S100A8 and A9 have interesting roles in oxidative stress, acting as scavengers for reactive oxygen and nitrogen species. It is thought that in this way, they may contribute to protecting phagocytes from intracellular ROS. However, as mentioned above, they can also interact with the intracellular portion of NADPH oxidase of phagocytes and increase ROS production. Extracellular S100A8 and A9 can act as ROS scavengers and therefore may decrease oxidative stress 271 288.
S100A8 and A9 are susceptible to cysteine oxidation and s-nitrosylation. These post- translational modifications can lead to completely different protein functions and can promote dimerisation, which in turn can alter the functionality. In fact, most known functions of S100
proteins are mediated by homo- or hetero-dimers 271. There is evidence of increased
oxidative stress in SSc; this may be as a result of inflammation or recurrent hypoxia- reperfusion 3 11 198 289. Whatever the cause, oxidative stress may result in post-translational modification of S100A8 and A9 resulting in increased dimerisation and a specific functional repertoire.
5.4.2 Specific granule proteins.
Lysozyme C is found in the primary and secondary neutrophil granules. Other granule proteins are found expressed on the plasma membrane surface at increased concentrations after degranulation, reflecting the fusion of granule membrane with the plasma membrane
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(e.g. CD11b, CD63, cytochrome b245, lactoferrin, fMLP receptor). One hypothesis for the increased membrane expression of lysozyme C in SSc is that this may represent a similar insertion of granule protein into the membrane and may reflect degranulation. Further experiments would be required to confirm this.
Neutrophil granules tend to discharge in a predefined order with increasing concentrations of activating or priming stimuli: secretory vesicles are mobilized first, followed by gelatinase-
containing granules, secondary granules and finally primary granules (Fig.47) 218.
It is also interesting to note that different priming agents have differential effects on neutrophil degranulation. For instance TNFα, PAF and LPS all cause degranulation of secretory vesicles, secondary and gelatinase-containing granules, whereas GMCSF is capable of mobilising secretory vesicles only. Interleukin-8 results in degranulation of only
secretory vesicles and secondary granules at priming doses 230.
Fig.47. Neutrophil granules. Granules are mobilised hierarchically with increasing stimulant concentrations: secretory vesicles first, followed by gelatinase-containing granules, secondary granules, and finally primary granules.
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Other markers of specific granule degranulation were identified in this study but were excluded from the analysis due to the unreliability of the data. Cytochrome b-245 light chain was increased in 3/4 patient samples but, it was excluded from analysis because variability compared to the internal control was greater than 63% (158%). In addition, the cytochrome b-245 heavy chain was not increased in SSc samples and the data was more reliable as the variation from internal control was only 15%. β-integrin and the fMLP receptor were both detected in the sample but only 1 peptide of each were detected and the information available was insufficient to allow meaningful quantification.
5.4.3 Cytoskeletal proteins.
L-plastin (LCP-1) is an actin-bundling protein that consists of two actin-binding domains and a head-piece that contains two calcium-binding EF hand motifs (Fig.48).
Fig.48. Structure of L-plastin. ACT=actin-binding domains. HP=head-piece. Shaded areas in head-piece represent calcium binding EF hand motifs.
L-plastin is calcium regulated and its actin cross linking functions are inhibited by rises in calcium in the physiological range. However, in addition to its actin-bundling activity, it is also involved in signal transduction. L-plastin is phosphorylated in response to stimulation with inflammatory cytokines, PMA, chemotactic peptides and immune complex ligation of FcγRII when bound to solid surfaces 254 290-293.
An intact cytoskeleton is essential for many signal transduction pathways, likely providing a scaffold for signal transduction molecules, keeping them within the vicinity of receptors. Focal adhesions are aggregates of actin, actin-binding proteins and signal transduction
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L-plastin phosphorylation occurs at a serine residue (Ser5) in the head-piece. Cell permeant peptides derived from the head-piece and containing the phosphorylated serine are capable of inducing neutrophil adhesion through activation of αMβ2 integrins. This can be abrogated by blocking the actions of phosphoinositol 3-kinase and protein kinase C. However, if the peptide was exogenously phosphorylated at serine 5, the peptide induced adhesion and this action was not blocked by kinase inhibitors. This integrin activation however, is not
accompanied by an increase in calcium mobilisation or ROS generation 233.
Activation of integrins involves two distinct processes; first, there is an increase in integrin diffusion as a result of release from cytoskeletal constraints allowing integrin clusters to form; secondly, there is a conformational change within the integrin resulting in increased affinity for the ligand. L-plastin has been implicated in both these roles. The cell permeant L-plastin
peptides described above were shown to mediate their effects on αvβ3 integrins through actin
depolymerisation, presumably to increase integrin diffusion, and by co-operating with Arg- Gly-Asp ligand to change the conformation of the integrin to a higher affinity form 234.
This is interesting in the light of data presented in Chapter 4 which describes a decrease in F-actin content but an increase in focal adhesions in SSc neutrophils compared to controls. Although other activating stimuli are associated with an increase in F-actin, L-plastin- dependent signalling results in a decrease in F-actin but an increase in focal adhesions as seen in SSc neutrophils. Taken together with the increase in L-plastin expression, this may imply that SSc neutrophils have been activated by a stimulus that acts through L-plastin and as a result is likely to increase integrin function and adhesion, though not necessarily through an increase in integrin expression. This reflects the SSc neutrophil phenotype which I have observed to be more “sticky” (see Chapter 3) and the effect of SSc serum on healthy neutrophils which induces cell-cell interactions and the formation of multi-cellular aggregates (see Chapter 7).
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An upregulation of membrane L-plastin has not previously been described and therefore the functional implications are not fully known. However, it is speculated that phosphorylation of L-plastin may regulate its molecular location, specifically, localisation to podosomes on the cell membrane 291.
Neutrophils of L-plastin knock-out mice have defective adhesion-dependent respiratory burst and hence a decreased capacity to kill S. aureus despite normal phagocytosis. This effect
was due to markedly decreased integrin dependent syk activation. However, unlike β2
integrin knockouts, L-plastin knockout murine neutrophils have normal adhesion to
fibrinogen, fibronectin and vitronectin 295. This result is, however, somewhat confusing since
β2 integrins do not bind to fibronectin or vitronectin. A recent study showed that engagement
of β2 integrins induce surface expression of β1 integrins, which do bind to extracellular matrix
components, but this does not explain the findings outlined above 296. Knockout models may
have their limitations since murine integrins may not faithfully represent the functions of human integrins and knock-outs may also disrupt epigenetic phenomena. The normal adhesion and spreading of L-plastin knockout neutrophils, does not rule out an important role for L-plastin in the regulation of integrin function, as many other interactions contribute to neutrophil adhesion. The neutrophils in this study were also stimulated with TNFα (1nM for 30min).
Phosphorylation of L-plastin has also been implicated in activation of neutrophil NADPH- oxidase but further studies are needed to confirm this 297.
It is interesting to note that integrin-linked protein kinase is increased in 2/4 patients with SSc. This protein is found in the podosomes and is involved in integrin-dependent signalling. It is implicated in cytoskeletal reorganization and inside-out integrin signalling. It phosphorylates the cytoplasmic domain of the β-integrins and may be implicated in integrin ligand avidity.
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5.4.4 Catalase expression.
Catalase is an important anti-oxidant in neutrophils. It catalyses the decomposition of hydrogen peroxide into water and oxygen. It is predominantly found in the organelles of neutrophils. To my knowledge, there are no reports of plasma membrane expression of catalase.
There are two important mechanisms in the neutrophil that protect them from oxidative damage. The first is catalase, the second is the cytosolic glutathione reductase system which reduces hydrogen peroxide by oxidising NADPH, itself regenerated by the hexose monophosphate shunt. Neutrophils express less glutathione and glutathione peroxidise than
monocytes and macrophages but higher concentrations of catalase 298. Experiments on
neutrophils from acatalasemic patients have revealed that the glutathione reductase system is sufficient to protect neutrophils from endogenous ROS, but insufficient to protect from
exogenous oxidative stress 299. Catalase in neutrophils affords them protection against
exogenous oxidative stress and the higher levels of catalase found in neutrophils compared to other inflammatory cells means that they are more resistant to extracellular oxidative damage 298.
In other cell types such as liver and endothelial cells, oxidative stress can induce the expression of catalase 300 301, but it is not known whether this is also the case in neutrophils. This could explain the apparent upregulation found in this proteomic study. An alternative explanation is that catalase is translocated to the plasma membrane under conditions of exogenous oxidative stress, but this remains to be confirmed. Finally, although my experiments would imply an increase in the plasma membrane expression of catalase, the possibility cannot be excluded that there is contamination of the membrane sample. Catalase is primarily located in the cytoplasmic compartment and is co-localised with lactate
dehydrogenase (LDH) 302. LDH was also found in the membrane fraction, indicating some
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contamination to be greater in the SSc neutrophils. All samples were treated in exactly the same manner so there is no reason to suspect that the increased contamination with catalase and LDH occurred for methodological reasons. It is more likely that these proteins have an increased association with the plasma membrane fraction in SSc neutrophils either due to a specific increase in plasma membrane expression or due the increased expression of other membrane proteins which lead to increased adhesion of LDH and catalase indirectly. Other cytoplasmic proteins are not differentially expressed between SSc and controls in the plasma membrane samples.
The overall signature of the neutrophil phenotype in SSc cannot be confirmed from these data, predominantly due to the small number of samples examined. However, the data do point to some membrane proteins of potential interest for examination using other techniques (e.g. flow cytometry) which will allow examination of a larger number of patients. There is insufficient evidence from these proteomic studies to confirm or refute in vivo
neutrophil priming in SSc. However, if further work can confirm that lysozyme C is a marker of degranulation and that lysozyme C is increased on the plasma membrane of SSc neutrophils, this will provide tantalising evidence of neutrophil priming in SSc. Priming is not an all or nothing response but rather a continuous spectrum of responses depending on the concentration of the priming agent. In addition, different priming agents have different effects on neutrophil phenotype. These factors, coupled with the heterogeneity of SSc, may go some way to explain the high degree of variation seen in SSc neutrophil protein expression.
Despite the obvious limitations of this approach, it does have some great strengths. It allows for an unbiased examination of the changes in membrane protein expression, and it has revealed important but unexpected changes, such as the down-regulation of membrane calgranulins and the up-regulation of L-plastin and catalase. These changes may give important clues to the factors in SSc which give rise to the change in neutrophil phenotype. For instance, the increase in catalase expression, if confirmed, could imply activation by
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exogenous oxidative stress which is well established in SSc. The increase in membrane L- plastin may reflect translocation to the membrane following phosphorylation. L-plastin is phosphorylated in response to interleukin-8, which also acts as a neutrophil priming agent resulting not only in specific granule degranulation but in the priming of ROS generation not only in response to fMLP but also to PMA which may tie in the functional changes seen in chapter 3. Interleukin-8 is raised in the serum and in lesional skin of patients with SSc and is associated with early disease 34 79 224.