In vitro investigations have revealed anti-inflammatory efficacy of melanocortin
treatments in human cells. The inhibitory actions of α-MSH on leukocyte endothelial interactions observed in the flow chamber model are supportive of the results obtained in the in vivo stroke model and builds confidence as to the potential to translate melanocortin treatments into a human inflammatory disease context. This work has also pinpointed these effects to specifically be mediated by melanocortin treatments acting on neutrophils rather than the endothelium. Whether the same holds true in the in vivo setting and at different time points following activation by different stimulus has however, yet to be determined. The chemotaxis assay has been used to further characterise the effect of melanocortin treatments on neutrophil functioning. In these pharmacological studies an important role for MC1 was revealed in inhibiting neutrophil chemotaxis to fMLP. This work thus shows that melanocortin treatments are not only active in modulating human cell leukocyte recruitment but can modulate multiple aspects of neutrophil functioning in response to different stimulus.
6.5. Future Directions
This work could be furthered to give a greater insight into role of individual MCs, by the use of blocking antibodies to the melanocortin receptors. HUVECs and neutrophils could again be treated separately with blocking antibodies for different MCs before giving non selective melanocortin treatments. Alternatively blocking antibodies could be used to study the endogenous production and influence of the melanocortins. However such work would rely on the specificity of such antibodies which is currently under some conjecture.
Another, more physiological relevant approach that could be pursued, to further investigate the role of MC subtypes in supressing neutrophil recruitment, would be to take advantage of natural MC mutations in humans. Loss of function MC1 mutations are relatively common in humans, arising in ~80 % of individuals with red hair and fair skin [323, 324]. Since MC1 mutations confer these easily observable physical characteristics, during blood donor recruitment people with this phenotype could be actively encouraged to participate. PCR genotyping for four common MC1 variants; D84E, R151C, R160W and D294H, could then be used to further inform results obtained from in vitro assays allowing an insight into the role of MC1 in neutrophil function.
Investigating the role of MC3 and MC4 using this strategy would however be a more complex task as these mutations are much rarer and are associated with an obese phenotype. In humans, the MC3 missense variants Thr6Lys and Val81Ile have been related with reduced in vitro expression of the receptor and diminished functionality [325] and is associated with childhood obesity, and elevated body fat percentage in comparison to non-carriers [326]. While genetic mutations of MC4 represent the biggest monogenic cause of childhood obesity, these are again rare mutation. In a study by Farooqi et al. from a group of 500 patients with severe childhood obesity, just 5.8 % displayed mutations in MC4 with only 6 individuals being homozygous [327]. Such investigations would be further hampered by the difficulties of extrapolating which findings are due to direct effects of MC dysfunction on immune functioning from those that are secondary to the metabolic dysfunction and the multitude of conditions associated with obesity.
While the flow chamber model used in the present investigation uses TNF-α (a key pro- inflammatory cytokine up regulated following stroke) to activate endothelial cells, this model could further tailored to more faithfully mimic stroke. Varying levels of glucose and oxygen deprivation could be used to activate endothelial cells, as previously performed in dermal microvascular endothelial cells by Lee et al.[328]. Furthermore brain microvascular cells could be seeded rather than the more readily available HUVECs. While the current model used demonstrates a proof of principle, confirming melanocortin action on leukocyte recruitment in human cells, this model also provides a useful tool for further research into the actions of the melanocortins.
The current investigations using the chemotaxis assay could also be built upon to analyse the chemotactic functioning of neutrophils in response to a number of different chemoattractants such as IL-8. Indeed following stroke a number of chemotactic factors are released by both resident and infiltrating immune cells, such as; Neutrophil Activating Protein 3 (NAP-3, CXCL1) [329], Macrophage Inflammatory Protein-1α (MIP- 1α, CCL3) [330], IL-8 (CXCL8) [331] and fractalkine (CXC3CL1)[332] each of which could be investigated in this assay. In the more complex in vivo situation these molecules create a pro-inflammatory milieu with multiple signals influencing leukocyte functioning. Examining more complex chemotactic solutions could provide an interesting insight into how different chemical signals could act to enhance or inhibit the chemotactic outcome. In order to model this complex chemical microenvironment experiments could be performed substituting the chemo-attractant in the bottom chamber with culture medium from cultured neuronal or glial cells that have been deprived of glucose and oxygen.
Another avenue of research could be to introduce a mixed population of leukocytes to the top level of the plate. Indeed many chemoatractants display differential chemotactic potencies and selectivity for different leukocyte populations, for instance MCP-1 (CCL2) attracts monocytes but not neutrophils whereas CXCL1 recruits neutrophils but not monocytes. The effect of melanocortin treatments on the composition of leukocyte populations recruited could be indicative of therapeutic value. While neutrophil recruitment is largely considered a pathological event in stroke and the arrival of monocytes leading to an M1 phenotype may enhance inflammatory responses, the recruitment of monocytes assuming an M2 phenotype marks the beginnings of
inflammatory resolution and is associated with the clearance of apoptotic cells and the release of IL-10 and pro-resolution factors. Differential expression of receptors on sub sets of monocytes mean that they may respond to different stimuli and be recruited differentially. For instance CCR2 is only expressed on inflammatory monocyte thus only the pro-inflammatory phenotype will respond to MCP-1. Assessing the effects of melanocortins on the composition of leukocyte recruitment could make an interesting progression of this project.
7.1. Summary of important results
Throughout this project a murine model of global cerebral I/R has been used to investigate the actions of the melanocortins on leukocyte recruitment in the brain. This BCCAo model was characterised by a rapid I/R induced recruitment of leukocytes in the pial microcirculation, which escalated as the length of reperfusion was extended. Neutrophil depleting experiments revealed the recruited cells consisted largely or even entirely of neutrophils. Coinciding with the enhanced neutrophil recruitment at 2 h, was an increase in circulating TNF-α and an activation of brain resident immune competent microglia.
Melanocortin treatments have been shown to inhibit leukocyte recruitment in a number of different models [127, 140, 151], however the effect of these peptides on the leukocyte recruitment cascade have until now not been investigated in the unique microcirculation found in the brain. During the course of this project the physiological roles and pharmacological potential of the MRS has been investigated in an inflammatory context relevant to stroke.
Treatments with the endogenous non-selective melanocortin agonist α-MSH revealed potent inhibitory actions on both rolling and adhesion of I/R induced leukocytes. These effects appeared to be via actions on vascular or circulating elements as no effect on microglial activation was observed, as would have been expected from previous in vitro studies [269].
Pharmacological investigations identified both MC1 and MC3 to suppress cerebral leukocyte recruitment. MC1 was found to be most important at reducing early leukocyte recruitment events however by 2 h of reperfusion MC3 mediated anti-inflammatory signalling appeared to predominate.
In addition to inhibitory actions on leukocyte recruitment melanocortin treatments reduced the expression of the pro-inflammatory cytokines TNF-α and MCP-1. However,