in Melicoff's research, whereas RBL-2H3 cells in Baram. Using immunoblotting it can be seen that expression of Syt2 is equivalent in all these cells but it could be possible that there are difference's in other areas, such as differences in sub-granule populations, within these mast cells that could have and effect on results. * need to re-write

Syt2 as regulator in Mast Cell Degranulation

As shown before the Syt-/- mice have an impaired B-Hxo release response (melicoff). The research also shows impairment in the passive cutaneous anaphylaxis (PCA) response, a model for allergic response, within mice. This was shown by mast cell deficient but Syt2-sufficent mice being rescued with Syt2+/+ BMMC's or Syt2-/- BMMC's and the corresponding PCA response being recorded. The Syt2-/- rescue response was approximately one third the response of the Syt2+/+ rescue. These in vivo findings were further supported by in vitro findings in which Syt-/- and Syt+/+ cells were FceRI stimulated. This was performed with an antigen to cell ratio that was the same as recorded for the maximum secretory response in vivo. Measurements of Histamine and B-HXO activity were recorded after stimulation. The results of this again showed that Syt-/- had approximately one third the response of the Syt+/+ cells. The effect of the Syt2-/- on both constitutive and non-exocytotic release showed no difference to that of Syt+/+, indicating only regulated exocytosis is involved. These results strongly demonstrate a role for Syt2 as a major regulator of degranulation and thus the PCA reaction in vivo.

However, the major problem within this is that Syt2 is cannot be the sole regulator of regulated exocytosis. It indicates that other factors must be involved in regulated exocytotic release of histamine. This conclusion can be drawn due to the 'residual activity' that remains after Syt2 is knocked out and hence there is not a complete abolition of either the PCA reaction or Histamine release. Or, it could be that the method for measuring regulated exocytosis is unreliable and that the release of histamine/ Β-hexosaminidase is controlled via another method. Having stated this, it is unlikely that the method of measuring regulated exocytosis is poor as the IgE cross stimulation method is a well-established method of stimulating deregulation (Rivera 2006).

In adrenal chromaffin cell vesicles it has been shown that Syt-7 can compensate for Syt1 -/- (Schonn et al. 2008). This indicates that it is possible that a form of compensation may occur within the Syt isoforms that could occur in mast cells. This could explain the minimal regulated exocytosis function seen by Melicoff. Within the RBL-2H3 cells, Baram noted Syt isoforms 2, 3 and 5 from the RNase protection assay, however, only 4 iosforms were tested for in this experiment and subsequently it has been shown that Syt7 can be found in lysosomal compartments. Therefore it could be that only certain types of Syt can compensate for Syt2 -/- or that Syt7 is a universal compensator. To test which isoform is responsible for compensation, it is proposed that the testing of other isoforms of Syt knockouts along with Syt2-/- and then using the histamine measurement as used by Melicoff. This would allow it to be determined which Syt isoforms, along with Syt 2 would cause complete abolition of Histamine release and hence which isoforms control regulated exocytosis.

Synaptotagmin regulation in docking and fusion

Immunomediator secretion can be seen as an example of regulated exocytosis just like at the nerve terminal, i.e. both process are immediate. The time delay noted in the effects of mast cell products (eg histamine) is due to the fact that the products operate on a transcriptional level and time response is not due to 'slow' exocytosis. Syt has been shown to play a key role at the level of docking and fusion within these processes. Docking refers to the recognition and lining up of and the vesicle and target membrane in a fusion-ready state. Fusion refers to the actual merging of the vesicle membrane and target membrane to allow release of vesicle contents. Syt is theorized to have a role in both of these processes.

Firstly, Syt-1 has only recently been shown to have role in docking secretory vesicles. Research shows that in those cells in mouse embryonic chromaffin cells with Syt1null there is a greatly decreased number of docked vesicles per measured section compared to wild type cells (roughly 75% decrease). All other proteins involved in docking were the same with or without Syt1. This is a similar effect to that in the Snap-25 null-cells. Next by adding soluble C2AB (C2 domains of Syt) to the wild-type cells, led to a Syt-1 null phenotype. This is due to C2AB out competing endogenous Syt1 for binding to Syntaxin-1/SNAP-25 so docking cannot occur. This indicates that Syt1 binding to the Syntaxin-1/SNAP-25 complex is a factor for effective vesicle docking. The research also shows that Munc18-1 is not directly involved in docking but has a down stream function which is postulated to be allowing acceptation of the correct SNARE complex. It should be noted that in this research, Syt1null cells still did not have a complete loss of docking, just a significant decrease. This supports the idea of compensation within the Syt isoforms because, there is still some docking or 'residual activity'. Thus this suggests that although this process is not fully understood, it could be a possible area for regulation and needs further investigation before a conclusive answer can be drawn.

Syt-1 has also been shown to have an established role within vesicle fusion. Experiments have shown that reducing t-SNARE binding activity to Syt1 causes a corresponding decrease in rate of secretion on time spent open by the fusion pore. This suggests that the fusion pore is regulated in some way by the syt1 and SNARE interactions, and controlled by CA2+ binding to the C2B domain of Syt.(chapman ref96). At the level of fusion it has been shown that Syt must interact directly with the SNARE protein in order to 'drive fusion' (chapman). This is shown by replacing neuronal SNARE's with yeast SNARE's (can't interact with Syt) and which lead to the yeast SNARE's being able to interact with membranes and liposomes but a complete inability to stimulate fusion.

In Chapman's review of the role of Syt in fusion, he postulates that from evidence with Syt1 null mice, unbound Syt1 acts as a clamp for fusion whereas the Ca2+ bound form acts to enhance exocytosis. Hence, Syt1 clearly acts as a Ca2+ dependent regulator.

As the creation of fusion pore is one the final stages of fusion, and Syt1 acts to regulates at this stage, it is highly possible that within mast cells a similar process, albeit possibly a different form of Syt, could therefore be regulating immunomediator secretion at this stage.

Another interesting point that arises in Chapman's review is the ability of the Syt isoforms to hertro- and homo- oligomerise with each other.

Synaptotagmin regulation at the structural level

It has been established that Ca2+ binding to the C2B domain of Syt1 is essential for release of neurotransmitter in the synapse. This was shown via Syt1 via ref27 & 28. Sudhof 2009 has further added to this evidence, by performing point mutations (Aspartate to Alanine) that abolish Ca2+ binding at the C2 domains. This has shown that although the C2A domain is not necessary for neurotransmitter release; it is the regulator of the C2B domain binding. This shows that Ca2+ affinity for C2A affects the cooperativity of Ca2+ induced neurotransmitter release. This shows that any structural changes within the C2 domains can have profound effects on the regulation of exocytosis in the synapse. However, it has to be questioned whether this can be replicated within mast cells, this is because the type of exocytosis within the synapse is different to that of mast cells and also the isoform of syt varies within mast cells.

Syt as a novel drug target

Based on the evidence set out above it seems that Syt at present is not good candidate as a drug target, however, in the future this may change. Melicoff has shown that by knocking-out Syt2 in Mast cells, this can cause a large decrease in Histamine release; a major factor in chronic inflammatory disease. Whilst this alone would seem to make a ideal drug target, there are numerous problems.

Conclusion/Areas of Further study

From research it is highly likely that a complex of lipids, SNARE's, other proteins and Syt operate together and changes to any of these affect function of entire complex.